The invention relates to a new improved catalyst to produce random copolymers of one or more iso-olefin monomers and one or more para-alkylstyrene monomers. The invention also relates to an improved continuous slurry polymerization process to produce random copolymers using the improved catalyst system. The process is carried out in an anhydrous polymerization system containing a mixture of the monomers in a polar solvent along with a Lewis acid and a stabilizing initiator.

The invention relates to an improved method for production of copolymers of isobutylene useful in rubber compounds.
Isobutylene-isoprene polymers have been well known since the 1930s. They have good impermeability to air and a high level of damping when stretched or compressed. These polymers are used extensively throughout the tire and pharmaceutical industries. The copolymers are made by a cationic slurry polymerization process at approximately xe2x88x9295xc2x0 C. using a catalyst comprising a Lewis Acid and an initiator. Suitable Lewis Acids and initiators are well documented by Kennedy, J. P. and Marechal, E., xe2x80x9cCarbocationic Polymerizationxe2x80x9d, Kreiger Publishing Company, 1991. Lewis Acids such as the aluminum alkyls and aluminum chloride are used extensively in both laboratory experiments and commercial scale production. Initiators such as water and anhydrous HCl are used extensively.
Isobutylene-para-methylstyrene (IPMS) polymers are also well known. They are made in a similar process to isobutylene-isoprene polymers using similar initiation systems and are also used in the tire and pharmaceutical industries. However, there are a number of difficulties with IPMS polymerization, as compared to isobutylene-isoprene copolymerization, and these difficulties are exacerbated by higher levels of para-methylstyrene (PMS) co-monomer. These difficulties include: instability of reaction temperatures and flash gas (reactor liquid composition); instability of Mooney viscosity control; lower than desirable conversion of monomer to product; higher than desirable warm-up rates due to rubber fouling, particularly around the reactor circulation pump; lower than desirable operability limitation on slurry concentrations; shorter reactor run lengths under comparable conditions; higher slurry viscosity under comparable conditions; and poorer and more erratic response of reactor to control parameters. Because of these difficulties it has historically been much more difficult and costly to produce IPMS copolymers than conventional isobutylene-isoprene copolymers. Currently, these undesirable process characteristics are managed by limiting throughput, PMS content, or a combination of the two.
It would be desirable to have a method for production of IPMS copolymers that reduces the magnitude of one or more of these complications, thereby reducing the current limitations on throughput and/or PMS content.
The invention relates to a new catalyst system for a continuous slurry polymerization process to produce random copolymers of one or more iso-olefin monomers and one or more para-alkylstyrene monomers. The process is carried out in an anhydrous polymerization system containing a mixture of the monomers in a polar solvent along with a Lewis acid and a stabilizing initiator. This polymerization system is capable of forming an in-situ electron pair donor using a stabilizing initiator. The stabilizing initiator according to this invention has the formula:
wherein:
R1 is an alkyl, alkenyl, aryl, aralkyl, or aralkenyl group containing up to 30 carbon atoms but not less than 3 carbon atoms unless R1 contains at least one olefinic unsaturation,
R2 and R3 are alkyl, aryl, or aralkyl groups containing up to 30 carbon atoms and can be the same or different,
X is a halogen or a carboxy, hydroxyl, or alkoxyl group, preferably a halogen, and n is a positive whole number.
The invention concerns a catalyst system and process for production of isoolefin copolymers containing a para-alkylstyrene comonomer. An improved catalyst system and process has been discovered which affords many unexpected advantages for commercial slurry polymerization of IPAS copolymers generally, and in particular IPMS copolymers. The invention is particularly useful in production of isoolefin-para-alkylstyrene (IPAS) copolymers having a higher PAS content, particularly isobutylene-para-methylstyrene (IPMS) copolymers having a higher PMS content (e.g. 10-20 weight percent PMS). This new catalyst system includes a Lewis acid with a new stabilizing initiator as described later in more detail.
The discussion and examples below are focused on preferred embodiments of the broad invention. In a particularly preferred embodiment, the copolymers produced contain isobutylene as the isoolefin and para-methylstyrene as the para-alkylstyrene comonomer. Discussion of these preferred embodiments should not be construed so as to limit the broad invention, which is applicable generally to copolymers of one or more isoolefin and one or more para-alkylstyrene (PAS) monomers. To the extent that our description is specific, this is done solely for the purpose of illustrating certain preferred embodiments and should not be taken as restricting the invention to these embodiments.
In accordance with the present invention applicants have discovered an improved polymerization system for copolymerizing an iso-mono-olefin having from 4 to 7 carbon atoms and para-alkylstyrene monomers. In accordance with a preferred embodiment of the invention, the process produces copolymers containing between about 80 and 99.5 wt. % of the isoolefin such as isobutylene and between about 0.5 and 20 wt. % of the para-alkylstyrene such as para-methylstyrene. In accordance with another embodiment, however, where glassy or plastic materials are being produced as well, the copolymers comprise between about 10 and 99.5 wt. % of the isoolefin, or isobutylene, and about 0.5 and 90 wt. % of the para-alkylstyrene, or para-methylstyrene.
