Methods, recombinant host cells and kits are disclosed-for the production of members of specific binding pairs (sbp), e.g. antibodies, using display on the surface of secreted replicable genetic display packages (rgdps), e.g. filamentous phage. To produce a library of great diversity recombination occurs between first and second vectors comprising nucleic acid encoding first and second polypeptide chains of sbp members respectively, thereby producing recombinant vectors each encoding both a first and a second polypeptide chain component of a sbp member. The recombination may take place in vitro or intracellularly and may be site-specific, e.g. involving use of the loxP sequence and mutants thereof. Recombination may take place after prior screening or selecting for rgdps displaying sbp members which bind complementary sbp member of interest.

The present invention relates to methods for producing members of specific binding pairs (sbp). In particular, the present invention relates to methods for producing members of specific binding pairs involving recombination between vectors which comprise nucleic acid encoding polypeptide chain components of sbp members.
Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds. The light chains exist in two distinct forms called kappa (K) and lambda (xcex). Each chain has a constant region. (C) and a variable region (V). Each chain is organized into a series of domains. The light chains have two domains, corresponding to the C region and the other to the V region. The heavy chains have four domains, one corresponding to the V region and three domains (1,2 and 3) in the C region. The antibody has two arms (each arm being a Fab region), each of which has a VL and a VH region associated with each other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognising the antigen and providing an antigen binding site (ABS). In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDR""s are the most variable part of the variable regions, and they perform the critical antigen binding function.
The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation and selection.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment (Ward et al., Nature 341:544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; and (vi) F(abxe2x80x2)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region.
Although the two domains of the Fv fragment are coded for by separate genes, it has proved possible to make a synthetic linker that enables them to be made as a single protein chain (known as single chain Fv (scFv));
Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci., USA 85:5879-5883 (1988)) by recombinant methods. These scFv fragments were assembled from genes from monoclonals that had been previously isolated.
Bacteriophage have been constructed that express and display at their surface a large biologically functional binding molecule (e.g. antibody fragments, and enzymes and receptors) and which remain intact and infectious. This is described in WO 92/01047, the disclosure of which is herein incorporated by reference. Readers of the present document are urged to consult WO 92/01047 for detailed explanation of many of the procedures used in the experiments described herein. The applicants have called the structure which comprises a virus particle and a binding molecule displayed at the viral surface a xe2x80x9cpackagexe2x80x9d. Where the binding molecule is an antibody, an antibody derivative or fragment, or a domain that is homologous to an immunoglobulin domain, the applicants call the package a xe2x80x9cphage antibodyxe2x80x9d (pAb). However, except where the context demands otherwise, where the term phage antibody is used generally, it should also be interpreted as referring to any package comprising a virus particle and a biologically functional binding molecule displayed at the viral surface.
pAbs have a range of applications in selecting antibody genes encoding antigen binding activities. For example, pAbs could be used for the cloning and rescue of hybridomas (Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-3837 (1989)), and in the screening of large combinatorial libraries (such as found in Huse et al., Science 246:1275-1281 (1989)). In particular, rounds of selection using pAbs may help in rescuing the higher affinity antibodies from the latter libraries. It may be preferable to screen small libraries derived from antigen-selected cells (Casali et al., Science 234:476-479 (1986)) to rescue the original VH/VL pairs comprising the Fv region of an antibody. The use of pAbs may also allow the construction of entirely synthetic antibodies. Furthermore, antibodies may be made which have some synthetic sequences e.g. CDRs, and some naturally derived sequences. For example, V-gene repertoires could be made in vitro by combining un-rearranged V genes, with D and J segments. Libraries of pAbs could then be selected by binding to antigen, hypermutated in vitro in the antigen-binding loops or V domain framework regions, and subjected to further rounds of selection and mutagenesis.
The demonstration that a functional antigen-binding domain can be displayed on the surface of phage, has implications beyond the construction of novel antibodies. For example, if other protein domains can be displayed at the surface of a phage, phage vectors could be used to clone and select genes by the binding properties of the displayed protein. Furthermore, variants of proteins, including epitope libraries built into the surface of the protein, could be made and readily selected for binding activities. In effect, other protein architectures might serve as xe2x80x9cnouvellexe2x80x9d antibodies.
The technique provides the possibility of building antibodies from first principles, taking advantage of the structural framework on which the antigen binding loops fold. In general, these loops have a limited number of conformations which generate a variety of binding sites by alternative loop combinations and by diverse side chains. Recent successes in modelling antigen binding sites augurs well for de novo design. In any case, a high resolution structure of the antigen is needed. However, the approach is attractive for making e.g. catalytic antibodies, particularly for small substrates. Here side chains or binding sites for prosthetic groups might be introduced, not only to bind selectively to the transition state of the substrate, but also to participate directly in bond making and breaking. The only question is whether. the antibody architecture, specialised for binding, is the best starting point for building catalysts.
Genuine enzyme architectures, such as the triose phosphate isomerase (TIM) barrel, might be more suitable. Like antibodies, TIM enzymes also have a framework structure (a barrel of xcex2-strands and xcex1-helices) and loops to bind substrate. Many enzymes with a diversity of catalytic properties are based on this architecture and the loops might be manipulated independently on the frameworks for design of new catalytic and binding properties. The phage selection system as provided by the present disclosure can be used to select for antigen binding activities and the CDR loops thus selected, used on either an antibody framework or a TIM barrel framework. Loops placed on a e.g. a TIM barrel framework could be further modified by mutagenesis and subjected to further selection.
One class of molecules that could be useful in this type of application are receptors. For example, a specific receptor could be displayed on the surface of the phage such that it would bind its ligand. The receptor could then be modified by, for example, in vitro mutagenesis and variants having higher binding affinity for the ligand selected. The selection may be carried out according to one or more of the formats described below.
Alternatively, the phage-receptor could be used as the basis of a rapid screening system for the binding of ligands, altered ligands, or potential drug candidates. The advantages of this system namely of simple cloning, convenient expression, standard reagents and easy handling makes the drug screening application particularly attractive. In the context of this discussion, receptor means a molecule that binds a specific, or group of specific, ligand(s). The natural receptor could be expressed on the surface of a population of cells, or it could be the extracellular domain of such a molecule (whether such a form exists naturally or not), or a soluble molecule performing a natural binding function in the plasma, or within a cell or organ.
Another possibility, is the display of an enzyme molecule or active site of an enzyme molecule on the surface of a phage (see examples 11, 12, 30, 31, 32 and 36 of WO 92/01047). Once the phage enzyme is expressed, it can be selected by affinity chromatography, for instance on columns derivatized with transition state analogues. If an enzyme with a different or modified specificity is desired, it may be possible to mutate an enzyme displayed as a fusion on bacteriophage and then select on a column derivatised with an analogue selected to have a higher affinity for an enzyme with the desired modified specificity.
Although throughout this application, the applicants discuss the possibility of screening for higher affinity variants of pAbs, they recognise that in some applications, for example low affinity chromatography (Ohlson, S. et al Anal. Biochem. 169, p204-208 (1988)), it may be desirable to isolate lower affinity variants.
pAbs also allow the selection of antibodies for improved stability. It has been noted for many antibodies, that yield and stability are improved when the antibodies are expressed at 30xc2x0 C. rather than 37xc2x0 C. If pAbs are displayed at 37xc2x0 C., only those which are stable will be available for affinity selection. When antibodies are to be used in vivo for therapeutic or diagnostic purposes, increased stability would extend the half-fife of antibodies in circulation.
Although stability is important for all antibodies and antibody domains selected using phage, it is particularly important for the selection of Fv fragments which are formed by the non-covalent association of VH and VL fragments. Fv fragments have a tendency to dissociate and have a much reduced half-life in circulation compared to whole antibodies. Fv fragments are displayed on the surface of phage, by the association of one chain expressed as a gene m protein fusion with the complementary chain expressed as a soluble fragment. If pairs of chains have a high tendency to dissociate, they will be much less likely to be selected as pAbs. Therefore, the population will be enriched for pairs which do associate stably. Although dissociation is less of a problem with Fab fragments, selection would also occur for Fab fragments which associate stably. pAbs allow selection for stability to protease attack, only those pAbs that are not cleaved by proteases will be capable of binding their ligand and therefore populations of phage will be enriched for those displaying stable antibody domains.
The technique of displaying binding molecules on the phage surface can also be used as a primary cloning system. For example, a cDNA library can be constructed and inserted into the bacteriophage and this phage library screened for the ability to bind a ligand. The ligand/binding molecule combination could include any pair of molecules with an ability to specifically bind to one another e.g. receptor/ligand, enzyme/substrate (or analogue), nucleic acid binding protein/nucleic acid etc. If one member of the complementary pair is available, this may be a preferred way of isolating a clone for the other member of the pair.
The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv""s (scFv), ed because heavy and light chain variable domains, normally on two separate proteins, are covalently joined by a flexible linker peptide. Alternative expression strategies have also been successful. Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to g3p.
