Recombinant human immunodeficiency virus antigens capable of immunologically identifying the presence of early anti-HIV antibodies are stably expressed in a number of cell lines. These antigens have several clinically important applications as non-hazardous tools in the detection of human immunodeficiency virus exposure/infection, and in screening methods for HIV infection in idiopathic chronic lymphopenia (ICL). These techniques are improved over existing immunologically based and PCR based detection methods, as they provide for the detection of infection/exposure in samples determined to be negative by conventional forms of these types of assays that do not detect anti-HIV gp160 antibodies that react to conformational epitopes of HIV. The invention finds particular application in the detection of human immunodeficiency virus exposure/infection in infants. The earlier detection of the described methods is provided through the preserved immunoreactivity of the described recombinant conformationally intact human immunodeficiency virus that is capable of detecting a class of xe2x80x9cearlyxe2x80x9d anti-human immunodeficiency virus antibody not previously detectable by standard Western Blot or ELISA methods. The human immunodeficiency virus gp160 envelope antigen comprises one of the specific recombinant antigens examined with clinical human samples in these improved screening methods.

1. Field of the Invention
The present invention relates generally to the fields of diagnostic and screening tests. More particularly, it concerns improved methods for the detection of early anti-HIV antibody in a sample, as well as early diagnostic tests to detect human immunodeficiency virus exposure or infection in infants. An additional aspect of the invention concerns methods for detecting HIV in idiopathic chronic lymphopenia patients. The invention also relates to the field of recombinant proteins, as particular recombinant proteins and recombinant protein composite preparations of several human immunodeficiency virus strains are disclosed. The invention also relates to the field of commercial diagnostic and prognostic assay plates and kits, as assay plates designed to detect an early anti-HIV antibody that include the described recombinant human immunodeficiency virus antigen or preparations of human immunodeficiency virus infected cell isolates are described.
2. Background of the Invention
Patients infected with human immunodeficiency virus (HIV) are known to eventually mount a humoral immune response to the virus. The production of anti-HIV antibody is one marker used to detect this response. In some studies, anti-HIV antibody has been reported to be more reliable for diagnosis than either HIV culture or HIV antigen detection in patient samples1,2. Consequently, anti-HIV antibody detection tests are the most common method of diagnosis of infection. Both EIA and Western blot assays are currently used in the detection of anti-HIV antibody.
Unfortunately, a degree of unreliability continues to exist with the use of conventional anti-HIV antibody screening methods, such as by conventional EIAs. Thus, a further confirmatory test, such as a Western Blot (WB) or fixed-cell immunofluorescence assay, have become recommended additional testing procedures.
Despite these and other additional precautionary testing measures, a number of studies report the existence of a seemingly silent period of HIV infection during which antibody to the virus is not detectable even after exhaustive testing. This period reportedly extends from the point of infection to the time infection is detectable through conventional sero-conversion assays. This silent period has been reported to persist anywhere from a few months to as much as two and one-half years before infection is detectable by conventional EIAs and Western blot assays.
While not always successful, culturing of peripheral blood lymphocytes to amplify HIV does provide for detection of the virus when anti-HIV antibody cannot be detected by conventional EIA or WB. However, several recent studies using PCR-based HIV detection methods continue to report the existence of PCR(+)positive, sero(xe2x88x92)negative cases in high-risk populations10-17. Nevertheless, PCR usually does detect infection before conventional sero-conversion methods, with the aforedescribed period of silent infection being reduced by approximately one month, at least in some cases18.
The rate of HIV transmission in negatively tested blood, using conventional testing methods, continues to persist at a relatively constant rate25. For example, HIV-1 transmission from seemingly xe2x80x9cseronegativexe2x80x9d blood using EIA conventional methods, continue to be reported21-23. Donated organs also constitute a source of HIV disease transmission, with HIV infection being diagnosed in recipients of organs from individuals whom, again, test HIV seronegative by conventional assays24.
Retrospective studies have reported that early donor education and self-exclusion measures has reduced the rate of disease transmission26. However, such exclusion methods together with antibody testing, while hopefully reducing the probability of at least some false negative results27, provides only a partial and imperfect solution to the problem in at least a small subset of reported HIV cases.
Some studies report the presence of HIV specific T-cells in high risk individuals testing negative with conventional EIA, WB, and PCR based detection techniques28,29. Other reports have identified the existence of B-cells which produce HIV-specific antibodies in vitro that are present in EIA-negative, WB-negative, high-risk subjects30. While these approaches present possible alternatives, for testing, they are relatively complex and difficult procedures, and are thus impractical for large-scale clinical screening. The expense and time associated with this type of testing again leaves a need in the medical arts for a reliable and practical HIV screening and detection approach.
Early HIV infection of infants is a particularly troublesome problem. Current technology renders it difficult to diagnose whether an infant less than 18 months of age is infected, absent development of overt clinical symptoms. Conventional HIV serological tests for anti-HIV antibody are inadequate for detecting infection in an infant because the antibody detected is not necessarily that of the infant, but is that of the HIV-positive mother. This maternally derived antibody typically persists for up to 21 months in the infants system34.
Neither IgA or IgM antibody cross the placenta. Hence, studies in children have emphasized the detection of IgA and IgM as indicators of infant HIV infection. In one study, both HIV-specific IgA and IgM were found in infants up to 12 months of age born to sero-positive mothers, with twice as many samples yielding IgA anti-HIV compared to IgM (66% vs. 33%)35 using conventional screening assays (WB, EIA).
Currently, approximately 50% of infected infants can be identified at birth, approximately 90% by 3 months of age, and almost all by 6 months of age using combination HIV culture, PCR, IgA antibody tests, and p24 antigen tests38. However, the fact that HIV can be detected in only one-half of infected infants at the time of birth again points to the continued need for improved early HIV detection in infants.
The present invention, in a general and overall sense, concerns recombinant HIV envelope proteins and peptides, and early anti-HIV antibody immunoreactive fragments thereof, that are capable of immunologically binding to early anti-HIV antibodies.
As used in the description of the present invention, early anti-HIV antibodies are defined as the first anti-human immunodeficiency virus antibodies that are induced in a human infected with the HIV virus, these antibodies being capable of recognizing conformational epitopes of HIV gp160 antigen and which are not detectable by current EIA or Western Blot assay using HIV gp160 target antigen that has not have retained conformational epitopes.
The invention further provides for an improved HIV detection and screening method by allowing for the identification of early anti-HIV antibody. These early anti-HIV antibodies previously went undetected using conventional assays because the target antigens historically employed in these assays lacked sufficiently preserved conformational epitopes necessary for early antibody recognition.
The compositions and assays of the present invention comprise improved HIV target antigens that include the conformational epitopes of an HIV envelope protein, specifically the HIV gp160. The early anti-HIV antibodies detectable using the described antigen do not recognize primary sequence epitopes, and hence they go undetected with conventional serological tests that employ at least partially denatured HIV target antigen (EIA and Western blot).
Because PCR-based assays provide little improvement over conventional EIA and Western Blot serological-based assays for detecting HIV, it is expected that the presently described EIA""s will also provide an improved method over PCR based detection and screening methods.
Recombinant Proteins and Peptides of Human Immunodeficiency Virus
In some embodiments of the invention, a recombinant protein comprising a recombinant human immunodeficiency virus envelope protein capable of immunologically binding an early anti-HIV antibody is provided. For example, these recombinant proteins in particular embodiments may be further defined as having a molecular weight of about 160,000 Daltons as determined by SDS/PAGE. In these particular forms, the recombinant HIV protein is an HIV envelope protein, HIV gp160.
The recombinant HIV envelope proteins of the invention may further be described by reference to the process by which they are prepared. For example, in one embodiment, the process comprises preparing a nucleic acid sequence or a restriction fragment of the human immunodeficiency virus nucleic acid sequence that encodes a human immunodeficiency virus envelope protein, such as gp160; inserting the restriction fragment into a vector capable of transfecting a eukaryotic cell to provide a recombinant vector; transfecting eukaryotic cell capable of expressing the human immunodeficiency virus envelope protein capable of binding an early anti-HIV antibody to provide a recombinant eukaryotic cell; culturing the recombinant mammalian cell under conditions suitable for expression of the recombinant HIV protein; and collecting recombinant HIV envelope protein capable of binding early anti-HIV antibody.
An example of a mammalian cell capable of expressing the HIV envelope protein nucleic acid sequence as described here is the CEM cell line. Such CEM cell lines are available through the American Type Culture Collection (ATCC). An example of the vector capable of transfecting a mammalian cell is a retroviral vector, such as pLNSX. Other carriers, such as an adenovirus or a plasmid, may also be used to transfer the HIV envelope protein encoding nucleic acid sequence. The recombinant retroviral vector in a particular embodiment of the process is the pLNSX-env.