The polymerization system of the invention contains a mixture of a Lewis acid catalyst, an initiator, and a polar solvent. The copolymerization reactor is maintained substantially free of impurities which can complex with the catalyst, the initiator, or the monomers, and the polymerization reaction is conducted under conditions to limit or avoid chain transfer and termination of the growing polymer chains. Anhydrous conditions are highly preferred and reactive impurities, such as components containing active hydrogen atoms (water, alcohol and the like) must be removed from both the monomer and solvents by techniques well-known in the art.
The polymerization reaction temperature is conveniently selected based on the target polymer molecular weight and the monomer to be polymerized as well as standard process variable and economic considerations, e.g., rate, temperature control, etc. The temperature for the polymerization is between xe2x88x9210xc2x0 C. and the freezing point of the polymerization system, preferably from xe2x88x9225xc2x0 C. to xe2x88x92120xc2x0 C., more preferably from xe2x88x9240xc2x0 C. to xe2x88x92100xc2x0 C., depending on polymer molecular weight. Reaction pressure will typically be about 200 kPa to about 1600 kPa, more typically about 300 kPa to about 1200 kPa, and preferably about 400 kPa to about 1000 kPa. In order to avoid moisture condensation the reaction should be carried out under a dry inert gas atmosphere, preferably carbon dioxide or nitrogen gas, or with a liquid seal.
Isomonoolefin and PAS, particularly isobutylene and PMS, are readily copolymerized under cationic conditions. The copolymerization is carried out by means of a Lewis Acid catalyst. Suitable Lewis Acid catalysts (including Friedel-Crafts catalysts) for the process according to the present invention thus include those which show good polymerization activity with a minimum tendency to promote alkylation transfer and side reactions which can lead to branching and the production of cross-links resulting in gel-containing polymers with inferior properties. The preferred catalysts are Lewis Acids based on metals from Group IIIa, IV and V of the Periodic Table of the Elements, including boron, aluminum, gallium, indium, titanium, zirconium, tin, vanadium, arsenic, antimony, and bismuth. The preferred metals are aluminum, boron and titanium, with aluminum being the most preferred. In the practice of the method of this invention, weaker acids are preferred as they lead to less alkylation and branching and higher monomer conversion rates.
The Group IIIa Lewis Acids have the general formula RmMXp, wherein M is a Group IIIa metal, R is a monovalent hydrocarbon radical selected from the group consisting of C1 to C12 alkyl, aryl, alkylaryl, arylalkyl and cycloalkyl radicals; m and p are integers from 0 to 3; X is a halogen independently selected from the group consisting of fluorine, chlorine, bromine, and iodine, preferably chlorine; and the sum of m and p is equal to 3. Nonlimiting examples include aluminum chloride, aluminum bromide, boron trifluoride, boron trichloride, ethyl aluminum dichloride (EtAlCl2 or EADC), diethyl aluminum chloride (Et2AlCl or DEAC), ethyl aluminum sesquichloride (Et1.5AlCl1.5 or EASC), trimethyl aluminum, and triethyl aluminum.
The Group IV Lewis Acids have the general formula MX4, wherein M is a Group IV metal and X is a ligand, preferably a halogen. Nonlimiting examples include titanium tetrachloride, zirconium tetrachloride, or tin tetrachloride.
The Group V Lewis Acids have the general formula MXy, wherein M is a Group V metal, X is a ligand, preferably a halogen, and y is an integer from 3 to 5. Nonlimiting examples include vanadium tetrachloride and antimony pentafluoride.
Particularly preferred Lewis acids may be any of those useful in cationic polymerization of isobutylene copolymers including: AlCl3, EADC, EASC, DEAC, BF3, TiCl4, etc. with EASC and EADC being especially preferred.
Catalyst efficiency (based on Lewis Acid) in the reactor is maintained between 10000 lb. of polymer/lb. of catalyst and 300 lb. of polymer/lb. of catalyst and preferably in the range of 4000 lb. of polymer/lb. of catalyst to 1000 lb. of polymer/lb. of catalyst by controlling the molar ratio of Lewis Acid to stabilizing initiator.
According to the invention, the Lewis Acid catalyst is used in combination with a stabilizing initiator. The stabilizing initiators, of which 2-chloro-2,4,4-trimethyl-pentane (TMPCl) is a preferred example, are those initiators which are capable of being precomplexed in cold methyl chloride or other suitable solvent with the chosen Lewis Acid to yield a stable complex which is in equilibrium with a carbenium ion pair which directly forms a propagating polymer chain in the reactor. These initiators yield a fast, simple initiation of polymerization in the reactor as opposed to the slow stepwise initiations involving many polar complexes and non-propagating ion pairs characteristic of the catalyst systems conventionally used in commercial cationic slurry polymerization of isobutylene copolymers. This polymerization system is capable of forming an in-situ electron pair donor with a stabilizing initiator having the formula:
wherein:
R1 is an alkyl, alkenyl, aryl, aralkyl, or aralkenyl group containing up to 30 carbon atoms but not less than 3 carbon atoms unless R1 contains at least one olefinic unsaturation,
R2 and R3 are alkyl, aryl, or aralkyl groups containing up to 30 carbon atoms and can be the same or different,
X is a halogen or a carboxy, hydroxyl, or alkoxyl group, preferably a halogen selected from fluorine, chlorine, bromine, and iodine, most preferably chlorine, and
n is a positive whole number.