More recent cloning has been performed with xe2x80x98phagemidxe2x80x99 vectors which have ca. 100-fold higher transformation efficiencies than phage DNA. These are plasmids containing the intergenic region from filamentous phages which enables single-stranded copies of the phagemid DNA to be produced, and packaged into infectious filamentous particles when cells harbouring them are infected with xe2x80x98helperxe2x80x99 phages providing the phage components in trans. When phagemids contain gIII fused to an antibody gene (e.g. pHEN-1), the resulting fusion protein is displayed on the phagemid particle (Hoogenboom et al., Nucleic Acids Res. 19(1S):4133-4137 (1991)). Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Efficient strategies have been developed for cloning antibody genes, a factor which becomes most important when dealing with large numbers of different antibody fragments such as repertoires.
The cloning vector fd-DOG-1 was used in early work with phage antibody repertoires in which scFv fragments were derived from spleen mRNA of mice immunised with the hapten oxazalone (Clackson et al., Nature 352:624-628 (1991)). Making antibody fragments using phage display libraries. VH and VL domains were separately amplified then linked at random via a short DNA fragment encoding the scFv linker peptide to produce a library of approximately 105 different clones. This was panned against the immunising antigen to select combinations of VH and VL which produced functional antibodies. Several binders were isolated, one in particular having an affinity not far below that of the best monoclonal antibodies produced by conventional hybridoma technology.
In a mouse, at any one time there are approximately 107 possible H chains and 105 possible L chains, making a total of 1012 possible VH:VL combinations when the two chains are combined at random (these figures are estimates and simply provide a rough guide to repertoire size). By these figures, the above mouse library sampled only 1 in 107 of the possible VH:VL combinations. It is likely that good affinity antibodies were isolated in the work described in the preceeding paragraph because the spleen cells derived from an immunised donor in which B cells capable of recognising the antigen are clonally expanded and producing large quantities of Ig mRNA. The low library complexity in this experiment is partly due to the intrinsically low transformation efficiency of phage DNA compared to plasmid (or phagemid).
Marks et al. (Marks et al., By-Passing Immunization: Human Antibodies from V-Gene Libraries Displayed on Phage. J. Mol. Biol. 222:581-597 (1991)) and W092/01047 describe construction of an antibody repertoire from unimmunised humans cloned in the phagemid pHEN-1. This library, consisting of 3xc3x97107 clones has so far yielded specific antibodies to many different antigens. These antibodies tend to have the moderate affinities expected of a primary immune response, demonstrating that usable antibodies to a range of structurally diverse antigens can indeed be isolated from a single resource.
New binders can be created from clones isolated from phage antibody libraries using a procedure called xe2x80x98chain-shufflingxe2x80x99. In this process one of the two chains is fixed and the other varied. For example, by fixing the heavy chain from the highest affinity mouse anti-OX phage antibody and recloning the repertoire of light chains alongside it, libraries of 4xc3x97107 were constructed. Several new OX-binders were isolated, and the majority of these had light chains that were distinct from those first isolated and considerably more diverse. These observations reflect the fact that a small library is sufficient to tap the available diversity when only one chain is varied, a useful procedure if the original library was not sufficiently large to contain the available diversity.
The size of the library is of critical importance. This is especially true when attempting to isolate antibodies from a naive human repertoire, but is equally relevant to isolation of the highest affinity antibodies from an immunised source.
It is clear that while phage display is an exceptionally powerful tool for cloning and selecting antibody genes, we are tapping only the tiniest fraction of the potential diversity using existing technology. Transformation efficiencies place the greatest limitation on library size with 109 being about the limit using current methods. Rough calculations suggest that this is several orders of magnitude below the target efficiency; more rigourous analysis confirms it.
Perelson and Oster have given theoretical consideration to the relationship between size of the immune repertoire and the likelihood of generating an antibody capable recognising a given epitope with greater than a certain threshold affinity, K. The relationship is described by the equation:
P=exe2x88x92N(p[K])
Where P=probability that an epitope is not recognised with an affinity above the threshold value K by any antibody in the repertoire,
N=number of different antibodies in the repertoire, and
p[K]=probability that an individual antibody recognises a random epitope with an affinity above the threshold value K
In this analysis p[K] is inversely proportional to affinity, although an algorithm describing this relationship precisely has not been deduced. Despite this, it is apparent that the higher the affinity of the antibody, the lower its p[K] and the larger the repertoire needs to be to achieve a reasonable probability of isolating that antibody. The other important feature is that the function is exponential; as shown in FIG. 1, a small change in library size can have either a negligible or a dramatic effect on the probability of isolating an antibody with a given p[K] value, depending upon what point on the curve is given by the library size.
WO 92/01047 and W092/20791 (also incorporated herein by reference) describe how the limitations of transformation efficiency (and therefore the upper limit on library size) can be overcome by use of other methods for introducing DNA into cells, such as infection. In one configuration, heavy and light chain genes are cloned separately on two different replicons, at least one of which is capable of being incorporated into a filamentous particle. Infectious particles carrying one chain are infected into cells harbouring the complementary chain; infection frequencies of  greater than 90% can be readily achieved. Heavy and light chains are then able to associate post-translationally in the periplasm and the combination displayed on the surface of the filamentous particle by virtue of one or both chains being connected to g3p. For example, a library of 107 heavy chains is cloned as an unfused population in a phagemid, and 107 light chains are cloned as g3 fusions in fd-DOG-1. Both populations are then expanded by growth such that there are 107 of each heavy chain-containing cell and 107 copies of each light chain phage. By allowing the phage to infect the cells, 107xc3x97107=1014 unique combinations can be created, because there are 107 cells carrying the same heavy chain which can each be infected by 107 phage carrying different light chains. When this is repeated for each different heavy chain clone then one ends up with up to 1014 different heavy/light combinations in different cells. This strategy is outlined in FIG. 2, which shows the heavy chain cloned as g3 fusions on phage and the light chains expressed as soluble fragments from a phagemid. Clearly, the reverse combination, light chains on phage, heavy chain on phagemid, is also tenable.
In the configuration shown in FIG. 2, fd-DOG xe2x80x98rescuesxe2x80x99 the phagemid so that both phage and phagemid DNA is packaged into filamentous particles, and both types will have paired heavy and light chains on their surface, despite having the genetic information for only one of them. For a given antigen or epitope, the vast majority of the heavy and light chain pairings will be non-functional (i.e. will not bind that antigen or epitope), so that selection on antigen will have the effect of vastly reducing the complexity of the heavy and light chain populations. After the first round of selection the clones are re-assorted, for example by infecting fresh host cells and selecting for both replicons. After several rounds of antigen selection and recovery of the two replicons, the considerably reduced heavy and light chain populations can be cloned onto the same replicon and analysed by conventional means. Selection from the, say, 1014 combinations produces a population of phages displaying a particular combination of H and L chains having the desired specificity. The phages selected however, will only contain DNA encoding one partner of the paired H and L chains. Selection for the two replicons may be as follows. Vectors of the H chain library may encode tetracycline resistance, with vectors of the L chain library encoding ampicillin resistance. The sample elute containing the population is divided into two portions. A first portion is grown on e.g. tetracycline plates to select those bacteriophage containing DNA encoding H chains which are involved in the desired antigen binding. A second portion is grown on e.g. ampicillin plates to select those bacteriophage containing phagemid DNA encoding L chains which are involved in the desired antigen binding. A set of colonies from individually isolated clones e.g. from the tetracycline plates are then used to infect specific colonies e.g. from the ampicillin plates. This results in bacteriophage expressing specific combinations of H and L chains which can then be assayed for antigen binding.
One technical problem with the use of separate replicons for VL and VH chains is so-called xe2x80x98interferencexe2x80x99 between filamentous phage origins of replication carried on different replicons as a result of competition for the same replication machinery.
Procedures have been described which work on the principle of first reducing the complexity of a repertoire then recloning one or both chains of the reduced population (WO92/20791). The present invention provides a different approach.
Much of the terminology discussed in this section has been mentioned in the text where appropriate.
This describes a pair of molecules (each being a member of a specific binding pair) which are naturally derived or synthetically produced. One of the pair of molecules, has an area on its surface, or a cavity which specifically binds to, and is therefore defined as complementary with a particular spatial and polar organisation of the other molecule, so that the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate, IgG-protein A.
This describes a first polypeptide which will associate with at least a second polypeptide, when the polypeptides are expressed in free form and/or on the surface of a substrate. The substrate may be provided by a bacteriophage. Where there are two associated polypeptides, the associated polypeptide complex is a-dimer, where there are three, a trimer etc. The dimer, trimer, multimer etc or the multimeric member may comprise a member of a specific binding pair.
Example multimeric members are heavy domains based on an immunoglobulin molecule, light domains based on an immunoglobulin molecule, T-cell receptor subunits.
This describes a biological particle which has genetic information providing the particle with the ability to replicate. The particle can display on its surface at least part of a polypeptide. The polypeptide can be encoded by genetic information native to the particle and/or artificially placed into the particle or an ancestor of it. The displayed polypeptide may be any member of a specific binding pair e.g. heavy or light chain domains based on an immunoglobulin molecule, an enzyme or a receptor etc.