In other embodiments of the recombinant human immunodeficiency virus protein, the protein is described as comprising a recombinant gp160 human immunodeficiency virus glycoprotein or fragments of the gp160 protein, such as the gp120 or gp41 fragment of the gp160 protein.
The recombinant HIV envelope proteins and peptides of the invention are further described in some embodiments as comprising recombinant proteins and peptides prepared from substantially purified and non-denatured lysates of mammalian cells transfected with vectors expressing recombinant human immunodeficiency virus, as well as recombinant proteins/peptides comprising the substantially purified and non-denatured expression products of these transfected mammalian cells.
In yet other embodiments, the recombinant protein/peptide preparations comprise a composite of recombinant human immunodeficiency virus gp160 proteins, the composite comprising recombinant gp160 protein from more than one human immunodeficiency virus strain.
Mammalian cell lines that are transfected with retrovirus expression vectors carrying the gp160 encoding-human immunodeficiency virus gene have been prepared with gp160-encoding nucleic acid fragments obtained from several different strains of the human immunodeficiency virus obtained from HIV-infected patients. For example, recombinant HIV envelope protein has been prepared from HIV213 virus strain.
In a particular embodiment, the protein/peptide gp160 composite preparation comprises the recombinant gp160 protein expression product of at least three different HIV strains found to be immunoreactive with the early HIV antibody detected in a representative number of serum samples determined to be seronegative by conventional EIA and Western Blot techniques. By way of example, three such HIV strains are HIV213, HIVC and HIVAC-1. These HIV strains have been deposited with the American Type Culture Collection depository (12301 Park Lawn Drive, Rockville, Md. 20852). The deposit information is as follows:
HIV213xe2x80x94ATCC VR 2247
HIVAC-1xe2x80x94ATCC VR 2246
HIVTP-1xe2x80x94ATCC VR 2245
Target antigen may be prepared from either the expression product obtained from eukaryotic cells transfected with retroviral or other vectors carrying these gp160 encoding nucleic acid sequences or fragments thereof, or from substantially nondenatured lysates of such transfected eukaryotic cells.
As used in the description of the present invention in the description of the human immunodeficiency virus recombinant protein/peptide preparations and cell lysates, the phrase xe2x80x9csubstantially non-denaturedxe2x80x9d in used to define a protein having a preserved configurational integrity of the human immunodeficiency virus envelope gp160 protein or a portion thereof sufficient to bind early anti-HIV antibody. Conformation of the protein/peptide used as target antigen is important in providing this early antibody recognition.
The present inventor provides here procedures that preserve sufficient conformational integrity of the protein/peptide to allow early anti-HIV binding recognition. It is anticipated that given the disclosure here, other similar protein/peptide preparation processes may be devised that result in useful target antigen compositions for early anti-human immunodeficiency virus screening. All such modified procedures, insofar as they represent minor or insignificant modification of the procedures and specific materials described herein, are therefore intended by the inventor to be embraced within the scope of the present invention.
While a number of different HIV strains were examined by the present inventor, other HIV viral strains not specifically mentioned or examined here may also be employed in the preparation of the various HIV proteins/peptides of the invention. It is expected that other HIV viral strains may be used to provide the defined substantially preserved conformational epitopically intact HIV proteins capable of early anti-HIV antibody recognition. The particularly noted HIV strains used to create recombinant protein/peptide target antigen were selected based on an observed activity to bind early anti-HIV antibody in human patient serum or plasma samples determined to be seronegative by conventional antibody testing procedures. Hence, additional such representative strains may be identified and selected using the procedures outlined herein, and subsequently processed again according to the procedures described in detail here in providing recombinant HIV antigen also useful in screening and diagnosing early anti-HIV antibody and HIV infection in a patient sample.
In a particular embodiment, the recombinant protein/peptide of the invention comprises a sufficiently conformationally intact HIV envelope protein capable of immunologically binding an early anti-HIV antibody, said antigen being isolatable as an expression product from a mammalian or other eukaryotic cell transfected with an expression vector having a sequence encoding a gp160 HIV envelope protein. The HIV gp160 envelope protein, by way of example, may be that expressed by a eukaryotic cell infected with a viral strain HIVC, HIV213, or HIVAC-1. It is also expected that other HIV isolate strains that are capable of expressing the gp160 antigen, or instead the gp41 or gp120 HIV envelope antigen, may be used in conjunction with the present invention as well.
Purified Preparations of HIV Envelope Proteins
Further aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of the described recombinant HIV envelope protein preparations. The term xe2x80x9cpurified envelope proteinxe2x80x9d as used herein, is intended to refer to a protein composition that is isolatable from eukaryotic cells, either in the form of a solubilized population of HIV infected cells or as a protein from recombinant-expressing cells. These purified envelope proteins are further defined as essentially non-denatured and capable of binding with early anti-HIV antibody. The HIV envelope protein is purified to any degree relative to its naturally-obtainable state. In some embodiments, the purified antigen state is relative to purity of the recombinant antigen as it exists in a whole mammalian cell lysate. A purified HIV envelope protein therefore also refers to a recombinant envelope protein, free from the environment in which it may naturally occur in intact recombinant-expressing mammalian cells.
Generally, xe2x80x9cpurifiedxe2x80x9d will refer to an HIV envelope protein composition wherein various non-HIV envelope components, such as other cell components, have been removed, and which composition substantially retains its antigenicity and/or capacity to interact with early anti-HIV antibody. Where the term xe2x80x9csubstantially purifiedxe2x80x9d is used, this will refer to a composition in which the human immunodeficiency virus envelope protein forms the major component of the composition, such as constituting about 50%, about 60%, about 80% or about 95% of the protein in the composition or more.
Various methods for quantifying the degree of purification of the HIV protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the activity of the preparation for detecting early anti-HIV antibody by assessing the number of different polypeptides within a fraction by SDS/PAGE analysis. In some embodiments, the recombinant HIV protein is to be purified to homogeneity expressed as an about 80% purity from a crude cell lysate so as to be identifiable in a single band of an SDS page gel stained with comassie blue. SDS page gel analysis of a protein preparation is well known to those of ordinary skill in the art.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. A specific example presented herein is the purification of recombinant gp/60 expressed mammalian cells which are solubilized using affinity binding to anti-gp41 monoclonal antibody bound to wells of plastic EIA plates.
Some of the recombinant HIV protein preparations from transfected mammalian cell lysate of the present invention have a purity of 80%. This number represents a ratio of, for example, 8 gp160 HIV antigen molecules to two non-gp160 molecules. Preparations of lower or higher purity are also useful in detecting the early anti-HIV antibody.
The preferred purification method disclosed hereinbelow contains several steps and represents the best mode presently known by the inventors to prepare a substantially purified recombinant HIV envelope protein and still maintain its native conformation. This method is currently preferred as it results in the substantial purification of the recombinant HIV protein, as assessed by SDS-PAGE determinations and ability to bind to early antibodies, while simultaneously coating EIA plates. This preferred mode of recombinant protein purification from HIV transfected mammalian cells involves the execution of certain steps in the order described hereinbelow. However, as is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified recombinant HIV envelope protein, such as the recombinant gp160 HIV envelope protein.
As mentioned above, although preferred for use in certain embodiments, there is no general requirement that the HIV transfected mammalian cell lysate always be provided in their most purified state. Indeed, it is contemplated that less substantially purified cell lysates, which are nonetheless enriched in recombinant HIV envelope protein, such as gp160 relative to the natural state, will have utility in certain embodiments. These include, for example, the detection of early anti-HIV antibody. Partially purified HIV transfected mammalian cell lysate fractions for use in such embodiments may be obtained by subjecting an HIV transfected mammalian cell lysate extract to one or a combination of the steps described herein, the HIV recombinant envelope preparations may also be obtained as a secreted product from cells carrying an HIV envelope antigen (e.g. gp160) encoding nucleic acid sequence.
Recombinant Vectors
The recombinant vectors of the invention may comprise any vehicle capable of transfecting a eukaryotic cell. By way of example, such vehicles include retrovirus vectors and adenovirus vectors. Plasmids may also be used to transfer HIV nucleic acid fragments encoding the HIV envelope proteins of the invention. In some embodiments, the retroviral vector comprises a nucleic acid sequence encoding a human immunodeficiency virus envelope protein, such as the gp160, or that encodes an early anti-HIV antibody immunoreactive fragment of the human immunodeficiency virus envelope protein.
In particular embodiments, the nucleic acid sequence encoding the human immunodeficiency virus envelope protein comprises an SalI-XhoI restriction fragment of a human immunodeficiency virus nucleic acid sequence. By way of example, the inventor has prepared SalI-XhoI restriction fragments of several human immunodeficiency viruses, particularly HIV strains C, 213 and AC-1, and blunt-end ligated the fragment into a retroviral vector, such as the pLNSX retroviral vector.
Mammalian Cell Lines