Multifunctional initiators (n greater than 2) are employed where the production of branched copolymers is desired, while mono- and di-functional initiators are preferred for the production of substantially linear copolymers.
As previously indicated, the initiator of the type contemplated by the invention may be tert-esters or tert-ethers producing in situ electron pair donors upon the addition of the Lewis acid in order to promote copolymerization of the monomers in a continuous slurry polymerization process. Suitable initiators are cumyl esters of hydrocarbon acids, and alkyl cumyl ethers. Representative initiators, for example, comprise compounds such as 2-acetyl-2-phenylpropane, i.e., cumyl acetate; 2-methoxy-2-phenyl propane, i.e., cumylmethyl-ether; 1,4-di(2-methoxy-2-propyl)benzene, i.e., di(cumylmethyl ether); the cumyl halides, particularly the chlorides, i.e., 2-chloro-2-phenylpropane, i.e., cumyl chloride; 1,4-di(2-chloro-2-propyl)benzene, i.e., di(cumylchloride); 1,3,5-tri(2-chloro-2-propyl)benzene, i.e., tri(cumylchloride); the aliphatic halides, particularly the chlorides, i.e., 2-chloro-2,4,4-trimethylpentane, 2,6-dichloro-2,4,4,6-tetramethylheptane; cumyl and aliphatic hydroxyls such as 1,4-di((2-hydroxyl-2-propyl)-benzene) and 2,6-dihydroxyl-2,4,4,6-tetramethyl-heptane and similar compounds. These stabilizing initiators are generally tertiary or allylic alkyl or benzylic halides and may include polyfunctional initiators. Preferred examples of these stabilizing initiators include: TMPCl, TMPBr, cumyl chloride as well as xe2x80x98di-xe2x80x99 and xe2x80x98tri-xe2x80x99 cumyl chloride or bromide.
The selected solvent or solvent mixture should provide a solvent medium having some degree of polarity in order for the polymerization to proceed at a reasonable rate. To fulfill this requirement a mixture of nonpolar and polar solvent can be used but one or a mixture of polar solvents is preferred. Suitable nonpolar solvent components includes hydrocarbons and preferably aromatic or cyclic hydrocarbons or mixtures thereof. Such compounds include, for instance, methylcyclohexane, cyclohexane, toluene, carbon disulfide and others. Appropriate polar solvents include halogenated hydrocarbons, normal, branched chain or cyclic hydrocarbons. Specific compounds include the preferred liquid solvents such as ethyl chloride, methylene chloride, methyl-chloride, CHCl3, CCl4, n-butyl chloride, chlorobenzene, and other chlorinated hydrocarbons. Methyl chloride produces exceptional results and therefore, is especially preferred. To achieve suitable polarity and solubility, it has been found, that if the solvent, or diluent, is mixed, the mixture is preferably at least 70% polar solvent, on a volume basis.
As is normally the case, product molecular weights are determined by reaction time, temperature, concentration, the nature of the reactants, and similar factors. Consequently, different reaction conditions will produce different products. Synthesis of the desired reaction product will be achieved, therefore, through monitoring the course of the reaction by the examination of samples taken periodically during the reaction, a technique widely employed in the art and shown in the examples or by sampling the effluent of a continuous reactor.
The reactors which may be utilized in the practice of the present invention include conventional reactors and equivalents thereof such as batch reactors, stirred tank reactors, fluidized bed reactors, and series tank or tubular reactors, and the like. The reactor will contain sufficient amounts of the catalyst system of the present invention effective to catalyze the polymerization of the monomer containing feedstream such that a sufficient amount of polymer having desired characteristics is produced. The reaction conditions will be such that sufficient temperature, pressure and residence time are maintained effective to maintain the reaction medium in the liquid state and to produce the desired polymers having the desired characteristics.
Typically, the catalyst (Lewis Acid) to monomer ratio utilized will be those conventional in this art for carbocationic polymerization processes. For example, catalyst to monomer mole ratios will typically be about 1/60,000 to about 1/50, more typically about 1/10,000 to about 1/100, and preferably about 1/2000 to about 1/200. This mole ratio will be calculated by determining the number of Lewis acid catalyst sites in the Lewis acid catalyst. This can be done by using conventional analytic testing techniques such as elemental analysis, nuclear magnetic resonance (NMR) spectroscopy (e.g., aluminum NMR), and absorption spectroscopy. Once the number of Lewis acid sites per unit of catalyst is known, the mole ratio is calculated in a conventional manner.
The preferred molar ratio of Lewis Acid to stabilizing initiator is in the range of 20/1 to 1/1, more preferably in the range of 10/1 to 2/1.
The monomer feedstream is preferably substantially anhydrous, that is, it contains less than 50 ppm, and more preferably less than about 30 ppm, and most preferably less than about 10 ppm, by weight of water. Such low levels of water can be obtained by contacting the feedstream, prior to the reactor, with a water absorbent (such as NaH, CaCl2, CaSO4, molecular sieves, alumina and the like) or by the use of distillation drying.
The monomer feedstream is typically substantially free of any impurity which is adversely reactive with the catalyst under the polymerization conditions. For example, the monomer feed preferably should be substantially free of bases (such as caustic), sulfur-containing compounds (such as H2S, COS, and organo-mercaptans, e.g., methyl mercaptan, ethyl mercaptan), N-containing compounds, oxygen containing bases such as alcohols and the like.