The particle may be a virus e.g. a bacteriophage such as fd or M13 or other viruses.
This describes a replicable genetic display package in which the particle is displaying a member of a specific binding pair at its surface. The package may be a bacteriophage which displays an ,antigen binding domain at its surface. This type of package has been called a phage antibody (pAb).
This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any protein having a binding domain which is homologous to. an immunoglobulin binding domain. These proteins can be derived from. natural sources, or partly or wholly synthetically produced.
Example antibodies are the immunoglobulin isotypes and the Fab, F(ab1)2, scFv, Fv, dAb, Fd fragments.
This describes a family of polypeptides, the members of which have at least one domain with a structure related to that of the variable or constant domain of immunoglobulin molecules. The domain contains two xcex2-sheets and usually a conserved disulphide bond (see A. F. Williams and A. N. Barclay, Ann. Rev Immunol. 6:381-405 (1988)).
Example members of an immunoglobulin superfamily are CD4, platelet derived growth factor receptor (PDGFR), intercellular adhesion molecule. (ICAM). Except where the context otherwise dictates, reference to immunoglobulins and immunoglobulin homologs in this application includes members of the immunoglobulin superfamily and homologs thereof.
This term indicates polypeptides having the same or conserved residues at a corresponding position in their primary, secondary or tertiary structure. The term also extends to two or more nucleotide sequences encoding the homologous polypeptides.
Example homologous peptides are the immunoglobulin isotypes.
In relation to a sbp member displayed on the surface of a rgdp, means that the sbp member is presented in a folded form in which its specific binding domain for its complementary sbp member is the same or closely analogous to its native configuration, whereby it exhibits similar specificity with respect to the complementary sbp member. In this respect, it differs from the peptides of Smith et al, supra, which do not have a definite folded configuration and can assume a variety of configurations determined by the complementary members with which they may be contacted.
In connection with sbp members or polypeptide components thereof, this is referring not only to diversity that can exist in the natural population of cells or organisms, but also. diversity that can be created by artificial mutation in vitro or in vivo.
Mutation in vitro may for example, involve random mutagenesis using oligonucleotides having random mutations of the sequence desired to be varied. In vivo mutagenesis may for example, use mutator strains of host microorganisms to harbour the DNA (see Example 38 of WO 92/01047). The word xe2x80x9cpopulationxe2x80x9d itself may be used to denote a plurality of e.g. polypeptide chains, which are not genetically diverse i.e. they are all the same.
A domain is a part of a protein that is folded within itself and independently of other parts of the same protein and independently of a complementary binding member.
This is a specific combination of an xcex1-helix and/or xcex2-strand and/or xcex2-turn structure. Domains and folded units contain structures that bring together amino acids that are not adjacent in the primary structure.
This describes the state of a polypeptide which is not displayed by a replicable genetic display package.
This describes a gene which does not express a particular polypeptide under one set of conditions, but expresses it under another set of conditions. An example is a gene containing an amber mutation expressed in non-suppressing or suppressing hosts respectively.
Alternatively, a gene may express a protein which is defective under one set of conditions, but not under another set. An example is a gene with a temperature sensitive mutation.
This describes a codon which allows the translation of nucleotide sequences downstream of the codon under one set of conditions, but under another set of conditions translation ends at the codon. Example of suppressible translational stop codons are the amber, ochre and opal codons.
This is a host cell which has a genetic defect which causes DNA replicated within it to be mutated with respect to its parent DNA. Example mutator strains are NR9046mutD5 and NR9046 mut T1 (see Example 38 of WO92/01047).
This is a phage which is used to infect cells containing a defective phage genome and which functions to complement the defect. The defective phage genome can be a phagemid or a phage with some function encoding gene sequences removed. Examples of helper phages are M13KO7, M13K07 gene III no. 3; and phage displaying or encoding a binding molecule fused to a capsid protein.
This is a DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a recombinant DNA molecule.
This is a vector derived by modification of a phage genome, containing an origin of replication for a bacteriophage, but not one for a plasmid.
This is a vector derived by modification of a plasmid genome, containing an origin of replication for a bacteriophage as well as the plasmid origin of replication.
This describes a rgdp or molecule that associates with the member of a sbp displayed on the rgdp, in which the sbp member and/or the molecule, have been folded and the package assembled externally to the cellular cytosol.
A collection of naturally occurring nucleotides e.g. DNA sequences which encoded expressed immunoglobulin genes in an animal. The sequences are generated by the in vivo rearrangement of e.g. V, D and J segments for H chains and e.g. the V and J segments for L chains. Alternatively the sequences may be generated from a cell line immunised in vitro and in which the rearrangement in response to immunisation occurs intracellularly. The word xe2x80x9crepertoirexe2x80x9d is used to indicate genetic diversity.
A collection of nucleotide e.g. DNA, sequences within clones;
or a genetically diverse collection of polypeptides, or specific binding pair members, or polypeptides or sbp members displayed on rgdps capable of selection or screening to provide an individual polypeptide or sbp members or a mixed population of polypeptides or sbp members.
A collection of nucleotide e.g. DNA, sequences derived wholly or partly from a source other than the rearranged immunoglobulin sequences from an animal. This may include for example, DNA sequences encoding VH domains by combining unrearranged V segments with D and J segments and DNA sequences encoding VL domains by combining V and J segments.
Part or all of the DNA sequences may be derived by oligonucleotide synthesis.
This is a sequence of amino acids joined to the N-terminal end of a polypeptide and which directs movement of the polypeptide out of the cytosol.
This is a solution used to breakdown the linkage between two molecules. The linkage can be a non-covalent or covalent bond(s). The two molecules can be members of a sbp.
This is a substance which derived from a polypeptide which is encoded by the DNA within a selected rgdp. The derivative polypeptide may differ from the encoded polypeptide by the addition, deletion, substitution or insertion of amino acids, or by the linkage of other molecules to the encoded polypeptide. These changes may be made at the nucleotide or protein level. For example the encoded polypeptide may be a Fab fragment which is then linked to an Fc tail from another source. Alternatively markers such as enzymes, flouresceins etc may be linked to e.g. Fab, scFv fragments.
According to one aspect of the present invention there is provided a method for producing specific binding pair (sbp) members, which method comprises:
causing or allowing recombination between (a) first vectors comprising nucleic acid encoding a population of a first polypeptide chain of a specific binding pair member and (b) second vectors comprising nucleic acid encoding a population of a second polypeptide chain of a specific binding pair member, at least, one of said populations being genetically diverse, the recombination resulting in recombinant vectors each of which comprises nucleic acid encoding a said first polypeptide chain and a said second polypeptide chain. The sbp member may be xe2x80x9cmultimericxe2x80x9d. The sbp member may be a single chain, e.g. a scFv antibody fragment, as disclosed herein.
The first vectors may each encode a fusion of a said first polypeptide chain and a component of a replicable genetic display package (rgdp), the recombination resulting in recombinant. vectors each of which comprises nucleic acid encoding a said fusion and a said second polypeptide chain. The fusion and a said second polypeptide chain. The recombinant vectors may be capable of being packaged into rgdps using said rgdp component.
One or other or both of the populations of first and second polypeptide chains may be genetically diverse. Where both are genetically diverse, the recombinant vectors will represent an enormously diverse repertoire of sbp members. Either or both of the populations may be genetically diverse but restricted compared with the full repertoire available, perhaps by virtue of a preceding selection or screening step. A library of nucleic acid encoding a restricted population of polypeptide chains may be the product of selection or screeningxe2x80x2using rgdp display.
According to another aspect of the invention there is provided a method of producing multimeric specific binding pair (sbp) members, which method comprises:
(i) expressing from a vector in recombinant host organism cells a population of a first polypeptide chain of a specific binding pair member fused to a component of a replicable genetic display package (rgdp) which thereby displays said polypeptide chains at the surface of rgdps, and combining said population with a population of a second polypeptide chain of said specific binding pair member by causing or allowing first and second polypeptide chains to come together to form a library of said multimeric specific binding pair members displayed by rgdps, said population of second polypeptide chains not being expressed from the same vector as said population of first polypeptide chains, at least one of said populations being genetically diverse and expressed from nucleic acid that is capable of being packaged using said rgdp component, whereby the genetic material of each said rgdp encodes a polypeptide chain of a said genetically diverse population;
(ii) selecting or screening rgdps formed by said expressing to provide an individual sbp member or a mixed population of said sbp members associated in their respective rgdps with nucleic acid encoding a polypeptide chain thereof;
(iii) obtaining nucleic acid from a selected or screened rgdp, the nucleic acid obtained being one of (a) nucleic acid encoding a first polypeptide chain, (b) nucleic acid encoding a second polypeptide chain, and (c) a mixture of (a) and (b);
(iv) producing a recombinant vector by causing or allowing recombination between (a) a vector comprising nucleic acid obtained in step (iii) encoding a first polypeptide chain and a vector comprising nucleic acid encoding a second polypeptide chain, or (b) a vector comprising nucleic acid encoding a first polypeptide chain and a vector comprising nucleic acid obtained in step (iii) encoding a second polypeptide chain.
The recombination may take place intracellularly or in vitro, although it is preferable that it takes place in recombinant host cells. This is discussed elsewhere, but briefly this may involve introducing a library of vectors including nucleic acid encoding first (or second) polypeptide chain components of sbp member into host cells harbouring a library of vectors comprising nucleic acid encoding second (or first) polypeptide chain components of sbp members.