The present invention in another aspect provide recombinant mammalian cells that express the recombinant human immunodeficiency virus envelope proteins and peptides. Any variety of mammalian cells or cell lines may be used, as long as they are capable of expressing the recombinant HIV protein capable of immunologically detecting the early anti-HIV antibody. By way of example, a cell line that may be used is a CEM human cell line, particularly described as CEM human T-cells.
Methods for Preparing Recombinant HIV Envelope Protein and Peptide
Methods for preparing the recombinant HIV envelope are also provided. In one particular embodiment, the method comprises transfecting a mammalian cell with a retroviral vector genetically engineered to include a sequence encoding a human immunodeficiency viral envelope protein. These transfected mammalian cells are then subject to a screening process, such as by antibiotic resistance to G418 for example, and clones selected that express the highest amount of the human immunodeficiency virus envelope protein. By way of example, clones expressing the highest amounts of HIV envelope protein could be selected using an anti-HIV gp160 monoclonal antibody (e.g., DZ33, Medarex, Inc.).
One particular clone expressing the HIV envelope protein gp160 isolated by the present inventors is Clone 7. Clone 7 was obtained by transfection of a CEM cell line with LNSX retroviral vector containing the env gene of HIV213 strain. This clone 7 was examined by flow cytometry, and shown to express high levels of the recombinant HIV gp160 protein (FIG. 5).
Other clones that express a fusion protein of gp160 obtained from more than one of the HIV strains may also be obtained. Such would be achieved, for example, by constructing a retroviral vector that included a gp160 encoding fragment of nucleic acid obtained from the desired HIV isolates, and then transfecting a mammalian cell line with said construct.
Any of the herein described recombinant HIV antigens may be used alone or in combination as a target antigen for the detection of early anti-HIV antibody. Combinations of these recombinant antigens from the noted viral strains have proven to be particularly efficacious for detecting the early anti-HIV antibody in the widest range of patient samples examined to date.
Methods/Processes of Producing Recombinant HIV Envelope Protein/Peptide
The invention also provides for methods/processes of producing a recombinant HIV antigen. In one embodiment, the method comprises obtaining a nucleic acid fragment encoding a human immunodeficiency virus gp160 envelope protein; ligating the nucleic acid fragment into a vector to provide a recombinant vector; transfecting a mammalian cell with the recombinant vector to provide a transfected mammalian cell; and collecting recombinant gp160 protein. The recombinant gp160 protein may be collected by virtue of solubilizing the infected mammalian cell in a solubilizing agent that does not destroy the early anti-HIV antibody detecting capability of the antigen, such as digitonin, and then removing the cellular components in the solubilized preparation. Alternatively, the recombinant HIV antigen may be collected as a secreted product from recombinant eukaryotic cells, such as from a yeast cell that has been genetically engineered such that it secretes the gp160 HIV envelope protein.
A method that was used to collect recombinant gp160 that was used as target antigen to detect early anti-HIV antibody in a sample was through the use of 1.0% digitonin as a solubilizing agent on CEM cells infected with HIV. This procedure is described at Example 3. The recombinant HIV gp160 antigen was captured onto an ELISA plate by pouring the whole recombinant cell lysate into wells of the plate, which will be coated with mouse anti-gp41 muAB. The plate was then washed to remove cellular components and other antigens. In this fashion, the desired HIV gp160 antigen was isolated on the plate.
In a particular embodiment, the method or preparing a recombinant gp160 envelope protein comprises: obtaining a nucleic acid fragment encoding a gp160 HIV envelope protein; inserting said fragment into a vector capable of transfecting a mammalian cell; and transfecting a mammalian cell capable of expressing the gp160 envelope protein with said vector. The method may include the further step of collecting the recombinant gp160 protein.
Assay Plates
In some embodiments, the wells of the assay plates may first be coated with an anti-gp41 or anti-gp160 antibody. This would immobilize HIV gp160 antigen to the plastic in the presence of a mild solubilizing buffer, such as from about 0.1% to about 10% digitonin (particularly about 1% digitonin). Such an approach is particularly efficacious in preparing assay plates with wells made of plastic.
The assay plates in other embodiments of the invention comprise a multiplicity of microtiter wells, and in some embodiments, polystyrene microtiter wells. These wells would be coated with about 500 ng/well of the recombinant HIV envelope protein, or recombinant HIV antigen or HIV-infected whole cells or cell lysates thereof.
Early Anti-HIV Antibody Detection Assays
In yet another embodiment, the invention provides for improved anti-HIV antibody detection assays using the aforedescribed native or recombinant HIV proteins/peptides as target antigen. These improved assays, particularly ELISA sandwich-type assays, provide for the detection of early anti-HIV antibodies in sandwich-type assays, provide for detection of early anti-HIV antibody in samples that test anti-HIV antibody negative using conventional EIA and WB techniques.
The format of the EIA may be described as employing plates that are directly coated with the HIV antigen (the antigen being either recombinant HIV envelope protein expressed from HIV-transfected mammalian cells, or a lysate of substantially non-denatured, solubilized HIV-infected mammalian cells that express the HIV envelope protein), or plates that are designed to function as an antibody capture sandwich assay.
Immunoassays
As noted, it is proposed that the recombinant gp160 of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of anti-gp160 conformational epitope-reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs) known to the art. However, it will be readily appreciated that the utility of the gp160 preparations described herein are not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays or procedures.
In some embodiments of the ELISA assay, native gp160 or appropriate peptides incorporating gp160 antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one will desire to bind or coat a nonspecific protein such as bovine serum albumin (BSA), casein, solutions of milk powder, gelatin, PVP, superblock, or horse albumin onto the well that is known to be antigenically neutral with regard to the test antisera. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. Following an appropriate coating period (for example, 3 hours), the coated wells will be blocked with a suitable protein, such as bovine serum albumin (BSA), casein, solutions of milk powder, gelatin, PVP, superblock, or horse albumin, and rinsed several times (e.g., 4 or 5 times) with a suitable buffer, such as PBS. The wells of the plates may then be allowed to dry, or may instead be used while they are still wet.
After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from 1 to 4 hours, at temperatures preferably on the order of 20xc2x0 to 25xc2x0 C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human IgG, IgM or IgA. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease, alkaline phosphatase, or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2xe2x80x2-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
In each of the microtiter wells will be placed about 10 xcexcl of the test patient sample along with about 90 xcexcl of reaction buffer (e.g., PBS with about 1% digitonin or other mild protein solubilizing agent). Control wells of the ELISA plate will include normal sera (human sera without early anti-HIV antibody), early anti-HIV antibody collected from HIV patient subjects who had not sero-converted as assessed using Western blot, and late anti-HIV antibody obtained from patients that have seroconverted using conventional anti-HIV antibody detection techniques.
Early HIV Infection Detection in Infants
In a particular embodiment, the invention provides for a method for detecting early IgM or IgA anti-HIV antibody in a young child. These methods are improved over existing techniques, as they provide for the earlier detection of HIV infection in infants under the age of 12 months.
Early Anti-HIV Antibody Detection Kits
In yet another aspect of the invention, a kit is envisioned for early anti-HIV antibody detection. In some embodiments, the present invention contemplates a diagnostic kit for detecting early anti-HIV antibodies and human immunodeficiency virus infection. The kit comprises of reagents capable of detecting the early anti-HIV antibody immunoreactive with the native or recombinant HIV antigen described here.
In some embodiments, the kit may also comprise a container means comprising a secondary antibody capable of detecting the early anti-HIV antibody which is immunoreactive with the recombinant HIV envelope antigen.
The HIV antigen reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, plastic beads or plates, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent. In yet other embodiments, the kit may further comprise a container means comprising an appropriate solvent.
In some embodiments, the kit comprises a container means that includes a volume of a second antibody, such as goat anti-human IgG or IgM conjugated with alkaline phosphatase or other anti-human Ig secondary antibody, and a second container means that includes a volume of a buffer comprising a non-denaturing solubilizing agent, such as about 1% digitonin.
The kit may in other embodiments further comprise a third container means that includes an appropriate substrate, such as PNPP for alkaline phosphatase, or 9-dianisidine for peroxidase. A fourth container means that includes an appropriate xe2x80x9cstopxe2x80x9d buffer, such as 0.5 m NaOH, may also be included with various embodiments of the kit.
The kit may further include an instruction sheet that outlines the procedural steps of the assay, and will follow substantially the same steps as the typical EIA format known to those of skill in the art.
Idiopathic Chronic Lymphopenia Screening
In yet another aspect of the invention, methods for screening samples for evidence of HIV in idiopathic chronic lymphopenia are provided. In some embodiments, the method comprises obtaining a biological fluid sample from a patient; exposing said sample to a native or recombinant human immunodeficiency virus envelope protein which is capable of binding early anti-HIV antibodies under conditions sufficient to allow immunocomplex formation between the labeled recombinant protein and any antibody present in the patient sample, so as to provide an incubation mixture; and identifying the presence of immunocomplex formation in the incubation mixture, via some label, wherein the presence of labeled immunocomplex formation provides a screen for HIV in idiopathic chronic lymphophenia.