The order of contacting the monomer feedstream, catalyst, cocatalyst (if any), and solvent is not critical to this invention. Preferably, the stabilizing initiator and Lewis Acid are pre-complexed by mixing together in cold methyl chloride or other suitable cold polar solvent, immediately before injection into the continuous reactor through a catalyst nozzle in the standard way.
The stabilizing initiator and Lewis Acid are allowed to precomplex by mixing together in cold methyl chloride at temperatures between xe2x88x9250xc2x0 C. and xe2x88x9298xc2x0 C. with a contact time between 0.5 seconds and several hours, preferably between 1 second and 5 minutes before injection into the reactor.
The overall residence time in the reactor can vary, depending upon, e.g., catalyst activity and concentration, monomer concentration, reaction temperature, and desired molecular weight, and generally will be between about one minute and five hours, and preferably between about 10 and 60 minutes. The principle variable controlling residence time is the monomer feed diluent rate.
The new catalyst system and process affords many unexpected advantages for commercial slurry polymerization of IPAS copolymers generally, and in particular IPMS copolymers. This new catalyst system comprised of an aluminum alkyl such as EASC and TMPCl initiator has been unexpectedly demonstrated to greatly mitigate or completely overcome the difficulties as enumerated above in previous attempts to produce IPMS elastomers using the established commercial catalyst systems previously used. The improvements obtained with this new catalyst have been demonstrated both in laboratory slurry polymerizations and in commercial plant scale tests.
Copolymers of isobutylene and p-methylstyrene were discovered (U.S. Pat. No. 5,162,445) and have been under development for more than 10 years. These relatively new isobutylene-based copolymers have many advantages over conventional butyl rubbers (isobutylene/diene copolymers, such as isobutylene/isoprene), which makes them useful and desirable in a wide range of applications. They retain the low permeability and high damping characteristics of conventional butyl rubbers but because of the absence of the backbone unsaturation present in butyl rubbers, IPMS elastomers possess outstanding ozone resistance. The absence of backbone unsaturation coupled with the presence of enchained aromatic rings also imparts greatly improved aging and environmental resistance so that they are useful under more extreme conditions and in harsher environments than most other elastomers. The enchained aromatic rings can also impart greatly improved resistance to degradation due to ultraviolet light as compared to other isobutylene-based elastomers.
While IPMS copolymers have many desirable properties and may be usefully and advantageously utilized in many applications, commercial production of various IPMS copolymers has presented many challenges which have previously forced them to be produced under conditions which have been far more costly and difficult than those employed in the production of conventional butyl rubbers. The problems have centered upon polymerization reactor control and fouling, and the removal and disposition of the unconverted PMS in the copolymers. These problems have been found to be greatly exacerbated as the PMS level in the copolymers is increased. For example, even though IPMS copolymers containing 10-15 weight percent PMS have been shown to give improved performance in certain large volume applications such as tire sidewalls, attempts to produce these polymers in commercial volumes have been impractical or uneconomic.
Mass fouling in reactors producing IPMS copolymers has been found to be much more severe than in reactors producing conventional butyl, especially around the reactor circulation pump. This fouling rate gets progressively worse as PMS level is raised and/or as level of hydrocarbon in the feed and slurry concentration (weight of polymer in reactor/weight of liquid plus polymer in reactor) in the reactors is raised. Fouling rates also increase as production rates and heat load on the reactor are increased. As fouling progresses, reactor circulation and hence the ability to remove heat from the reactor are reduced. At some point the ability to remove heat is reduced an amount sufficient to require removal of the reactor from service for cleaning. In normal plant operations, there is typically a rotation sequence between reactors producing product and those out of service for cleaning. Therefore fouling rate in the producing reactors must be reduced sufficiently to permit time to clean the other reactors. Excessive fouling rates must be offset by uneconomic steps such as reductions in level of hydrocarbon in the feed and slurry concentration and/or production rate and heat load. Normally butyl rubbers can be produced at slurry concentrations of about 28-30% and reactor production rates of 6-7 klb./hr or more with acceptable reactor run lengths
In contrast, comparable reactors and catalyst systems producing IPMS copolymers containing less than 5 weight percent PMS have been limited to slurry concentrations of less than 18% and production rates of less than 5 klb./hr in order to achieve acceptable reactor run lengths. Allowable slurry concentrations and production rates continue to decrease as PMS content is increased, eventually leading to unsuitable if not impossible operating conditions.
Furthermore, conventional butyl processes are easily stabilized, whereas uncontrollable cycling of reactor flash gas and reactor temperature in IPMS processes frequently result in products that do not meet desired specifications and at higher production costs.
It has been discovered that changing the catalyst system to a stabilizing initiator, such as TMPCl, in conjunction with a Lewis acid, such as EASC, enables excellent stable reactor control to be achieved during production of IPMS copolymers (even at a higher PMS content, such as 10-20 weight percent) and mitigates the mass fouling rates so that acceptable run length can be achieved at much higher feed hydrocarbon and slurry concentrations and higher production rates with little off-spec production so that production costs can be greatly reduced. This new catalyst system also enables production to be accomplished at higher conversion with less unconverted PMS and so mitigates the problems associated with the unconverted PMS.