Following the recombination the polypeptide fusions (first polypeptide chains fused to a rgdp component) and the second polypeptide chains may be expressed, producing rgdps which display at their surface said first and second polypeptide chains and which each comprise nucleic acid encoding a said first polypeptide chain and a said second polypeptide chain, by virtue of the packaging of the recombinant vectors into rgdps. This expression may therefore produce an extremely diverse library of sbp members displayed on rgdp. In one embodiment, the rgdps displaying sbp member are pAbs (i.e. phage displaying antibodies or antibody fragments or derivatives), and those which bind antigen of interest may be selected using their binding capability. Since each pAb contains within it nucleic acid encoding both polypeptide chains of the antibody displayed on its surface, pAbs selected by binding to an antigen of interest will provide nucleic acid encoding an antibody which binds that antigen. The nucleic acid may be isolated from the selected pAbs and used in subsequent obtention of desired antibodies, after any amplification and cloning required in a given case.
The recombination may be promoted by inclusion in the vectors of sequences at which site-specific recombination will occur. This enables accurate design of the resultant recombinant vectors. For instance, a sequence at which site-specific recombination will occur may be position in the nucleic acid which encodes a polypeptide linker which joins the two domains of a single chain sbp member. The single chain sbp member may consist of an immunoglobulin VH domain linked to an immunoglobulin VL domain. VH and VL domains may associate to form an antigen binding site. The resultant recombinant vector may then comprise nucleic acid encoding a single chain Fv derivative of an immunoglobulin resulting from recombination between first and second vectors. (Note: a single chain sbp member, such as a scFv fragment or derivative of an antibody, may be considered to be multimeric (dimeric) because it consists of two polypeptide chain domains, such as VL and VH of an antibody.)
The sequences at which site-specific recombination will occur may be loxP sequences obtainable from coliphage P1, with site-specific recombination catalysed by Cre-recombinase, also obtainable from coliphage P1. The site-specific recombination sequences used may be derived from a loxP sequence obtainable from coliphage P1.
The Cre-recombinase used may be expressible under the control of a regulatable promoter.
In order to increase the efficiency of the method, increasing the proportion of productive recombination leading to the resultant recombinant vectors desired, each vector may include two site-specific recombination sequences each of which is different from other. The sequences should then be such that recombination will take place between like sequences on different vectors but not between the different sequences on the same vector.
Site-specific recombination sequences which are different may recombine inefficiently on the same vector. Preferably, recombination takes place preferentially between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.
Each of the first vectors and each of the second vectors may include a first site-specific recombination sequence and a second site-specific recombination sequence different from the first, site-specific recombination taking place preferentially between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.
The first site-specific recombination sequence may be loxP obtainable from coliphage P1 and the second site-specific recombination sequence a mutant loxP sequence, or vice versa. Potentially, both the first and second site-specific recombination sequences may be mutants, as long as the first sequence will not recombine with the second sequence as efficiently as first sequences will recombine with each other and second sequences will recombine with each other. Others include loxP 1, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8. Suitable sites may be selected on the basis of ability for like sites to recombine on different vectors preferentially over unlike sites on the same vector.
A third site-specific recombination sequence may be used in addition to and different from the first and second. Provided the third site-specific recombination sequence has a frequency of recombination with the first site-specific recombination sequence which is low compared with the frequency of recombination between first site-specific recombination sequences and a frequency of recombination with the second site-specific recombination sequence which is low compared with the frequency of recombination between second site-specific recombination sequences, the presence of the third site will not interfere with successful recombination between first sites and between second sites. The third site may be used in a further recombination step following the first, e.g. to transfer recombined sequences encoding first and second polypeptide chains of an sbp member from the recombinant vector into a further vector, e.g. for expression and/or fusion to nucleic acid encoding a component of and rgdp. Alternatively, the third site may be used in xe2x80x9cchain shufflingxe2x80x9d.
Thus, the present invention provides a method comprising causing or allowing recombination between (a) first vectors comprising nucleic acid encoding a specific binding pair (sbp) member and (b) second vectors, the vectors comprising site-specific recombination sequences and the site-specific recombination sequences of the first vectors flanking the nucleic acid encoding a specific binding pair member. The first vectors may comprise nucleic acid encoding a genetically diverse population of sbp members, as disclosed. As discussed above, the second vectors may comprise nucleic acid for expression of the sbp member following recombination and may comprise nucleic acid for expression of a fusion of the sbp member and a component of a rgdp.
While the first and second site-specific recombination sequences may flank the nucleic acid encoding the sbp member, a third site-specific recombination sequence (as discussed) mayxe2x80x2separate nucleic acid encoding each of two chains of the sbp member. A vector comprising such a construct may be provided by recombination between (i) vectors comprising nucleic acid encoding a first polypeptide chain flanked by two site-specific recombination sequences wherein one is a said first site-specific recombination sequence and the other is a said third site-specific recombination sequence and (ii) vectors comprising nucleic acid encoding a second polypeptide chain flanked by two site-specific recombination sequences wherein one is a said third site-specific recombination sequence and the other is a said second site-specific recombination sequence and further comprising a said first site-specific recombination sequence, recombination taking place preferentially between first site-specific recombination sequences on different vectors and between third site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a third site-specific recombination sequence on the same vector.
Where three site-specific recombination sequences are used they may be selected from the group consisting of loxP, loxP 511, loxP 1, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8.
A suitable mutant loxP sequence is loxP 511.
The first vectors may be phages or phagemids and the second vectors plasmids, or the first vectors may be plasmids and the second vectors phages or phagemids.
In one embodiment, the recombination is intracellular and takes place in a bacterial host which replicates the recombinant vector preferentially over the first vectors and the second vectors. This may be used to enrich selection of successful recombination events. The intracellular recombination may take place in a bacterial host which replicates plasmids preferentially over phages or phagemids, or which replicates phages or phagemids preferentially over plasmids. For instance, the bacterial host may be a PolA strain of E. coli or of another gram-negative bacterium. PolA cells are unable to support replication of plasmids, but can support replication of filamentous phage and phagemids (plasmids containing filamentous phage intergenic regions). So, for instance, if the first vectors are plasmids containing a first marker gene, and the second vectors are phage or phagemids containing a second marker gene, selection for both markers will yield recombinant vectors which are the product of a successful recombination event, since recombination transferring the first marker from plasmid must take place in order for that marker to be replicated and expressed.
Nucleic acid from one or more rgdp""s may be taken and used in a further method to obtain an individual sbp member or a mixed population of sbp members, or polypeptide chain components thereof, or encoding nucleic acid therefor.
The present invention also provides a kit for use in carrying out methods provided, having:
(i) a first vector having a restriction site for insertion of nucleic acid encoding or a polypeptide component of an sbp member, said restriction site being in the 5xe2x80x2end region of the mature coding sequence of a phage capsid protein, with a secretory leader sequence upstream of said site which directs a fusion of the capsid protein and sbp polypeptide to the periplasmic space of a bacterial host; and
(ii) a second vector having a restriction site for insertion of nucleic acid encoding a second said polypeptide chain,
at least one of the vectors having an origin of replication for single-stranded bacteriophage, the vectors having sequences at which site-specific recombination will occur.
The kit may contain ancillary components needed for working the method.
Also provided by the present invention are recombinant host cells harbouring a library of first vectors each comprising nucleic acid encoding a first polypeptide chain of a sbp member fused to a component of a secretable replicable genetic display package (rgdp) and second vectors each comprising nucleic acid encoding a second polypeptide chain of a sbp member, the first vectors or the second vectors or both being capable of being packaged into rgdps using the rgdp component, and the vectors having sequences at which site-specific recombination will occur.
According to another aspect of the present invention there is provided a population of rgdps each displaying at its surface a sbp member and each containing nucleic acid which encodes a first and a second polypeptide chain of the sbp member displayed at its surface and which includes a site-specific recombination sequence.
According to another aspect of the invention there is provided a population of rgdps each displaying at its surface a sbp member and each containing nucleic acid which comprises a combination of (i) nucleic acid encoding a first polypeptide chain of a sbp member and (ii) nucleic acid encoding a second poypeptide chain of a sbp member, the population containing 1010 or more combinations of (i) and (ii). Such a population exceeds in size the maximum which is achievable using available techniques. The present invention enables production of enormously diverse libraries or populations of rgdps displaying sbp members. The nucleic acid encoding a first polypeptide chain of a sbp member may have, for instance, 107 different sequences throughout the population. Where the nucleic acid encoding a second polypeptide chain of a sbp member also has such a genetic diversity throughout the population, the number of different combinations of nucleic acid encoding first and second polypeptide chains is immense.
Embodiments of the present invention will now be described in more detail by way of example only and not by way of limitation, with reference to the figures.