EIA test using nondenatured HIV antigen for early detection of HIV infection

Board of Regents, The University of Texas System


Provided may be a method of manufacturing a semiconductor device. The method may include forming a plurality of isolation patterns including conductive patterns on a semiconductor substrate and forming gaps between the isolation patterns, forming active patterns filling the gaps on the semiconductor substrate, forming a gate insulation layer on the isolation patterns and the active patterns, and forming gate patterns on the gate insulation layer.

1. Field
Example embodiments relate to a method of manufacturing a semiconductor device, and more particularly, to a method of manufacturing a semiconductor device including an isolation process.
2. Description of the Related Art
In general, unit transistors, e.g., unit cells, of a semiconductor device may be electrically insulated using an isolation layer. The isolation layer may include a field oxidization layer formed by selectively oxidizing a semiconductor substrate, for example, a silicon substrate, using Local oxidation of silicon (LOCOS) or a trench isolation layer formed by filling an isolation layer in a trench prepared by etching a semiconductor substrate to a fixed depth.
The field oxidization layer may be undesirable in terms of integration, and thus, may not be used as semiconductor devices are highly integrated devices. Instead, the trench isolation layer may be used in most cases. Because the trench isolation layer is formed on the trench prepared by etching a semiconductor substrate to a fixed depth, a semiconductor device may be highly integrated in one plane.
However, in the trench isolation layer, a depth of the trench may be larger for insulation between unit cells and a width (breadth or diameter) of the trench may be smaller to allow for higher integration. In particular, if a depth of the trench is not large, the unit cells may not be electrically insulated. An insulation layer may be filled in the trench when the trench insulation layer is formed. However, when a depth of the trench is larger and a width of the trench is smaller, the insulation layer may not be properly filled in the trench. When the insulation layer is not properly filled in the trench, the unit cells may not be electrically insulated.

Method of manufacturing semiconductor device including isolation process


A safety retainer for retaining an electrical device connected to an electrical receptacle against inadvertent disconnection includes a cover plate having opposed raised bars secured to the cover plate adjacent the electrical connector of the receptacle and a retainer strap looped beneath the raised bars and across the electrical device to secure the electrical device in place when connected to the receptacle, the cover plate being unbroken between the electrical connector and the peripheral edge of the cover plate so as to provide a completely integrated cover plate over the receptacle.

1. Field of the Invention
The present invention relates generally to safety retainers for electrical devices and pertains, more specifically, to a safety retainer for retaining an electrical device connected to an electrical receptacle against inadvertent disconnection from the electrical receptacle.
2. Description of the Prior Art
Conventional electrical receptacles usually include at least one, and usually more than one, electrical connector having a connector element with sockets for receiving complementary pins projecting from an electrical plug on the electrical device to be connected to the receptacle. A cover plate covers the receptacle and includes apertures through which the connector elements are accessed for connection to the electrical device. It has become accepted widely, in the installation of various low-voltage systems, such as intruder alarm systems, smoke and fire alarm systems, communications systems, and the like, to connect a small transformer directly to an available receptacle, usually found in a wall, and rely upon the electrical connection between the pins and the sockets to maintain the mechanical as well as the electrical connection between the transformer and the receptacle.
In many installations, a supplemental mechanical connection, often coupled to an existing screw which holds the cover plate in place, is employed to secure the transformer in place against inadvertent disconnection. Local building codes generally require that the integrity of the cover plate be maintained; hence, these supplemental mechanical connections should not disturb the integrity of the cover plate. While a wide variety of retention devices are available for securing electrical connectors and electrical devices in place when connected at an electrical receptacle, these available devices often are cumbersome, require somewhat complex installation procedures, and do not permit ready removal, should such removal become necessary for maintenance purposes. Those retention devices which are more simple in construction and use often do not maintain adequate and reliable securement.

Safety retainer for an electrical receptacle


To provide a convenient selection of origin and/or destination of a route, in an on-line map service, the user is permitted to select and view a map and select any point from the displayed map as one of the points necessary for computation of a route. In an example, the user selects a start point, and the client device provides pixel coordinates of the first selected point. The user then selects the other point from the map, and the client device provides coordinates of the other selected point. A GIS program at the client converts the coordinates to map units and communicates with the server in order to access map information, compute a route, and send back route information for presentation to the user. The resulting route information, for example, may take the form of a map display highlighting the route or textual instructions for traveling the route.

People often use maps to plan and guide them during travel. Where the origin and destination are known or selected, a route can be traced on a street map and/or presented as textual directions. Traditionally, travel agencies, car rental firms and automobile/travel clubs provided paper maps or map booklets of region(s) to be traveled along the route, with highlighting to indicate the recommended travel route.
Map applications were developed for personal computers that offered similar capabilities utilizing a local database of map information. The computer's user interface allowed the user to input points of origin and destination, and possibly points of interest along the way. The application program would retrieve information from the database to construct a display of a map, overlaid with a route between the origin and destination. The personal computer typically offered an option to print out a copy of the resulting route map. Similar route mapping capabilities have been developed for on-board vehicle navigation systems and/or handheld GPS type navigation devices.
More recently, on-line services, like Mapquest.com, have offered map and direction capabilities via the Internet. Such services utilize a client-server communication paradigm. Essentially, the mapping database resides on a server coupled to the public Internet. A user establishes a communication session through the Internet, and client browser software enables communications with the server. Via the client browser, the user can identify a point or location, and the server returns map data for displaying the selected location. If the user operates the terminal device to identify a point on the map, the browser sends the selection to the server and the server returns a related map display, e.g. a version of the map zoomed in on the selected point. The user may also input origin and destination information, and the server will return map data showing the route between the points and textual directions for travel between the points.
The user interface capabilities of the on-line services for mapping routes and/or providing travel directions, however, have been limited. Existing on-line services, designed to map and plot routes require the user to enter some amount of address information. This address information could be in the form of a full address or may be just a zip code or city/state combination. The point is that some sort of address information is required, for both the starting point of the route (origin) and the ending point (destination) of the route. While such an approach is convenient at times, there are situations where a user may prefer a quicker or more convenient solution for inputting information about one or the other or both of these key points of reference.

GIS point-to-point routing for directions


A method for carrying multiple services includes: transmitting services to bearer channels at a physical port of an XDSL chipset according to different requirements of the services on service latency; and mapping the services on the bearer channels into different latency paths according to preset relations between the bearer channels and the latency paths. Network apparatus for carrying multiple services are also provided. The embodiments of the present invention enable different services to be transmitted on different latency paths, by which the requirements of different services on both transmission latency and transmission quality are fulfilled. The embodiments of the present invention also remedy the defect in ADSL and VDSL technologies that XDSL chipset does not support multiple latency paths, which improves the application of XDSL technology and further improves user satisfaction.

In the late 1990s, new services including online surfing, video multicast, Video on Demand (VoD), Voice over Internet Protocol (VoIP), etc, emerged one after another along with the rapid development of Internet, and the new services have imposed higher requirements on network bandwidth. Varieties of access network technologies have been developed to satisfy the demand of users for bandwidth, e.g., Integrated Services Digital Network (ISDN), coaxial cable modem, Asymmetrical Digital Subscriber Loop (ADSL), Very High Speed Digital Subscriber Loop (VDSL), etc.
Among the emerging new access network technologies, ADSL provides a high-speed data transmission on twisted pairs based on the existing narrow band telephony network, which is favorable for conventional operator to support new services with a low cost and a high efficiency, hence the ADSL technology is very promising. Though ADSL technology appeared more than 10 years ago, it is still the mainstream broadband access technology and will probably remain popular for another couple of years.
Being challenged by other new technologies, ADSL technology does not stop evolving but keeps on developing and perfecting the technology itself. The authority that issues the ADSL standards, since the issuance of the first ADSL standard G.992.1 in 1999, has issued ADSL2 standard G.992.3 and ADSL2+ standards G.992.5. The development of ADSL technology is directly reflected most in the ever increasing transmission bandwidth: at first the download transmission rate supported by ADSL technology was 6144 k and the upload transmission rate was 896 k, now ADSL2+ supports an upload transmission rate of millions of bits and a download transmission rate of tens of million bits.
Another mainstream broadband access technology other than ADSL technology is VDSL technology. VDSL technology also provides broadband services based on twisted pairs of narrowband telephony network. The frequency spectrum of VDSL technology is wider. Therefore the data transmission rate provided by VDSL technology can be as high as 200 Mb. However, while providing services, VDSL technology requires a shorter distance between a central office and end users and consequently certain modifications of the existing networks. Therefore VDSL technology has not been applied in large scale. Greater development is expected in a few years.
A conventional method for carrying multiple services at an XDSL physical port of a single latency path achieves multiple services recognition only, i.e., on either single Virtual Channel (VC) or multiple VCs, the services are transmitted on the same latency path, thus the transmission latency and quality of different services are also the same, which makes it impossible to fulfill different requirements of different services on transmission latency and transmission quality, e.g., VoIP service requires short latency and tolerates low transmission quality to some extent while video multicast or VoD service is very sensitive to frame loss in the transmission. When the transmission latency at the XDSL port is short, the requirements of voice services on time latency will be satisfied while the requirements of video services on frame loss rate can not be satisfied. When the transmission latency at the XDSL port is long, the requirements of video services on frame loss rate will be satisfied while the requirements of voice services on time latency can not be satisfied. As a consequence, when the XDSL port supports a single latency path, the requirements of varieties of services are all taken into consideration while setting the maximum transmission latency at the XDSL port, which results in a poor quality in general for all services and eventually low satisfaction of users.