Without limiting the scope of the invention, it is believed that many of the benefits of the process of this invention are derived from use of the stabilizing initiators. These initiators are believed to promote a much simpler and faster initiation by elimination of most if not all of the intermediate polarized complexes and non-propagating ion pairs formed when conventional initiators are used.
In a particularly preferred embodiment, the TMPCl is made by dissolving isobutylene dimer in methyl chloride and then adding anhydrous HCl to form the alkyl chloride. Excess HCl is then purged by nitrogen and the resulting solution of TMPCl in methyl chloride is used as the initiator stream in a continuous plant to make IPMS polymers. In the commercial-type process, the TMPCl stream is mixed with a cold methyl chloride stream and an aluminum alkyl stream to form the catalyst system. This stream is then injected into the stirred tank butyl type reactor to initiate the polymerization of the IPMS elastomer under much more controllable and economic conditions than has previously been possible.
The catalyst system of this invention which comprises a stabilizing initiator, such as TMPCl, in conjunction with a Lewis acid, such as EASC, eliminates the required sequence of initiation steps and all of the intermediate associated polar complexes and non-propagating ion pairs to greatly reduce the electro-viscous effects caused by carged particles and greatly speeds up the initiation process. With the TMPCl/EASC catalyst system the final initiating complex is fed directly into the reactor and the chain-propagating ion-pair forms directly. Hence, the catalyst system of this invention results in the observed, lower slurry viscosity and permits running reactors at higher feed HC levels and slurry concentrations.
While butyl-type reactors may be thought of as well-mixed stirred tanks on a reactor residence time scale, they are pump-around heat exchangers with non-homogenous zones beneath the pump where feed is introduced and around the nozzle where the catalyst stream is injected (normally on the side of the bottom head). The monomerxe2x80x94rich zone beneath the pump can be particularly troublesome because feed may be introduced with as high as 40% monomer concentration whereas the steady-state monomer level in the reactor may be  less than 1%. Despite the high circulation rates in butyl reactors and the considerable efforts made to achieve good mixing, it still takes some time to dilute the incoming rich-feed to the reactor steady-state conditions and all rate processes can be much faster in this monomer rich zone. It is desirable to achieve and maintain homogenous complexation, initiation and polymerization rates throughout the reactor and in particular to avoid too much and too rapid reactions in the monomer rich zone around the feed inlet. Too rapid reactions in that zone produce a hot zone in which mass-fouling can occur. This situation is much easier to avoid and control in butyl reactors because the feed contains a retarder (isoprene) which slows down and moderates the initiation and polymerization rates in the feed rich zone. In IPMS elastomer reactors, the feed does not contain a moderating but rather an accelerating comonomer so mass-fouling around the pump is much harder to avoid and control. With the usual catalyst systems (HCl/AlCl3 , HCl/EADC, etc.) all of the step wise initiation reactions occur more rapidly in this monomer-rich zone and so it becomes the zone in which many initiations occur and is also a zone where electro-viscous effects are high because it is a zone rich in polarized complexes and ion-pairs. This combination of high viscosity and high rates can result in rapid mass-fouling in this area and necessitate reducing the feed hydrocarbon level and slurry concentration in IPMS reactors.
These problems are amplified as PMS comonomer level is increased because the styrenic monomers complex more rapidly and strongly with the Lewis acids (they are better bases, or electron-donors) than do the olefinic monomers. A particular problem with PMS is that it inherently contains a minor amount of meta-methylstyrene (MMS) as an impurity. Even a minor amount of MMS leads to chain terminations due to formation of a non-propagating indanyl ion pair. Since the process and catalyst of this invention permit lower PMS concentration at greater polymerization efficiency, less MMS is introduced in to the polymerization medium thus reducing the probability of undesirable chain termination.
The new TMPCl/Lewis acid catalyst system greatly mitigates this problem and moves the initiation away from the feed rich zone because the initiating complex (and chain propagating ion pair in equilibrium with it) is introduced directly into the reactor and doesn""t have to form preferentially in the feed rich zone. The catalyst system according to this invention then results in the more desired homogenous initiation and polymerization throughout the reactor and greatly mitigates the mass fouling rate in the rich feed zone around the pump. This reduction in the mass fouling rates enables feed hydrocarbon and slurry concentrations to be raised while still maintaining acceptable run lengths, thus enabling IPMS reactors to be operated under more favorable and economic conditions.
The catalyst system of this invention also greatly ameliorates the reactor control problems characterized by oscillating reactor flash gas and temperature. This instability has made production of IPMS polymers having a higher PMS content very difficult in the past and resulted in unacceptably high amounts of product having properties outside the desired specifications.
Without limiting the scope of the invention, it is believed that the new catalyst system has greatly simplified and accelerated the initiation process by eliminating the sequence of chemical reactions required prior to the formation of a propagating ion pair with previous catalyst systems. Reactor operations are more controllable when initiation is fast and simple as it is with a catalyst system according to this invention. With a TMPCl/Lewis acid catalyst system, reactors respond rapidly to changes in catalyst rate so that standard reactor computer control systems are able to rapidly correct any drifts in flash gas and maintain stable reactor control.