Methods for producing members of specific binding pairs


An apparatus and method for automatically detecting and recovering from a fault in a microprocessor-based system. The apparatus and method utilizes a leaky bucket routine and an event handler procedure. The method may further use Object Oriented techniques that abstracts differences between hardware and software faults to allow for the development of a common framework.

The present invention relates generally to fault detection and recovery and, more particularly, relates to a system and method for automatically detecting and recovering from software and/or hardware faults in microprocessor-based systems.
Microprocessor-based systems are used in an increasing number of applications, in part, because present day microprocessors are inexpensive and extremely powerful. Many of these systems are sophisticated and have complex software for driving the operation of the microprocessor and other hardware components. Since many of these systems, such as a router in a computer network, must operate continuously and unattended, the systems must be designed to operate in the presence of faults. These faults can be hardware faults or software faults resulting from hardware or software malfunctions.
In most microprocessor-based systems, fault detection and recovery is not implemented. In those rare cases where fault detection and recovery is implemented, the implementation is relatively primitive and informal. Specifically, it is typically left to the discretion of the hardware and software developers to design fault detection and recovery into their software processes which creates many problems. For example, any fault detection and recovery that does exist is tightly coupled and intertwined with the software process so re-use is difficult or impossible. This is especially true since software and hardware faults are typically handled by separate modules and not by one integrated module. Additional problems arise since many software processes are designed to exit when a fault occurs requiring the system to be manually restarted or rebooted to resume operation.
From the foregoing, it will be appreciated that a need exists for a more formal and comprehensive approach to hardware and fault detection and recovery. There is also a need for a fault detection and recovery method that can be easily re-used by any process or module in a system product. Finally, there is a need for fault recovery that is automatic in the sense that manual intervention is not required to recover from the fault.

This patent is subject to a terminal disclaimer.

System and method for fault detection and recovery


A method is disclosed for transmitting messages in a communications network with at least three communications nodes. At least one communications node always remains active and stores messages sent by a second communications node. If the second communications node switches to an idle state, the caching node is informed thereof by means of a sleep-event and takes over the sending of the messages for this node. By means of a wake-up event, the caching node obtains information that the second node has switched back into the active state and ceases sending the stored messages. Further nodes, which likewise can switch to the idle state at any time and possibly not receive messages sent by the second node during their own idle phase, thus do not notice anything about the idle state of the second node and operate on the assumption of an up-to-date status in the network at all times.

Particularly in motor vehicles, many different controllers are used today. In order to connect these individual electronic system components, bus systems are used, such as CAN (Controller Area Network) or FlexRay. In established bus systems, it is possible to transfer the controllers that are connected to the bus to a quiescent state with reduced power consumption when none of the controllers are required. In this case, however, it is then possible for all respective bus systems and controllers of a connected system to be put into the quiescent state or back into the active state again simultaneously. Even if only a single arbitrary controller is requested for communication on an arbitrary bus, all the others likewise need to be in an active state. Therefore, even controllers that are not required for communication in this period are consuming power.
As part of the reduction of CO2 emissions from vehicles, what is known as partial networking has therefore been developed. In this case, it is possible for individual controllers (node partial networking) or buses (bus partial networking) to be put to "sleep" or "awoken" as required. By way of example, it is thus possible for a trailer controller to remain switched off while there is no trailer on a vehicle, even when other controllers are required on a bus during this time. In this way, controllers consume power only when they are also really needed, and the total power consumption in the vehicle and hence CO2 emissions can be reduced.
EP 0 981 875 B1 describes such a bus system having a plurality of controllers that allows partial networking in order to reduce the average power consumption of an overall bus system having 30 controllers from 2.5 mA to approximately 1.6 mA.
A problem that arises with the introduction of such partial networking is that the controllers can now see any messages or message changes that are sent while they are switched on. Buffer-storing (caching) the values locally in the sleeping controller, that is to say buffer-storing the original or a copy thereof there in a buffer store, for example, as is generally frequently done in data processing, is not possible in this case. If messages were to be sent by other controllers in the system while a controller is sleeping, the controller would, upon being woken up, set out from the state that is stored in its buffer store (cache), which is possibly already outdated when it wakes up, however.

Method for transmitting messages in a communication network


A pivot assembly bearing includes a shaft, a sleeve provided coaxially with the shaft, and a pair of rolling bearings arranged to be separated from each other in an axial direction between the shaft and the sleeve. The sleeve includes a ventilation path configured to communicate a region surrounded by the shaft, the pair of rolling bearings, and the sleeve with the outside of the sleeve.

1. Field of the Invention
The present invention relates to a pivot assembly bearing and a hard disk drive device.
2. Description of the Related Art
A hard disk drive device is one of storage devices for computers. For example, in the hard disk drive device, a swing arm is supported by a pivot assembly bearing in a swingable manner. The pivot assembly bearing is configured such that a pair of rolling bearings is arranged between a shaft and a sleeve surrounding the shaft, and is fixed to a base plate with a screw.
In recent years, as an amount of recording data increases and a data reading/writing speed increases, it becomes increasingly important to maintain cleanliness inside the hard disk drive device. Examples of foreign matters generated in the hard disk drive device include particles due to a lubricant such as grease used for the rolling bearing of the pivot assembly bearing. The particles are minute amount of particles of oil in the lubricant discharged to the outside when a rolling element of the rolling bearing is rotated to stir the lubricant enclosed in the rolling bearing. When the particles float, the particles adhere to components of the hard disk drive device and exert an adverse effect on the hard disk drive device. To reduce the particles to be discharged, various pivot assembly bearings have been developed.
As such a pivot assembly bearing, for example, Japanese Laid-open Patent Publication No. 2013-48005 discloses a pivot assembly bearing (a pivot bearing unit for a hard disk actuator) having a shield cap with a stepped shape, the shield cap being arranged to face an outer ring of the rolling bearing constituting the pivot assembly bearing in an axial direction, in order to narrow a gap between the shield cap and the outer ring of the rolling bearing for reducing the amount of discharged particles.
Japanese Laid-open Patent Publication No. 1-277375 (the invention of a magnetic disc device) discloses a bearing structure for preventing diffusion of contaminants by providing a magnetic fluid seal containing a plurality of bearings between a shaft and an arm holder in a magnetic disc device including the arm holder rotatably held by the shaft via the bearings.
However, in the pivot assembly bearing disclosed in Japanese Laid-open Patent Publication No. 2013-48005, there is a large gap between the shield cap and a shielding member of the rolling bearing, so that a flow of air entering or exiting through the gap is generated when a strong air flow is caused inside the hard disk drive device due to rotation of the magnetic disc or a swing movement of the swing arm. In this case, the air flowed out from the inside of the pivot assembly bearing passes through a portion of the rolling bearing in which the lubricant is enclosed. Due to this, the lubricant in the rolling bearing may be discharged as particles along with the air that is flowed out. Thus, in terms of reducing the discharged particles, further improvement is required.
In the bearing structure disclosed in Japanese Laid-open Patent Publication No. 1-277375, an internal pressure is increased when the air inside the pivot assembly bearing is expanded due to heat caused by the operation of a motor or heat caused when the swing arm repeatedly swings for a long time, and a magnetic fluid of the magnetic fluid seal may be scattered.

Pivot assembly bearing and hard disk drive device


An improved lever latch ring for securing a cover panel having a plurality of tabs thereon to a container wherein the tabs extend beneath an outwardly extending rim forming an upper margin of the container and against an outer wall thereof. The improved lever latch ring includes an arcuate member having a lower horizontal portion that coextends the tabs. The lower horizontal portion is urged against the tabs by a lever latch connected to opposing ends of the arcuate member and when so urged confines the tabs against the outer wall and against a lower portion of the rim. A vertical portion is connected to the lower horizontal portion and extends upwardly therefrom in contact with an outermost portion of the cover panel extending intermediate the vertical portion and the rim. Engagement of the lever latch urges the vertical portion against the cover panel to press the cover panel against the rim. An upper horizontal portion is integrally connected to the vertical portion and extends over the rim and rests on the cover panel to support the lower horizontal portion subjacent the lower portion of the rim.

On Jan. 1, 1991 the United States Government adopted and put into effect The United Nation's Chapter 9 Recommendations for the Transport of Dangerous Goods. This action mandates changes in the heretofore accepted Department of Transportation Regulations for Hazardous Goods.
The change set forth in the United Nations Chapter 9 Recommendations altered the test height from which a container is dropped in determining the sealing integrity of the dropped container. The "drop test" previously required a container to be dropped forty eight inches whereas the United Nation's Recommendations requires the container to be dropped ninety inches. A "hydrostatic test" is also required by the United Nations Recommendations whereby containers must withstand an internal hydrostatic pressure of 37 PSI without leaking.
A container commonly subjected to this test and most widely used for the transportation of generally classified hazardous materials is a cylindrical bucket having a circular bottom a substantially cylindrical outer wall and a circular cover panel. The cover panel extends over a rim defining the upper margin of the bucket and is secured thereto by relatively rigid but malleable tabs that extend beneath the rim and against the outer wall. A securing ring, commonly referred to as a lever latch ring, circumscribes the rim outwardly thereof and is drawn in pressed engagement therewith to press an outermost portion of the cover panel in pressed abutment with the rim. The ring is defined by an arcuate, cross-sectionally C-shaped member substantially forming a circle and a lever latch connected to adjacent opposing ends of the arcuate member for drawing the ends together and thus the arcuate member inwardly toward the rim. The C-shaped, arcuate member is formed by a vertical portion circumscribing the rim and drawn into contact with the outermost portion of the cover panel, an upper horizontal portion extending from an upper margin of the vertical portion above the rim and a lower horizontal portion extending from a lower margin of the vertical portion below the rim. The upper and lower horizontal portions secure the vertical portion proximal the rim but do not exert any pressure on the cover panel or the tabs forming a portion thereof. The securing pressure provided by such lever latch rings is applied only at the outermost point of the cover panel and rim. The lower horizontal portion is actually spaced from the tabs and outer wall.
Though the lever latch ring set forth above has been used for years, it cannot prevent leakage when a hydrostatic pressure of thirty seven PSI is introduced within the container. In fact, it is difficult if not impossible for the aforesaid lever latch ring to sustain a hydrostatic pressure of twenty PSI without experiencing leakage. Obviously such lever latch rings and associated containers will not meet the Department of Transportation's new requirements for hazardous waste materials thus an improved container and lid engaging and sealing apparatus is required.