Method and network apparatus for carrying multiple services


Disclosed herein are botanical tinctures that can be variously formulated and packaged and used as dietary supplements and in the prevention and treatment of symptoms associated with disorders affecting digestion or the digestive tract (e.g., symptoms of functional gastrointestinal disorders, FGIDs).

Chinese medicine has evolved over thousands of years and incorporates the use of Chinese herbal medicines (CHMs) and various practices, such as acupuncture; tui na manipulative therapy; and qi gong and tai chi mind-body stretching exercises. In the United States and elsewhere, people are increasingly using CHMs as dietary supplements and engaging in other practices, such as those just mentioned, to complement pharmaceutical medications.


The storage life of cereal grains or other products is extended by reducing biological activity in the storage enclosure. Air is oxidized in a chamber which is external to the storage enclosure. The oxidized air is propelled from the oxidizing chamber to the storage enclosure. Oxidation of the air may be produced by heat, ultra-violet radiation, or catalyzation. Heating may be done in a block provided with anodes for dissipating electrostatic charges. After being heated, the air is cooled before it is propelled to the storage enclosure. When air is oxidized by ultra-violet radiation, it is processed by an ozone decomposition system before it is admitted to the storage enclosure.

The present invention refers to a process, an installation and a chamber for reducing the biological activity in storage spaces, such as silos, storage sheds, grain stores, cargo holds in grain transport ships or the like, and particularly for the storage and preservation of grains or other perishable foodstuffs.
The storage of large quantities of agricultural products and principally cereals is problematic with regard to the preservation of the product throughout the period of storage. Losses due to the deterioration of stored grains has been a considerable problem on a worldwide scale and it should be observed that losses of this nature are presently of the order of 30% of the total product stocked in Brazil, about 12% in the USA and as high as 50% in some countries of the African continent.
Cereals are characterized by a high potential energy content. Their availability in thousands of tons makes storage locations an excellent nutrition medium for fungi, bacteria, acarids and insects, provided that the ambient conditions such as temperature and humidity (absolute humidity of the grain and relative humidity of the atmosphere) are favorable. The processes used up to the present involve a storage system which controls such parameters so as to reduce biological activity to a minimum. The controls act on the following variables:
Reduction of the absolute humidity of the grain by previous drying.
Constant airing of the storage ambient so as to reduce the fungi population by entertainment of the spores and to reduce the temperature increased as a result of biological activity.
Utilization of gases such as nitrogen (N2), carbon dioxide (CO2) and other gases with the basic purpose of reducing the concentration of oxygen and thus producing a reduction in the aerobic organisms population as well as the metabolism of the grain itself.
Use of toxic chemical products to reduce the amount of microorganisms and insects by intoxication.
All these processes have limited efficiency and some present health risks to the consumers, particularly those related to the use of toxic chemical products.
According to the most recent scientific works regarding the inter-relation of the various variables within a mass of cereals, fungi have been identified as one of the most important elements in the matrix of the echo-system. The following works can be cited as being of special importance: 1) Sinha R.N., Van Bronswijk J.E.M.H., Wallace H.A.H., C.M.A. Journal, Aug. 1, 1970, Vol. 103, 300-301 2) Sinha R.N., Van Bronswijk J.E.M.H., Environm. Entomology 2: 142, 1973 3) Sinha R.N., Journal of Economic Entomology, Vol 64, 1, 3-6. 4) Mori M., Onoue Y., Takahashi T., Chounan S., Moriya K., J Antibact. Antifung. Agents Vol. 15 N.degree. 12, P 601 -605, 1987. 5) Griffiths A.D., Hodson A.C., Christensen C.M., Journal of Economic Entomology, Vol. 52, N.degree. 3, pag. 516-518. 6) Sinha R.N., Harasymek L., Enviromental Entomology Vol 3, N.degree. 2, pags. 244, 246. 7) Tadros, M.S., Vestnik Ceskoloveniske Spolecnosti Zoologicke, Svazek XXXIX, Cislo 4, 1975, Str. 293-296.
The above works show that the elimination of fungi in the stored mass results in the reduction over a period of time of the population of insects and acarids. It is important to note that not only fungivorous organisms depend on fungi but also all larva which, after hatching of the eggs, need fungi to be able to create their digestive systems.
A device for oxidizing particles in suspension in air is already known from Brazilian patent PI 8302255 (U.S. Pat. No. 4,877,990) and European patent 281.551 of the present inventor. Such device creates the generation of high power density within ducts provided in a refractory ceramic block by means of a resistance wire passing through such ducts. These devices are suitable for small and medium sized ambients but are not specially adequate for large areas, such as large stores for the stocking of grain.
Brazilian patent application PI 8502832 of the present inventor discloses a system for the preservation of agricultural products by sterilizing the air. That system uses U-shaped pipes or tubes inside silos, each tube being formed with various orifices in both of its vertical arms and one of the arms having in its interior an apparatus of the type disclosed in Brazilian patent PI 8302255 (U.S. Pat. No. 4,877,990). Such a system, however, was found to have the following disadvantages:
reduced preservation capacity per apparatus: the maximum amount with which satisfactory results of air sterilization were obtained is limited to about 200 kg of stocked product per apparatus;
grain damage: heat generation within the silo tends to dehydrate the stocked grain, thus prejudicing its preservation; and
necessity to adapt the interior of the silos: the same number of U-shaped pipes as of the necessary apparatuses has to be installed within the silos.
Another disadvantage of the prior art grain storage systems is related to the generation of static electricity in view of friction between individual grains, in particular at the time of storage, and this may generate discharges that can cause explosions should there be any inflammable gases in the silo or shed, or even serious injury or death to persons within the silo.
Considering the prior art, the object of the present invention is to eliminate fungi from storage ambients so as to permit agricultural products to be preserved without the use of chemicals, as well as to permit the storage of the product with high absolute humidity content, in a totally new manner and without damaging the sorted grain.
A further object of the invention consists in eliminating the electrostatic charge in the stored product so as to avoid the resulting disadvantages and risks.
The process by which the above objects may be reached is characterized by the following steps:
gradually extracting the air from the ambient; ambient;
oxidating air within a chamber located outside the ambient;
forcing the processed air into the ambient.
The above process may be applied in closed, open or mixed circuit, that is to say, the air may circulate permanently without exchange with the atmosphere, or fresh processed air may be admitted to the ambient together with total removal of the air previously within the ambient, or further, part of the air may be circulated and the rest admitted from outside.
The use of as closed circuit, however, has the advantage that the air of the storage ambient is gradually sterilized with respect to the presence of microorganisms until almost total sterilization is obtained. Apart from this, a closed circuit permits one to obtain a hydrostatic balance between the air and the stored product.
The new concept presently proposed consists in introducing to the storage ambient regular volumes of sterilized air, sterilization being obtained by subjecting the air to elevated temperatures, of the order of 500.degree. C,. and subsequently cooling the air before it is pumped into the storage ambient. The air is heated in such a manner that heat is not dissipated within the storage ambient, so as to permit temperature and humidity conditions to be maintained within the desired parameters.

Process, installation and chamber for reducing biological activity in an enclosure, particularly for a storage space


The invention includes an image sensor, a shutter which opens/closes by making a front curtain and a rear curtain travel, a light source which illuminates the image sensor, a control unit which performs control to make the front and rear curtains travel with a predetermined time shift to form a slit-like opening, and to make the light source emit light a predetermined period of time after the start time of traveling of the front and rear curtains, a detection unit which detects the deviation between the position of an image of the recorded slit-like opening and a predetermined reference position, a computation unit which calculates the error between the traveling start timing of the front and rear curtains on the basis of the detected deviation, and a correction unit which corrects the calculated error between the traveling start timing of the front and rear curtains.