Furthermore, with the previous complicated sequence of initiation steps, all of the complexes and ion-pair equilibria involved were sensitive to temperature and to changing ratios of monomers (i.e., especially ratios of PMS and its inherent impurity, meta-methylstyrene (MMS)) so any perturbation in reactor temperature or flash gas affected all these equilibria to amplify the change. Thus, the reactor response to catalyst rate changes was typically slow and inconsistent depending upon temperature and how the various intermediate equilibria in the multiple initiation steps were being affected, resulting in a failure to achieve an acceptable level of computer control (i.e., reactor response to catalyst rate changes was different at different times as a result of process variables that can neither be detected or controlled).
In addition, because of the rapid reactor mass fouling rate, reactors had to be operated at very low slurry concentrations and heat loads to achieve the run lengths required to allow washing and turnaround to be accomplished in the time available before the fouled reactor had to be put back into production to replace another fouled reactor. Reactor heat loads needed to be so low that reactor polymerization temperature was the frozen film temperature which is dependent upon reactor hydrocarbon level. The fact that reactor polymerization temperature is governed by the frozen film temperature at low heat loads coupled with the fact that the previous catalyst systems were strongly activated by rising temperature typically caused uncontrollable cycles in reactor flash gas and temperature. These cycles usually caused IPM reactors to operate in an unstable, oscillating manner with computer control unsatisfactory, if not impossible, resulting in production of product failing to meet desired specifications.
The catalyst system of this invention has greatly ameliorated or entirely eliminated this reactor control instability. The reactor responds rapidly to catalyst rate changes with the new catalyst systems so small perturbations are easily corrected. Additionally, reactor heat loads can be increased due to a reduced fouling rate. This results in the reactor temperature being controlled by the heat load and heat transfer coefficient (i.e. required differential temperature between reactor heat removal systems and reactor polymerization temperature) instead of being controlled by the frozen film temperature, which continuously changes as a function of reactor hydrocarbon level. Thus, stable reactor flash gas and temperature, as well as uniform, IPMS copolymer having properties within the desired specifications can be produced with the TMPCl/EASC catalyst system, even at 10-20 weight percent PMS content.
Another benefit of the catalyst system of this invention relative to conventional catalyst systems is a significant increase in conversion. The catalyst system of this invention permits production of IPMS copolymers of the desired molecular weight at lower steady state hydrocarbon levels in the reactor than was previously possible, therefore mitigating the problems associated with the unconverted PMS monomer left in the polymer.
The reasons for the improved conversion afforded by the catalyst system according to the invention are not clear, but can speculated upon. It is known that one of the most important termination reactions in IPMS polymerization is the propensity to xe2x80x9cback bitexe2x80x9d on the enchained aromatic ring when a new monomer unit adds to a propagating styrenyl carbenium ion chain end (p-methyl styrenyl or especially m-methyl styrenyl carbenium ion) with ring closure to form a non-propagating indanyl carbenium ion. This indanyl ring formation termination reaction becomes less favored as Lewis acidity of the catalyst is reduced. Therefore, copolymer molecular weight under a given set of conditions tends to rise as acidity, or effectiveness to act as an alkylation catalyst, goes down. Hence, reduced acidity allows operation at higher conversion to produce the desired molecular weight. The HCl/Lewis acid catalyst systems used previously are more acidic than the TMPCl/EASC catalyst system. Therefore, the catalyst system according to this invention allows operation at higher conversion to ameliorate the problems associated with unreacted PMS.
Another reason for the improved conversion using a catalyst according to this invention is that the stabilizing initiator promotes less chain transfer activity than conventional catalyst systems used in IPMS polymerization. All initiators have some propensity to also act as chain transfer agents, thus reducing polymer molecular weight. Those initiators which are powerful (i.e. fast) initiators and poor chain transfer agents yield higher molecular weight product. The step-wise initiation sequence required with previous catalyst systems resulted in many transfer agents being present in the reactor simultaneously (i.e. HCl, tBuCl, TMPCl). Consequently, conventional processes typically yield lower molecular weight product than the process of this invention. The TMPCl/Lewis acid catalyst system eliminates the intermediate transfer agents; since the propagating ion pair is formed directly, it produces higher molecular weight under any set of conditions, thus permitting desirable operation of the copolymerization process at higher conversion of monomer to product and with higher molecular weight products.
The slurry catalyst system of this invention by its very nature includes a measurable amount of chain terminators. That is to say, the catalyst system of the invention is a non-living system. It is preferable, however, that the process and catalyst system contain a minimum amount or be substantially free of impurities which can complex with the catalyst being used or which can copolymerize with the monomers themselves. There are thus a great number of species which, if present in even trace amounts, will adversely affect cationic polymerization by reducing the polymer molecular weight thereof, broadening the molecular weight distribution, or in extreme cases even preventing polymerization entirely. These chain terminators are well known to those skilled in this art, and they function by reacting or complexing with the Lewis Acid polymerization catalyst or the propagating carbenium ion chain carriers. They are thus loosely referred to as xe2x80x9cpoisons,xe2x80x9d and they include a large number of Lewis bases such as water, alcohols, ethers, compounds containing carbonyl groups, nitrogen-containing compounds, such as amines, amides, nitrites, etc., and sulfur-containing compounds, such as mercaptans, thioethers, etc. These impurities or poisons also include many olefinic compounds which act as terminators or transfer agents in cationic polymerizations, such as butenes, cyclopentadiene, alpha-diisobutylene, m-methylstyrene, etc., as well as species containing cationically active halogens, such as allylic, tertiary, or benzylic halogens, and basic oxides.