Lever latch ring for securing a cover panel to a container


Solid-state highly brominated compounds such as solid brominated flame retardants with bromine contents of at least about 60 wt % often contain occluded bromine. Because of their high bromine contents, such compounds have relatively low solubility in common organic solvents. Despite this low solubility, the content of occluded bromine in such solid brominated compounds is effectively reduced by agitating a concentrated agitated slurry of the compound in an inert solvent at a suitable temperature for at least about 1 hour. Thereafter, solids with reduced content of occluded bromine are isolated or recovered from the slurry. Novel products having substantially reduced or eliminated occluded bromine contents and other very desirable characteristics are also provided.

Various solid, highly brominated aromatic organic compounds, i.e., compounds which contain at least about 60 wt % of total bromine, are useful, e.g., as flame retardants for thermoplastic and/or thermosetting polymeric resins, or for other flammable materials. Such compounds are usually formed by bromination of the unbrominated aromatic compound with excess bromine in the presence of a bromination catalyst and in a reaction mixture having a liquid phase which may be excess bromine itself. Unfortunately, many such highly brominated compounds formed in this manner, also have elemental bromine trapped within their particles or crystals. Such bromine is often referred to as occluded bromine. The presence of such bromine in the compound is undesirable because the entrapped bromine imparts color to the particles, and can lower the thermal stability of the flame retardant and of the polymer in which the flame retardant is used. Also during thermal processing of thermoplastic polymers containing such highly brominated compounds as flame retardants, bromine and/or HBr can be released and these substances are highly corrosive to materials used in the fabrication of such processing equipment.
Because these highly brominated aromatic compounds have poor solubility in common organic solvents, it is not economically feasible to purify the compounds by typical washing or recrystallization procedures. Too much solvent would be required.
U.S. Pat. Nos. 6,518,468; 6,603,049; 6,768,033; 6,958,423; and 6,974,887 describe, inter alia, commercially-used process technology for effectively reducing the occluded bromine content of decabromodiphenylethane product, one of the commercially important members of the highly brominated aromatic organic compounds with which this invention is concerned. That process technology involves oven-aging at elevated temperatures, previously ground and dried product.
U.S. Pat. No. 5,457,248 describes as one of its features, a high temperature aromatic solvent treatment of a brominated diphenylalkane to improve its color characteristics. It is pointed out that the aromatic solvent used in such high temperature treatment must not decompose at the temperatures at which the brominated diphenylalkane dissolves in the solvent. Such temperatures are stated to be, typically, in excess of 175° C. and often in excess of 200° C., e.g., usually about 200° C. to about 300° C. To perform such high temperature treatment, the brominated diphenylalkane is contacted with the aromatic solvent at a temperature sufficiently high that the brominated diphenylalkane is dissolved in the solvent, preferably forming a solution essentially free of solid diphenylalkane material. Preferred aromatic solvents are indicated to be non-fused aromatic compounds such as biphenyls, diphenylalkanes, (e.g. with C1 to C10 alkylene bridges), diphenyloxides, diphenylsufones, diphenylcarbonates, and the like, as well as fused aromatic solvents such as naphthalene or naphthalene derivatives such as alkyl naphthalenes. After complete dissolution of the brominated diphenylalkane, the solution is caused to be at a temperature at which the brominated diphenylalkane precipitates. Although it is stated that high product recoveries can be readily achieved, the primary figure given is 75% or more. Although recoveries ranging even up to about 90 to 95 wt % or more are mentioned, it appears that at least some loss of product is to be expected by use of the solvent process of this patent. Also, because many of the aromatic solvents, including the preferred diphenylalkanes and diphenyloxide, are solids at room temperature, special procedures are recommended to effect separation between the brominated diphenylalkanes and the aromatic solvent. One such procedure includes hot filtration to avoid solidification of the solvent itself along with the brominated product. Another such procedure includes allowing both the aromatic solvent and the brominated diphenylalkane to solidify. The resultant solid mixture can then be treated with another liquid organic solvent in which the solid solvent material, but not the brominated diphenylalkane product, is soluble. The brominated diphenylalkane product can then be filtered from the resulting medium. In conducting the high temperature aromatic solvent procedure of the patent, the special hot filtration procedure was used and in each such case the filtrate resulting from the high temperature filtration was washed with an ordinary organic solvent (methanol or acetone), the filtrate was reslurried in the same solvent and filtered, and this filtrate was washed with the same solvent. Obviously such special procedures add to the complexity and costs of the process.
Thus, procedures used heretofore for reducing occluded bromine content involved roasting the compounds at high temperatures or dissolving the compounds in a solvent at high temperature and then precipitating the compounds out of solution at lower temperatures. It would be of advantage if less complex and less costly procedures for achieving reductions in occluded bromine content could be found.

Processing of solid brominated aromatic organic compounds containing occluded bromine


Solid catalyst components for olefin polymerization which comprise a solid carrier components comprising a solid substance having the general formula EQU MgX.sub.2.(E).sub.n ( 1). wherein X is a halogen atom, E is a monoether compound and n is 0.01.about.4, at least a portion of said E being replaced by an eletron donative compound different from said monoether compound (E), and a titanium compound and these catalyst components can produce an olefin polymer having a high stereoregularity in a high polymer yield per unit of a catalyst component in olefin polymerization.

1. Field of the Invention
This invention relates to a solid catalyst component for olefin polymerization. More particularly, it is concerned with a solid catalyst component for olefin polymerization which can produce an olefin polymer with a high stereoregularity in a high yield of a polymer per unit of a solid catalyst component, a process for the production of the same and a process for the production of a polyolefin using the same.
2. Prior Art
It is well-known that Ziegler-Natta solid catalyst components bearing a titanium halide on a carrier such as magnesium chloride and the like have been used together with an organometallic compound and the like for olefin polymerization.
For producing a solid catalyst component, there has been suggested, for example , a process wherein magnesium chloride is dissolved in an ether compound such as tetrahydrofuran and the like and contacted with a titanium compound(Japanese Patent Kokai Application No. 151603/1982). Another known process is to contact a copulverized product of an ether compound such as tetrahydrofuran and the like with a titanium compound.
(Japanese Patent Kokai Application No. 89210/1986). When the solid catalyst component obtained according to these processes is used for olefin polymerization, there have been presented the problems of an insufficient polymerization activity, a low stereoregularity of a polymer and others.
For improving stereoregularity of a polymer, there have been suggested a process using a solid catalyst component containing an electron donor such as a phthalic acid ester and the like together with a silicon compound(Japanese Patent Kokai Application No. 811/1983), a process using a solid catalyst component containing an electron donor such as a diether compound and the like together with a silicon compound (Japanese Patent Kokai Application No. 336503/1991) and others. When the solid catalyst components obtained according to these processes are used for olefin polymerization, a large amount of a catalyst residue is retained in the polymer because of a low yield of a polymer per unit of the catalyst component and there is presented the problem in which the catalyst residue results in adverse influence such as deterioration of the polymer and so on.
Accordingly, there has been desired a solid catalyst which may give an improved yield of a polymer per unit of a catalyst component and decrease a catalyst residue capable of producing an an adverse influence such as deterioration of a polymer and so on.


A method of applying a laser metal formed build layer on a surface of an article is disclosed. A laser deposition head including a light source and a nozzle is positioned relative to the surface of the article by a distance that is about normal to the surface. The surface of the article is melted locally using the laser light source to form a melt pool. Powdered metal is injected into the melt pool using the nozzle. The melt pool is solidified to form the build layer having a desired microstructure on the surface of the article.

A component may become damaged over time due to erosion or impact by foreign object to the component. As a result, the damaged component may be repaired by adding material to the damaged component. In one example, the component may be a compressor blade, compressor vane, turbine blade, or turbine vane which has been damaged due to impact with a foreign object. Such blades or vanes may be repaired by adding layers of material to the damaged portion to rebuild the damage component. One method of repairing such damaged components is laser deposition or additive manufacturing.
Laser deposition is typically performed in layers at a fixed working distance between a laser head and a metal substrate of a workpiece. The laser head produces a melt pool in the metal substrate of a surface of the workpiece. Metal powder is then injected at a powder flow rate into the melt pool via a nozzle. The melt pool is cooled to produce a build layer having a specific microstructure.
As the thermal boundary conditions of the workpiece change, for example when the substrate geometry changes, it may become necessary to increase the powder flow rate to maintain the same cooling rate of the melt pools to produce the desired microstructure of the build layer. As an example, corners and edges of the workpiece may have less ability to act as a heat sink and, as such, may require an increased powder flow rate to prevent melt-back and improve draft angles.
However, fixed working distances are not able to maintain constant cooling rates in the laser deposition if the substrate geometry is changing. Increasing and/or decreasing powder flow rate would allow dynamic adjustment of the cooling rate of the melt pools. However, typically, the powder flow rate is adjusted at a powder feeder and the system response, as observed at the melt pool, is delayed by several seconds while the flow stabilizes. As such, powder flow rate has been typically a set and forget arrangement because it is not adequately adjustable within the timescales needed.