1. Field of the Invention
The present invention relates to a technique of detecting and correcting the traveling accuracy of the shutter of a camera.
2. Description of the Related Art
Conventionally, as disclosed in Japanese Patent Application Laid-Open No. 6-208157, there is known a camera having a function of measuring the traveling accuracy of shutter curtains by measuring the passage times of the shutter curtains using an LED as a light source and a phototransistor which receives light from the LED.
In the camera disclosed in Japanese Patent Application Laid-Open No. 6-208157, the light source and the light receiving unit need to be arranged outside the opening of the shutter used for recording, and hence the size of the shutter becomes large. In addition, since measurement is not made on a portion of the shutter which is actually used for recording, sufficient accuracy cannot be obtained.

Camera with shutter speed detection and correction


A compact current transformer is formed by arranging a pair of secondary windings on opposite sides of a single turn primary winding. A core is then wound about the primary winding and part of the secondary winding coils from a continuous roll of metal strap. The secondary windings are arranged on a pair of bobbins on opposite sides of the transformer core. A modular twin bobbin arrangement allows the secondary windings to be wound from a continuous source of transformer wire. The continuous wound core eliminates air gaps and improves magnetic transfer between the primary winding and the secondary winding. The continuous transformer wire eliminates the need for soldering or welding to connect between the two secondary windings.

The use of electronic trip units in low-ampere industrial-rated circuit breakers has often been forestalled because of size constraints on the discreet electrical and electromagnetic components within the circuit breaker enclosure. The current transformer used in conjunction with the electronic trip unit, has a two-fold requirement namely, to provide an input signal to the trip unit representative of the current flow within the associated protected power circuit while providing the necessary input power to the trip unit power supply. A predetermined maximum core volume is required within the current transformer to ensure that the current transformer does not become magnetically saturated upon the occurrence of overcurrent conditions when used within compact circuit breakers having variable ampere ratings while a predetermined minimum core volume insures that the core will become sufficiently magnetized at the lower steady-state operating current levels.
With earlier-designed electronic trip circuit breakers, such as described within U.S. Pat. No. 4,281,359 entitled "Static Trip Unit for Molded Case Circuit Breakers", for example, a standard trip unit circuit is employed over a wide range of ampere ratings while the size of the current transformer used to sense the input current to the trip unit circuit is correspondingly increased in proportion to the increased ampere rating.
When compact electronic trip unit circuit breakers employing various accessory devices, such as described in U.S. Pat. No. 4,754,247 entitled "Molded Case Circuit Breaker Accessory Enclosure", are used within industrial rated power distribution circuits, the size constraints of the circuit breaker enclosure limit the geometry of the current transformer core to a size just sufficient to provide operating power to the electronic trip unit circuit without becoming saturated at the higher ampere ratings due to the low inductance of the smaller core. Another problem involved with the use of small-sized current transformer cores is the lack of sufficient core inductance to provide the requisite core magnetization for transformer operation at the lower ampere ratings.
U.S. Pat. No. 5,515,597 entitled "Method for Assembling a Current Transformer" describes a compact core arrangement for current transformers and the like that is accomplished by winding the secondary coils around the completed magnetic core.
U.S. Pat. No. 5,015,983 entitled "Compact Circuit Interrupter Having Multiple Ampere Ratings" describes a compact current transformer arrangement using a metal core formed from laminations of silicon sheet steel positioned over a pair of secondary windings. The air gaps inherent with such laminated steel plates increase the core losses that are subsequently compensated for by increasing the core size and the amount of core material.
U.S. patent application Ser. No. 08/663,760 entitled "Compact Circuit Interrupter Having Multiple Ampere Ratings" describes a compact current transformer used within circuit breakers having electronic trip units. The continuous arrangement of a sheet of core material about the primary winding provides a transformer core without air gaps. When current transformers are formed about a pair of secondary winding coils the two coils are separately wound on individual bobbins and are later electrically connected together such that the directions of the wires in each of the coils is in opposite directions. One early example of a pair of miniature coil bobbins used within telephone receivers is found within U.S. Pat. No. 4,103,268 entitled "Dual Coil Hinged Bobbin Assembly".
It would be advantageous to arrange the separate bobbins in such a manner that the coils could then be wound from a continuous source of wire without requiring separate connection operations and orientation as is now required.
One purpose of the invention is to provide a unique bobbin arrangement that allows the two coils to be wound from a single source of wire to eliminate the inter-coil connection process.

Electronic circuit breaker having modular current transformer sensors


Methods, systems, and devices for wireless communications are described in which a user equipment (UE) may monitor for transmissions during an on-duration of a discontinuous reception cycle. The UE may receive one or more go-to-sleep indications from one or more transmission/reception points (TRPs), and may discontinue monitoring for transmissions from one or more of the TRPs responsive to the one or more go-to-sleep indications. Each TRP of the two or more TRPs may transmit a separate per-TRP go-to-sleep signal that indicates that the TRP will not transmit during a time period associated the go-to-sleep signal. In other cases, a TRP may transmit a cross-TRP go-to-sleep signal that has go-to-sleep indications for multiple TRPs.

Wireless communications systems are widely deployed to provide various types of communication content such as voice, video, packet data, messaging, broadcast, and so on. These systems may be capable of supporting communication with multiple users by sharing the available system resources (e.g., time, frequency, and power). Examples of such multiple-access systems include fourth generation (4G) systems such as Long Term Evolution (LTE) systems, LTE-Advanced (LTE-A) systems, or LTE-A Pro systems, and fifth generation (5G) systems which may be referred to as New Radio (NR) systems. These systems may employ technologies such as code division multiple access (CDMA), time division multiple access (TDMA), frequency division multiple access (FDMA), orthogonal frequency division multiple access (OFDMA), or discrete Fourier transform spread orthogonal frequency division multiplexing (DFT-S-OFDM).
A wireless multiple-access communications system may include a number of base stations or network access nodes, each simultaneously supporting communication for multiple communication devices, which may be otherwise known as user equipment (UE). In some wireless communications systems, a UE may support operations in a discontinuous reception (DRX) mode, where the UE may transition to a sleep mode and periodically wake up to monitor for data or control information from a base station in accordance with a DRX cycle. As such, the UE may save power since the UE may not have to constantly stay awake or constantly monitor for data or control information from the base station. In such systems, efficient mechanisms to transition a UE between sleep mode and wake up mode may be desirable to further reduce power consumption at the UE.

Techniques for signaling go-to-sleep for multiple transmission/reception points


A portable ramp for use in allowing access for the disabled, in a wheelchair, to enter an automobile, elevated area and the like or for similar entry use by a pet. The ramp is comprised of a platform member having folding legs and a ramp hingedly attached thereto with ramp elements, each having a bevelled end for selective length placement of ramp elements on a ground surface (with other non-used elements being folded or positioned within the elements being used for the ramp). With the ramp elements being foldable into each other and the platform legs being foldable thereinto, the ramp element and platform are foldable into a readily carryable attache configuration.

Wheelchairs, while providing the disabled with capability of movement, are restricted in ready access to elevated areas such as automobiles, buses, vans and the like, wherein, though the elevation is slight, the wheelchair wheels are generally unable to negotiate the height. Accordingly a plethora of elevation ramps have been devised to permit wheelchair users to negotiate step level heights. However, the problem with many of such ramps, even if they are simply constructed of folding components, is the overly large weight and volume they occupy even when folded, making them difficult to transport for possibly occurring uses. In addition many of the ramps are unstable, especially if the ground surfaces upon which they are supported is not substantially flat.


Systems and methods for multi-dimensional metrology and inspection of a specimen such as a bumped wafer are provided. One method includes scanning the specimen with partial oblique illumination to form an image of the structure, either through the normal collection angle or through an oblique collection angle. The method also includes integrating an intensity of the image and determining a height of the structure from the integrated intensity. The integrated intensity may be approximately proportional or inversely proportional to the height of the structure. In addition, the method may include scanning the specimen with bright field illumination to form a bright field image of the specimen. The method may also include determining a lateral dimension of the structure from the bright field image. Furthermore, the method may include detecting defects on the specimen from the bright field image or the obliquely-illuminated image.