Since this list of impurities or xe2x80x9cpoisonsxe2x80x9d is rather extensive, it is generally necessary to carry out careful and elaborate purification processes in order to achieve pure enough materials so as to enable production of high molecular weight polymers by cationic polymerization processes. Therefore, in producing the uniform, narrow molecular weight distribution copolymers and high molecular weight copolymers of the present invention, it is preferred that the para-alkylstyrene be at least 95.0 wt. % pure, and preferably 97.5 wt. % pure, most preferably 99.5 wt. % pure, that the isoolefin be at least 99.5 wt. % pure, and preferably at least 99.8 wt. % pure, and that the diluents employed be at least 99 wt. % pure, and preferably at least 99.8 wt % pure. Again, however, the xe2x80x9cpurityxe2x80x9d referred to in these preferred figures is understood to be the absence of the impurities or poisons discussed above, and therefore far larger quantities of inert materials are permissible therein. Thus, the isobutylene must not contain more than about 0.5% of such impurities or poisons, but may contain larger quantities of an inert material like butane, or methyl chloride.
It is also preferred to minimize or eliminate the presence of any proton scavenger in the process such that the catalyst system of this invention is substantially free of any proton scavenger. The nature of proton scavengers are well known and described in the Journal of Macromolecular Science Chemistry, vol. A 18, No. 1, 1982, pgs. 1-152 or Carbocationic Polymerization by Joseph P. Kennedy and Ernest Marechal at pgs. 32, 199, 449, 452 460 and 461 and incorporated by references herein.

Production of polyisobutylene copolymers


The invention relates to a novel thermal spray process for the deposition of coatings with a graded or layered composition and the coated articles produced thereby. More particularly, the invention relates to feeding mixtures of coating materials to a thermal spray device and continuously or intermittently changing the composition of the deposited coatings by changing the thermal spray operating parameters. The continuous or intermittent change in the composition of the coating material during deposition creates a graded or layered coating structure.

The invention relates to a novel thermal spray process for the deposition of coatings with a graded or layered composition on a substrate and the coated articles produced thereby. More particularly, the invention relates to feeding at least two coating materials to a thermal spray device and continuously or intermittently changing the composition of the deposited coatings by changing the thermal spray operating parameters. The change in the composition of the coating during deposition creates a graded or layered coating structure.
The family of thermal spray processes includes detonation gun deposition, high velocity oxy-fuel deposition and its variants such as high velocity air-fuel, plasma spray, flame spray, and electric wire arc spray. In most thermal coating processes metallic, ceramic, cermet, or some polymeric materials in powder, wire or rod form is heated to near or somewhat above its melting point and droplets of the material accelerated in a gas stream. The droplets are directed against the surface of a substrate to be coated where they adhere and flow into thin lamellar particles called splats. The coating is built up of multiple splats overlapping and interlocking. These processes and the coatings they produce have been described in detail in the following: xe2x80x9cAdvanced Thermal Spray Deposition Techniquesxe2x80x9d, R. C. Tucker, Jr., in Handbook of Deposition Technologies for Films and Coatings, R. F. Bunshah, ed., Second Edition, Noyes Publications, Park Ridge, N.J., 1994, pp. 591 to 642; xe2x80x9cThermal Spray Coatingsxe2x80x9d, R. C. Tucker, Jr. in Handbook of Thin Films Process Technology, Institute of Physics Publishing, Ltd., London, 1995; and xe2x80x9cThermal Spray Coatingsxe2x80x9d, R. C. Tucker, Jr., in Surface Engineering ASM Handbook, Vol. 5, ASM International, Materials Park, Ohio, 1994, pp 497-509.
In virtually all of the thermal spray processes two of the most important parameters controlling the structure and properties of the coatings are the temperature and velocity of the individual particles as they impact on the surface to be coated. Of these, the temperature of the particles is of greatest importance relative to the present invention. The temperature the particles achieve during the deposition process is a function of a number of parameters including the temperature and enthalpy (heat content) of the process gases, the specific mechanisms of heat transfer to the particles, the composition and thermal properties of the particles, the size and shape distributions of the particles, the mass flow rate of the particles relative to the gas flow rate, and the time of transit of the particles. The velocity the particles achieve is a function of a number of parameters as well, and some of these are the same as those that affect the particle temperature including the composition, velocity and flow rate of the gases, the size and shape distributions of the particles, the mass injection rate and density of the particles. Thus the thermal gas dynamics characteristics of the thermal spray process determine the quality of the resulting coating.