Variable working distance for laser deposition


A method of controlling a hydraulic system is preferably applied to common rail fuel injection systems. The problem in these systems is to control pressure in the common rail while at the same time maintaining the fluid supply to the rail in a way that precisely meets the dynamically changing consumption demands on the hydraulic system. In order to control the hydraulic system, the present invention contemplates the combination of a standard feedback controller with observer models of the various hardware items that make up the hydraulic system. Using this strategy, the system can generally be thought of as controlling fluid supply in an open loop type fashion based upon the consumption rates estimated by the various observer models, and utilizing a conventional feedback controller to make the slight pump adjustments needed to control pressure and to correct for any errors between the actual hardware performance and that predicted by the observer models.

The present invention relates generally to the control of hydraulic systems, and particularly to a model based pressure control strategy for a hydraulic system with a variable delivery pump.
Hydraulic systems, particularly those used in conjunction with an internal combustion engine, have been known for years. For example, Caterpillar Inc. of Peoria, Illinois has been successfully manufacturing and selling hydraulic fuel injection systems for many years. In the past, these systems typically included at least one common rail containing high pressure actuation fluid that was supplied to actuate a plurality of hydraulic devices such as hydraulically actuated fuel injectors and/or gas exchange valve actuators (engine brake, intake, exhaust). The high pressure common rail was supplied with pressurized actuation fluid by a fixed displacement pump. Control of pressure in the common rail was maintained by sizing the pump to always supply more than the needed amount of high pressure fluid and then utilizing a rail pressure control valve to spill a portion of the fluid in the common rail back to the low pressure reservoir. The control system strategy for these systems typically relied upon a feedback control loop in which the desired rail pressure was compared to the measured or estimated rail pressure, and the position of the rail pressure control valve was set as a function of the error signal generated by that comparison. A system of this type is illustrated, for example, in U.S. Pat. No. 5,357,912 to Barnes et al. While these hydraulic systems, and the control thereof, have performed magnificently for many years, there remains room for improvement.
One area in which these previous hydraulic systems could be improved is by decreasing the amount of pressurized actuation fluid that is spilled back to the low pressure reservoir without performing any useful work, such as actuating one of the hydraulic devices. In other words, energy is consumed and arguably wasted whenever the rail pressure control valve opened to allow pressurized fluid from the high pressure rail to leak back to the low pressure reservoir. In order to decrease the amount of energy consumed in controlling the pressure in the hydraulic system, one strategy has been to introduce a variable delivery pump and eliminate the previous rail pressure control valve. Such a hydraulic system is shown and described in co-owned U.S. Pat. No. 6,035,828 to Anderson et al. This system greatly reduces the amount of wasted energy since the pump is controlled to produce only the amount of actuation fluid necessary to maintain a desired rail pressure. Although this type of fluid supply and pressurization strategy has considerable promise, it still may suffer from at least one subtle drawback when it is controlled via a feedback loop based upon a comparison of the desired rail pressure to the actual rail pressure. Due at least in part to the fact that the fluid being consumed from the high pressure common rail can be rapidly and continuously changing, engineers have observed that the control system can be at least temporarily overwhelmed in this highly dynamic system. In other words, the system can sometimes demonstrate an inability to both maintain an adequate fluid supply to the hydraulic devices and do so at the desired pressure without unacceptable lags between the control system response and the fluid demands of the hydraulic devices.
The present invention is directed to these and other problems associated with hydraulic systems.
In one aspect, a method of controlling a hydraulic system includes at least some features of the previous control systems based upon a pressure error feedback control system. Thus, the method includes a step of generating a control variable at least in part by comparing a desired liquid pressure to an estimated liquid pressure. Next, the liquid consumption rate of the hydraulic system is estimated. Finally, the pump output rate is set as a function of the control variable and the estimated system consumption rate.
In another aspect, a method of controlling liquid pressure in a common rail hydraulic system for an engine includes a step of estimating engine speed, the viscosity of the liquid in the hydraulic system and the rail pressure of the hydraulic system. The injector consumption rate and the pump consumption rate are also estimated. A control rate is generated at least in part by comparing the desired rail pressure to the estimated rail pressure. Finally, the pump output rate is set as a function of the control rate plus the estimated injector consumption rate plus the estimated pump consumption rate.
In still another aspect, a common rail hydraulic system includes a variable delivery pump with an outlet. At least one hydraulic device has an inlet. A common rail has an inlet fluidly connected to the outlet of the variable delivery pump, and an outlet connected to the inlet of the at least one hydraulic device. A pump output controller is operably coupled to the variable delivery pump, and produces a pump control signal that is a function of a desired rail pressure, an estimated rail pressure and an estimated consumption rate of the hydraulic system.

Model based rail pressure control for variable displacement pumps


Altering messaging according to sentiment analysis by providing a baseline messaging tree including topics arranged according to a sentiment score, providing speaker prompts according to the baseline messaging tree, receiving real-time audience sentiment feedback during a messaging presentation, and adjusting the speaker prompts according to the baseline messaging tree and the real-time audience sentiment feedback.

Individual sentiment may be analyzed using facial expression, posture, and hand gesture data associated with the individual. The average or aggregate sentiment of a listening audience may be determined using similar data gathered for audience members.
Decision trees enable a mapping of potential decisions along a planned timeline enabling the progression of activities along one of a plurality of preplanned paths according to input data values at runtime for each decision node of the tree.
In presenting a message to an audience, maintaining audience engagement to the messaging and managing audience receptivity to upcoming messaging topics may depend upon the sentiment of the audience to current messaging topics and transitioning to new topics according to the current and desired sentiments. Successfully calling an audience to an action may depend upon the real-time sentiment of the audience regarding being called to action.

Altering messaging using sentiment analysis

International Business Machines Corporation


An electronic printing system having an image input for receiving digitally represented images from a source of input information such as an image input scanner; a controller having the capability of rotating an image in at least 90.degree. increments and which can provide such rotation in accordance with a pre-programmed set of commands; a user interface, connected to the controller, and for directing information about the orientation of the input image, and information about output orientation and placement in accordance with one of a plurality of selectable output orientation; and an output, such as a binary printer, to which the controller may direct reoriented image data.

The invention relates to electronic printers and printing systems, and more particularly, to accomplishment of certain jobs in such devices which provide automatic rotation of images.
In certain high speed electronic print publishing systems, such as for example, the Xerox Docutech Publishing System, first announced and made available to the public in October, 1990, rotation and placement of images and of multiple images on a single sheet or side of a sheet is desirable for several reasons. Such a capability makes possible operating features that enhance the production of document sets, including signatures, calendars, and certain duplex sets, that may be later bound.
The Xerox DocuTech provides a feature that allows specification of input orientation of an image and separately allows specification of output plex, independently of input orientation. Additionally, DocuTech allows printing signatures, which are always duplexed sheets, and requires specification of input orientation and plex and a limited selection of output orientation. Users, however, have difficulties in relating the selection of plex of a document with its image orientation. Accordingly, it is desirable to have full control of specifying input orientation and plex, as well as output orientation and plex, and provide operator understandable indicia of the same.


An object is to obtain a stable electric connection resistance under a mild crimping condition. The present invention is a terminal connector 12 that includes a crimp portion 30 to be crimped to an electric wire. The crimp portion 30 includes a base material, an aluminum layer or an aluminum alloy layer a surface on the base material, and a hard layer on a surface of the aluminum layer or the aluminum alloy layer. The hard layer is harder than the base material. The present invention may be an electric wire with a terminal connector 10 that includes the above terminal connector 12 and a covered electric wire 40 that includes a core wire 42 made of aluminum or aluminum alloy. The crimp portion 30 of the terminal connector 12 is crimped to the core wire 42.

1. Field of the Invention
The present invention relates to a terminal connector, an electric wire with a terminal connector, and a method of connecting a terminal connector and an electric wire.
2. Description of the Related Art
Conventionally, Japanese Unexamined Patent Publication No. 2010-3584 discloses a known method of connecting a terminal connector and an aluminum electric wire that includes an aluminum core covered by an insulating covering. An oxide film is likely to be formed on a surface of a core of the aluminum electric wire. A crimping section of the terminal connector is serrated to break the oxide film, and the oxide film formed on the surface of the aluminum electric wire is broken by the serration. In this configuration, the core is electrically conductively connected to the crimping section when the oxide film is broken to uncover the aluminum core. As a result, electrical connection resistance between the aluminum electric wire and the terminal connector can be reduced.
However, in the above-described connection method, although the oxide film is broken by the serration, the crimping section is still required to be crimped hard to obtain stable electrical connection resistance. When the crimping section is crimped hard, the terminal connector may be damaged or the crimped section may protrude from a rear end of a connector because the crimping section is extended in a front-rear direction. A connection method that can provide stable electrical connection resistance even under mild crimping condition has been expected.
The present invention has been achieved in view of the above. It is an object of the present invention to obtain stable electrical connection resistance even under the mild crimping condition.