1. Field of the Invention
The present invention generally relates to systems and methods for three-dimensional metrology and/or inspection of a specimen. Certain embodiments relate to systems and methods for metrology and inspection of a specimen such as a bumped wafer.
2. Description of the Relevant Art
Fabricating semiconductor devices such as logic and memory devices typically includes processing a specimen such as a wafer using a number of semiconductor fabrication processes to form various features and multiple levels of the semiconductor devices. For example, lithography is a semiconductor fabrication process that typically involves transferring a pattern to a resist arranged on a wafer. Additional examples of semiconductor fabrication processes may include, but are not limited to, chemical-mechanical polishing, etch, deposition, ion implantation, and electro-chemical plating. Multiple semiconductor devices may be fabricated in an arrangement on a wafer and then separated into individual semiconductor devices.
Prior to being separated into individual semiconductor devices, an entire wafer of semiconductor devices may be prepared for packaging. Packaging may include coupling a chip carrier such as a substrate to a semiconductor device. A chip carrier may provide mechanical strength, heat transfer, and environmental protection to a semiconductor device. In addition, a chip carrier may include electrical wiring or other electrical structures such that electrical connection may be made between a semiconductor device and another structure such as a card-on-board ("COB") structure for a computer system. Preparing a wafer of semiconductor devices for packaging may include forming structures on a surface of the semiconductor devices that may be used to couple a semiconductor device to a chip carrier. For example, relatively small solder balls may be arranged in an area on a semiconductor device. Such relatively small solder balls may be commonly referred to as "bumps," and a wafer on which such solder balls are formed may be commonly referred to as a "bumped wafer." The solder balls may be configured to physically attach the semiconductor device to a chip carrier and to make electrical connection to a chip carrier. For example, a device may be placed on a substrate, and the solder balls may contact and melt onto an array of metal pads on the substrate. Such a packaging method may be commonly referred to as face-down bonding, flip-chip bonding, or controlled collapse chip connections ("C4").
Bumps that are incorrectly formed on a wafer may cause significant problems during further processing such as probing of the wafer and during use of the device. For example, incorrectly formed bumps may damage probes on a probe card thereby causing tester downtime and incurring tester repair costs. Incorrectly formed bumps may also bridge power and ground contacts thereby resulting in excessive current draw through a probe card. Therefore, bumped wafers may typically be inspected and measured prior to probing. In addition, incorrectly formed bumps may not be detected during probing and may increase the potential for failure of a packaged device. Furthermore, probing may damage bumps especially on a wafer having a relatively high number of bumps per unit area that may require high vertical forces during testing to seat probes for proper contact. In this manner, inspection and metrology of a bumped wafer may also be performed after wafer probing.
Currently available methods for three-dimensional inspection, which may be commonly referred to as "defect detection," and metrology of bumped wafers generally include first acquiring data in the x-y plane and then acquiring data in the z plane. Essentially, such systems may be described as being serial in data acquisition (i.e., two-dimensional ("2D") data acquisition followed by three-dimensional ("3D") data acquisition). For bumped wafers, 2D defects may include, for example, missing bumps, improperly located bumps, bridged bumps, large-diameter bumps, and small-diameter bumps. Examples of 3D defects may include, but are not limited to, bumps that may be too tall or too short or that may have protruding vertical spikes or recessed vertical pits. Because currently available methods are generally not capable of simultaneous 2D and 3D data acquisition, such methods may be significantly slower than either 2D or 3D data acquisition. In addition, 3D data acquisition may be significantly slower than 2D data acquisition thereby resulting in substantially reduced sampling for 3D in comparison to sampling for 2D. As used herein, the term "sampling" generally refers to the number of points or locations inspected or measured on a single specimen during a process. In other words, if a relatively large percentage of a wafer may be scanned to acquire x-y data, then only a much smaller percentage of the wafer may be scanned to acquire additional z data.
One currently available system, the ABI-2000 automatic bump inspection system, available from KLA-Tencor, San Jose, Calif., is configured to scan a wafer using bright field, white light imaging with image data captured using a charge coupled device ("CCD") camera and a frame-grabber. A frame-grabber is a device that may be configured to receive a signal from a detector such as a CCD camera and to convert the signal (i.e., to digitize an image). In such a system, inspection and metrology algorithms are used to detect defects in the x-y plane and to make measurements in the x and y directions. Such a system is also configured to re-scan, or "revisit," select locations on the wafer to acquire 3D data for individual bumps. This revisiting uses laser scanning microscopy and incremental z movement of the wafer along with additional algorithms to detect bump defects in the z plane and to make bump measurement in the z-direction.
In another example, systems developed and manufactured by Robotic Vision Systems, Inc., Canton, Massachusetts, e.g., Model Nos. WS-1000 and WS-2000, incorporate 2D vision technology using a time delay integration ("TDI") camera and low-angle ring light or co-axial light. Such systems are configured to first scan a wafer to detect typical 2D bump defects and surface defects. Such systems are also configured to perform a second wafer scan using a laser triangulation technique to acquire 3D metrology measurements and to detect 3D bump defects. An example of a laser triangulation technique is illustrated in U.S. Pat. No. 6,181,472 to Liu, and is incorporated by reference as if fully set forth herein.
In yet another example, systems developed and manufactured by August Technology Corporation, Bloomington, Minn., e.g., the NSX bump inspection system, incorporate 2D vision technology using a high-resolution CCD camera and strobe illumination. Such systems are configured to first inspect a wafer to acquire 2D data for 2D defect detection of bumps and defect detection elsewhere on the surface of the wafer. For 3D data acquisition, relatively small, select regions of the wafer are revisited. At each of the smaller regions, the system may use automatic focusing at different vertical locations to determine the height of all bumps in the field of view.
Another example of a system for measuring 3D surface topography provides high resolution contour measurements of an object using interferometric methods. The system includes an optical head that is a moiré-type interferometer optical head employing co-sight detector phase shifting. Ronchi gratings are placed at image planes to produce moiré fringe patterns indicating the contour of the surface. The moiré images are relayed to detector planes, and CCD camera arrays view exactly the same image of the part except for the fringe pattern superimposed on the image. The images, and bucket images collected from a known reference surface needed to properly compensate the images and phase data, are used with a standard bucket phase algorithm. Output of the algorithm is analogous to the surface contour of the sample inspected. One example of such a system is illustrated in U.S. Pat. No. 6,268,923 B1 to Michniewicz et al., which is incorporated by reference as if fully set forth herein.
An additional example of a moiré-type system for 3D inspection of an object includes a source of light projected through a grid assembly. An image acquisition apparatus includes one or more cameras with an array of pixels in the form of a CCD camera. The system is used to simultaneously project at least three phase-shifted grids onto an object, to simultaneously take an image of each of the phase-shifted grids on the object, to gather an intensity value for each pixel of the images, and to compute the phase for each pixel of the image using the intensity images. The above steps are performed for a reference object and the object to be measured. The difference of height between the object and the reference object for every pixel is computed by using the respective phases for each pixel, and the relief of the object for each pixel is determined using the difference of height at every pixel. An example of such a system is illustrated in U.S. Patent Application Publication No. US 2002/0018118 A1 to Coulombe et al. and PCT International Publication No. WO 01/71279 A1 to Coulombe et al., which are incorporated by reference as if fully set forth herein.
There are several disadvantages to the moiré-type systems and methods for three-dimensional inspection of objects. For example, these systems and methods include complex system configurations and rely on data collected from a separate reference object. Therefore, the methods have a lower throughput than methods that do not require a separate reference. In addition, the systems measure and use a sine wave pattern to determine the surface contour or relief of the object. Therefore, the sensitivity of the system may vary for objects of different heights. For example, the system may be more sensitive to measurements that correspond to a slope of the sine wave pattern than to measurements that correspond to a crest or valley of the sine wave pattern. In addition, using such a sine wave pattern may yield inaccurate height measurements. For example, the period of the sine wave pattern may be selected to correspond to a range of expected heights. However, if the height of an object is outside of this range, the height of the object may be inaccurately reported with an error of one or more times the range of heights.
There are several additional disadvantages to currently available systems and methods for three-dimensional inspection of bumped wafers. For example, as described above, such systems are configured to scan a wafer more than once in series to acquire 2D and 3D data for inspection and metrology. In addition, configurations of such systems are incompatible with rapid scanning of relatively large areas of bumped wafers. Such disadvantages will generally lower a throughput of an inspection and metrology system. Furthermore, such systems may be relatively complex due to the use of separate 2D and 3D data acquisition and processing systems. This disadvantage will generally increase cost and reduce reliability of an inspection and metrology system.

Systems and methods for multi-dimensional metrology and/or inspection of a specimen


A leakage calibration and removal system and method estimates the complex in-phase and quadrature phase (I/Q) components of a leakage signal for each beam location in the sampled down-converted radar signal in a radar system (10). In a digital embodiment, the stored leakage calibration signal (264) is subtracted (206) from the sampled radar signal, and the resultant signal is processed (208, 210, 212) to detect targets. A leakage calibration process (250) is activated if a leakage signal test (214) indicates a problem for a sufficient number of consecutive scans (216), wherein for each beam location, M consecutive I/Q waveforms are averaged (252), known targets are removed (254, 256, 258), and the resulting signal is scaled (262) and stored (264) as a new leakage calibration signal if the variance is within acceptable limits (262). In a hybrid embodiment, the stored leakage signal (364) is converted to analog form (366), subtracted (301) from the analog down-converted radar signal (300), and scaled by a variable gain (303) before the complex I/Q components are sampled therefrom (302, 304). A leakage calibration process (350) is activated every Nth scan (316), wherein for each beam location, M chirp waveforms are averaged (352) and checked for variance (354). If the variance is within acceptable limits, the leakage signal is updated by a Kalman Filter (356), stored as the new leakage calibration signal (364), and the Kalman gain matrices are updated (358).