Terminal connector, electric wire with terminal connector, and method of connecting terminal connector and electric wire


Systems and methods for dynamically generating filtered audio responses based on the presence of one or more on-site participants are disclosed. In embodiments, a computer-implemented method comprises: identifying, by a computing device at a site, one or more on-site participants in a user session; receiving, by the computing device, input from a select participant of the one or more on-site participants; determining, by the computing device, context from the input; accessing, by the computing device, privacy settings of the select participant, wherein the privacy settings are associated with restricted participant data; determining, by the computing device, that the one or more participants match the restricted participant data associated with the context; and outputting, by the computing device, a filtered audio response, wherein the filtered audio response is based on the context of the input and the privacy settings of the select participant.

The present invention relates generally to computer-based personal assistants and, more particularly, to dynamically generating personal assistant responses based on the presence of one or more on-sites participants.
Virtual assistants such as personal assistants come in a variety of forms, and generally function as personal information managers. Personal assistants may be in the form of smart speakers or devices including voice recognition software and audio response capabilities. Some examples of personal assistants include Amazon Alexa™ and Google Home™. As smart devices gain in popularity, a variety of personal assistants have been developed which interface with one or more smart devices to provide information management as well as smart device control features. Personal assistants may be programmed to recognize verbal or text-based commands, and reply with an appropriate response. For example, a personal assistant may recognize a voice command or query of a user and respond with an appropriate audio answer to the command or query. Personal assistants may recognize individual users and respond to an individual user's commands/queries accordingly. For example, a personal assistant may access a music play list associated with a particular user's voice in response to a voice command from the user.

Personal assistant device responses based on group presence

INTERNATIONAL BUSINESS MACHINES CORPORATION


A reactor cell for use in organo-metallic chemical vapor deposition (OMCVD). Multiple precursor gases are combined in a supply tube (14). A deflector section (56) connected to the supply tube consists of three tubes (58, 60, 61) joined at right angles to each other and to the supply tube in order to maximize the turbulence in the gas flow and to thereby uniformly mix the gas. The last of the three tubes is perpendicularly joined to a tapered section (30) of rectangular cross-section that tapers outwardly toward a deposition chamber (22) of rectangular cross section containing a heated susceptor (18) holding the wafer (20) over which the uniformly mixed gas flows and cracks, thereby epitaxially depositing some of its constituents on the wafer.

Several different processes can be used to grow epitaxial layers of compound semiconductors and other materials. Chemical vapor deposition (CVD) enjoys great commercial importance because it grows layers relatively quickly and involves relatively inexpensive growth apparatus compared to molecular beam epitaxy (MBE). In CVD, one or more chemical species, each bearing an element to be incorporated into the grown material, flow in a gaseous phase over a heated wafer. The chemical species react at the surface, and the growth elements recombine on the wafer in the desired composition. For the growth of silicon, silane (SiH.sub.4) is the silicon precursor, and other precursors can be used for dopants, for example, phosphine (PH.sub.3) and arsine (AsH.sub.3) for the n-type dopants P and As. Vapor phase epitaxy (VPE) is an alternate name for CVD, which emphasizes that the new material can be grown to be epitaxial with the underlying wafer.
The interest in high-speed electronics and opto-electronics has prompted much research and development in the epitaxial growth of compound semiconductors such as GaAs and InP. These compound materials involve some somewhat exotic materials for which simple gaseous metal-hydride chemical precursors are not readily available. Furthermore, many interesting compound-semiconductor devices require the growth of exceedingly thin layers or of very abrupt junctions, of the order of manometers to tens of nanometers. MBE can satisfy most of these requirements although it cannot be used for P because the many forms of P prevents accurate control of its vapor pressure. The technology for CVD is thus desirable both for InP and for commercial environments demanding high production rates.
In organo-metallic chemical vapor deposition (OMCVD), at least some of the chemical precursors have a more complicated, organic form. For instance, for the growth of GaAs, as illustrated in the schematic diagram in FIG. 1, trimethylgallium ((CH.sub.3).sub.3 Ga) and arsine flow from respective supply tanks 10 in amounts regulated by respective valves 12. Trimethylgallium is in fact a liquid, but its vapor is obtained by bubbling a gas such as H.sub.2 through it. The gas flows combine through an unillustrated manifold into a common inlet port 14 to a generally illustrated reactor cell 16, which contains a graphite susceptor 18 on which is placed a wafer 20. The reactor cell 16 includes a main body wall 22, usually made of fused quartz and usually having a rectangular cross-section of the order of 15 cm.sup.2. An RF coil 24 external to the cell 16 controllably heats the graphite susceptor 18 and hence the wafer 20.
The combined gases flow over the wafer surface, and the arsine reacts and the trimethylgallium cracks on the surface of the heated substrate 20 to provide the As and Ga, which combine at the substrate surface in the crystalline arrangement of the substrate 20. That is, they are epitaxially deposited. A pump 26 maintains the gas flow and exhausts the gas but keeps the interior of the cell 16 at a fixed pressure. For the growth of InP, trimethylindium (CH.sub.3).sub.3 In) and phosphine can be used as precursors. Small-scale OMCVD reactors and complete growth systems usable with the invention are commercially available from Thomas Swan & Co., Ltd. of the United Kingdom, Spire Corporation of Massachusetts, and Emcore Corporation of New Jersey, among others.
Although OMCVD has at least partially solved the problem of precursors, the problem of control of thickness has been solved only for large commercial OMCVD reactors. See, for example, "Aixtrons's Low-Pressure MOVPE: Concepts for Improved Quality and Safety," III-Vs Review, volume 4, number 2, 1991, pp. 1, 3, 32, and 33. The thickness-control problem remains unsolved for less expensive, smaller reactors, as has been discussed by Thrush et al. in "MOCVD grown InP/InGaAs structures for optical receivers," Journal of Crystal Growth, volume 93, 1988, pp. 870-876 and by van de Ven et al. in "Gas phase depletion and flow dynamics in horizontal MOVCD reactors," Journal of Crystal Growth, volume 76, 1986, pp. 352-372. For the deposition of very thin layers, the gas flow must be uniformly maintained over the area of the wafer 20.
The lateral flow has been improved in the prior art by a number of schemes. In a first approach illustrated in plan view in FIGS. 2 and 3, the relatively small cylindrical inlet port 14 is joined to the relatively large main body 22 of the cell 16 by a long taper section 30. The susceptor 18 is placed in a trough 32 so that the wafer surface is near the lower side of the interior of the taper section 30. Additionally, a wedge 34 may be placed in the taper section 30 to force additional flow to the sides. The deposition can be made more uniform in the direction of gas flow by tilting the susceptor 18 by about 7.degree. about an axis perpendicular to the gas flow and with its surface toward the gas source so as to counter depletion of the reactants. The tilting can be accomplished by placing a fused quartz block 36 between the head of the trough 32 and the susceptor 18 so as to present a smooth surface over which the gas flows and then manually pushing a shim 38 to elevate the susceptor 18 to the desired angle. Additional uniformity can be obtained by a gas rotation technique in which the wafer 20 rests on a thin graphite disc disposed within the recess of the susceptor 18. A gas is externally supplied through channels in the susceptor 18 connected to a series of angularly offset nozzles in the recess to thereby continuously rotate the wafer 20 during the OMCVD deposition. In an alternative approach illustrated in FIGS. 4 and 5, a shower head 40 mostly fills the cross-section of the main body 22 of the cell 16. The head 40 contains a square array of holes 42 through which the gas flows with a significant pressure drop. Thereby, the gas is spread across the cell cross-section. Related approaches include baffles set in the gas flow.
Although the uniformity has been improved by these techniques, it has remained insufficient for advanced devices.

Deflected flow in chemical vapor deposition cell


A probe card assembly can comprise a support structure to which a plurality of probes can be directly or indirectly attached. The probes can be disposed to contact an electronic device to be tested. The probe card assembly can further comprise actuators, which can be configured to change selectively an attitude of the support structure with respect to a reference structure. The probe card assembly can also comprise a plurality of lockable compliant structures. While unlocked, the lockable compliant structures can allow the support structure to move with respect to the reference structure. While locked, however, the compliant structures can provide mechanical resistance to movement of the support structure with respect to the reference structure.

Test systems for testing electronic devices, such as semiconductor dies, are known. In some such test systems, electrically conductive probes are brought into contact with terminals of the electronic devices. Power and test signals are then provided to the electronic devices through the probes, and the responses of the electronic devices to the test signals are monitored through the probes. To establish reliable electrical connections between the probes and the terminals of the electronic devices, the probes typically must be generally aligned with the terminals. In some test scenarios, aligning the probes with the terminals includes adjusting an attitude (e.g., tilt, orientation, planarity, etc.) of the contact tips of the probes to correspond generally with an attitude of the terminals. Moreover, in some test scenarios, thermal gradients, mechanical loads placed on the probes, and other such causes can cause undesired movement of the probes. In some instances, these movements can cause the probes to become so misaligned with the terminals that electrical connections between some or all of the probes and some or all of the terminals of the electronic devices are lost during testing of the electronic devices. Embodiments of the present invention are directed to improvements in adjusting an attitude of a probe card assembly and providing mechanical stiffening of the probe card assembly.