A radar system measures the distance and/or velocity of a target by sensing the effects of the interaction of the target with a beam of either continuous or pulsed electromagnetic energy. In a linear frequency modulated continuous wave (LFM CW) radar system, the target is continuously illuminated with electromagnetic energy, the frequency of which is linearly modulated in time in accordance with a periodic pattern. The radar receiver measures the distance to the target from the difference in frequency between the received and transmitted signals. One problem with LFM CW radar systems results from leakage of a portion of the transmitted energy that is directly coupled to the receiver without first interacting with a target so as to alias as a stationary near range target. The strength of this leakage signal can be sufficiently great that sidelobes thereof mask the target return signals. Radar systems that incorporate a single antenna for both transmit and receive are particularly susceptible to such leakage problems.
Some prior art linear frequency modulated continuous wave (LFM CW) radar systems, including many automotive CW applications, use separate antennas for transmitting and receiving the radar signals. While separate antennas substantially reduces the problem of leakage, the primary difficulty with this method is that the use of separate transmit and receive antenna arrays can prohibitively increase the cost and size of the system.
Other prior art CW radar systems remove the leakage component in the received signal by mixing the received signal with a portion of the transmitted signal that has been shifted in phase by a fixed analog delay line. The analog delay line must be correctly matched to the leakage. The problem with this approach is that the delay of fixed analog delay line is not responsive to changes in the leakage that may result from temperature variations, etc. The problem with analog delay lines is compounded in multiple beam aperture (MBA) architecture radar systems, wherein each beam can have a distinct leakage path and correspondingly requires a separately delayed signal to compensate for the associated leakage. With an increasing number of beams, the corresponding number of delay lines and associated high speed switches--that switch to the correct delay line for the given beam number--can become prohibitively expensive and cumbersome.
Yet other prior art radar systems attempt to reduce the effect of the leakage signal in the final signal processing stage by either heavily weighting the data prior to Fourier Transform processing or else by ignoring the first N range cells of data from the Fourier Transform. The problems with stronger amplitude weighting of the Fourier transform to reduce the sidelobe levels is that this causes significant broading of the peaks, which can reduce the system's ability to recognize closely spaced targets and consequently targets close to the radar system (and the host vehicle). Clearly, the problem with ignoring the first N range cells is that for automotive collision prediction and avoidance radars the near range information is vital in estimating the time to collision, and the likelihood of collision.
Still yet other systems use a pulsed radar rather than a CW radar, whereby the receiver is gated to ignore the leakage signal. When applied to automotive collision prediction, a pulsed radar system requires very short radar pulses (<6 nanoseconds) to detect targets at very near range, which short pulses are difficult to transmit with sufficiently high power to detect far range targets. Accordingly, pulsed radar systems are currently not suited for detecting targets at both near and far ranges as necessary for automotive collision prediction.


A phased array antenna may include a substrate and a plurality of controllable phased array antenna elements carried thereby, a central controller for generating a mode selection signal and beam control signals, and at least one multi-mode element controller connected to at least one of the controllable phased array antenna elements and the central controller. The at least one multi-mode element controller may be operable in a desired operating mode from among a plurality of operating modes based upon the mode selection signal from the central controller. Furthermore, the at least one multi-mode element controller may also generate output signals for the at least one controllable phased array antenna element based upon the beam control signals from the central controller.

The present invention relates to the field of communications, and, more particularly, to phased array antennas and related methods.
Antenna systems are widely used in both ground based applications (e.g., cellular antennas) and airborne applications (e.g., airplane or satellite antennas). For example, so-called xe2x80x9csmartxe2x80x9d antenna systems, such as adaptive or phased array antennas, combine the outputs of multiple antenna elements with signal processing capabilities to transmit and/or receive communications signals (e.g., microwave signals, RF signals, etc.). As a result, such antenna systems can vary the transmission or reception pattern (i.e., xe2x80x9cbeam shapingxe2x80x9d or xe2x80x9cspoilingxe2x80x9d) or direction (i.e., xe2x80x9cbeam steeringxe2x80x9d) of the communications signals in response to the signal environment to improve performance characteristics.
A typical phased array antenna may include, for example, one or more element controllers connected to a central controller. Among other functions, the element controllers process beam control signals generated by the central controller (e.g., beam steering signals and/or beam spoiling signals) and provide output control signals for each of the phased array antenna elements. More particularly, each antenna element may have a controllable device associated therewith (e.g., a phase shifter, attenuator, and/or delay generator), and the output control signals may be used to control a phase, attenuation, or delay thereof. Thus, the transmission or reception pattern may be varied, as noted above.
Phased array antenna designs can vary widely due to the numerous environments and applications in which they are used. As a result, the design of various components used in phased array antennas may also vary widely from one antenna to the next. This is particularly true of element controllers, which are typically designed to perform very specific element control functions based upon the intended use of the antenna. Thus, a common prior art approach is to implement element controllers in application specific integrated circuits (ASICs), which, as the name implies, are designed for one particular antenna application.
A drawback of this approach is that even slight changes in system requirements may dictate the use of different ASIC element controllers from one antenna to the next. Yet, the design and testing of an ASIC can be both expensive and time consuming.
Attempts have been made in the prior art to provide more xe2x80x9cuniversalxe2x80x9d communications equipment that is adaptable to changing system requirements. One example is disclosed in U.S. Pat. No. 5,999,990 to Sharrit et al. entitled xe2x80x9cCommunicator Having Reconfigurable Resources.xe2x80x9d The communicator is for use in a communications system, such as in a base station or hand held transceiver unit, and includes a plurality of reconfigurable resource units (RRUs) that can each be dynamically altered to perform different processing tasks. A controller determines the processing tasks to be supported by the communicator and configures the RRUs accordingly. More particularly, the communicator determines if one of the RRUs is already configured to perform a desired processing task. If not, the controller obtains the necessary configuration file for the desired task and determines an amount of resources required by the configuration file as well as RRU availability to determine which RRU(s) will perform the desired task.
While such a communicator may provide certain advantages when used in a base station, it may not be well suited for use as an individual element controller. One reason is that it may require a relatively large amount of processing capability as well as memory space to implement, which may not be practical in an element control ASIC because of cost and power constraints. This problem would become particularly acute in phased array antennas including numerous phased array antenna elements and a respective element control ASIC for each element.
In view of the foregoing background, it is therefore an object of the present invention to provide a phased array antenna including one or more element controllers that may be operated in multiple operating modes.
This and other objects, features, and advantages in accordance with the present invention are provided by a phased array antenna which may include a substrate and a plurality of controllable phased array antenna elements carried thereby, a central controller for providing a mode selection signal and beam control signals, and at least one multi-mode element controller connected to at least one of the controllable phased array antenna elements and the central controller. The at least one multi-mode element controller may be operable in a desired operating mode from among a plurality of operating modes based upon the mode selection signal from the central controller. Furthermore, the at least one multi-mode element controller may also generate output signals for the at least one controllable phased array antenna element based upon the beam control signals from the central controller. By having a plurality of operating modes, the multi-mode element controller may advantageously be used in numerous phased array antennas.
More particularly, the at least one multi-mode element controller may include a memory for storing temperature compensation data or other element calibration, and a processor cooperating with the memory for generating the output signals based upon the beam control signals and the temperature compensation data. The output signals may be digital output signals, for example, and the at least one multi-mode element controller may define a number of bits for the digital output signals based upon the mode selection signal. For example, automatic rounding can be set according to the variable number of output control bits.
The phased array antenna may also include a controllable device connected between the at least one multi-mode element controller and the at least one controllable phased array antenna element for controlling at least one of a phase, attenuation, and delay based upon the output signals. Further, the plurality of operating modes may include a phase control operating mode, an attenuation control operating mode, and a delay control operating mode. In addition, the beam control signals may be beam steering signals and/or beam spoiling signals, for example.
The at least one multi-mode element controller may also include an output register for storing and outputting the output signals, and a bus interface for receiving the mode selection signal and beam control signals from the central controller. Additionally, the at least one multi-mode element controller may be implemented in an application specific integrated circuit (ASIC), for example.
A multi-mode element controller for a controllable phased array antenna element is also provided according to the present invention and may include an interface for receiving a mode selection signal and beam control signals from a central controller and a processor coupled to the interface. The processor may be operable in a desired operating mode from among a plurality of operating modes based upon the mode selection signal, and the processor may generate output signals for the controllable phased array antenna element based upon the beam control signals.
A method aspect of the invention is for controlling a phased array antenna element using a multi-mode element controller. The method may include generating a mode selection signal and beam control signals, selecting a desired operating mode for the multi-mode element controller from among a plurality of operating modes based upon the mode selection signal, and generating output signals for the at least one controllable phased array antenna element using the multi-mode element controller based upon the beam control signals.

Phased array antenna including a multi-mode element controller and related method