In a typical detonation gun deposition process, a mixture of oxygen and a fuel such as acetylene along with a pulse of powder of the coating material is injected into a barrel, such as a barrel of about 25 mm in diameter and over a meter long. The gas mixture is detonated, and the detonation wave moving down the barrel heats the powder to near or somewhat above its melting point and accelerates it to a velocity of about 750 m/sec. The molten, or nearly molten droplets of material strike the surface of the substrate to be coated and flow into strongly bonded splats. After each detonation, the barrel is generally purged with an inert gas such as nitrogen, and the process repeated many times a second. Detonation gun coatings typically have a porosity of less than two volume percent with very high cohesive strength as well as very high bond strength to the substrate. In the Super D-Gun(trademark) coating process, the gas mixture includes other fuel gases in addition to acetylene. As a result there is an increase in the volume of the detonation gas products which increases the pressure and hence greatly increases the gas velocity. This, in turn, increases the coating material particle velocity which may exceed 1000 m/sec. The increased particle velocity can result in an increase in coating bond strength, density, and an increase in coating compressive residual stress. In both the detonation gun and Super D-Gun coating processes, nitrogen or another inert gas can be added to the detonation gas mixture to control the temperature of the detonated gas mixture and hence the powder temperature. A number of parameters can be used to control both the particle temperature and velocity including the composition and flow rates of the gases into the gun.
In high velocity oxy-fuel and related coating processes, an oxygen, air or another source of oxygen is used to burn a fuel such as hydrogen, propane, propylene, acetylene or kerosene in a combustion chamber and the gaseous combustion products allowed to expand through a nozzle. The gas velocity may be supersonic. Powdered coating material is injected into the nozzle and heated to near or above its melting point and accelerated to a relatively high velocity, such as up to about 600 m/sec. for some coating systems. The temperature and velocity of the gas stream through the nozzle, and ultimately the powder particles, can be controlled by varying the composition and flow rate of the gases or liquids into the gun. The molten particles impinge on the surface to be coated and flow into fairly densely packed splats that are well bonded to the substrate and each other.
In the plasma spray coating process a gas is partially ionized by an electric arc as it flows around a tungsten cathode and through a relatively short converging and diverging nozzle. The partially ionized gas, or gas plasma, is usually based on argon, but may contain, for example, hydrogen, nitrogen, or helium. The temperature of the plasma at its core may exceed 30,000 K and the velocity of the gas may be supersonic. Coating material, usually in the form of powder, is injected into the gas plasma and is heated to near or above its melting point and accelerated to a velocity that may reach about 600 m/sec. The rate of heat transfer to the coating material and the ultimate temperature of the coating material are a function of the flow rate and composition of the gas plasma as well as the torch design and powder injection technique. The molten particles are projected against the surface to be coated forming adherent splats.
In the flame spray coating process, oxygen and a fuel such as acetylene are combusted in a torch. Powder, wire or rod is injected into the flame where it is melted and accelerated. Particle velocities may reach about 300 m/sec. The maximum temperature of the gas and ultimately the coating material is a function of the flow rate and composition of the gases used and the torch design. Again, the molten particles are projected against the surface to be coated forming adherent splats.
Thermal spray coating processes have been used for many years to deposit layered coatings. These coatings consist of discrete layers of different composition and properties. For example, the coating may be a simple duplex coating consisting of a layer of a metal alloy such as nickel-20 chromium (compositions herein are in weight percent unless otherwise noted) adjacent to the substrate with a layer of zirconia over it. In this case the undercoat of nickel-chromium may be used to enhance the mechanical or thermal shock resistance of the coating system or to protect the substrate from corrosion. An increase in mechanical or thermal shock resistance may be achieved by adding a third layer of coating consisting of a mixture of nickel-chromium and zirconia between the pure nickel-chromium and zirconia layers. Alternatively, perhaps even better thermal or mechanical shock resistance could be achieved by using two or more intermediate layers, each with an increasing amount of zirconia, thus approximating a continuously graded structure. Recently some graded coatings have been referred to as xe2x80x9cfunctionally gradedxe2x80x9d coating systems.
Prior to the present invention the general means of creating graded coating structures was to intermittently change the powder, wire or rod coating material composition being fed to the thermal spray device or devices if more than one was used to deposit the various layers. The coating deposition parameters and in some cases the coating thermal spray device were changed with each layer to obtain the desired coating structure of the particular layer composition. In most cases this meant that the coating process had to be stopped; the powder, wire, or rod feeders and composition changed; the deposition parameters such as electrical power, gas flows, and gas compositions changed; the process restarted; and new coating qualified before the coating of the substrate could be continued. All of this added very substantial time and cost to the coating process. Moreover, the time between coating layers generally tends to decrease the bond strength between layers and the over-all strength of the coating system.
It is an object of the present invention to provide a novel process for the production of thermal spray coatings that are graded in composition and/or properties.
It is a further object of the present invention to provide novel coatings that are graded in composition and/or density properties produced by the said novel process.
It is also a further object of the present invention to provide articles with graded coatings produced using the novel process of the invention.
It is a further object of this invention to provide a process for the deposition of graded coatings with greater cohesive strength than can be achieved using the multiple layer coatings of the present art.
It is also a further object of this invention to provide graded coatings with greater cohesive strength than that of the graded coatings of the present art. The higher cohesive strength of the coatings of this invention is believed to be the result of the smoother transition in composition and properties achieved with the process of this invention and the minimal time between the deposition of the layers of the coating.
The invention relates to a process for producing a graded thermal spray coated layer on a substrate comprising the feeding of a mixture of at least two coating materials to a thermal spray device and varying at least one of the deposition parameters of the thermal spray device during the deposition operating thereby varying the composition of the deposited coating material to produce a graded coated heterogeneous layer on the substrate. The thermal spray device for the process of this invention has parameters that can control or monitor the temperature of the depositing coating material and the velocity of the coating material particles.
The invention also relates to the deposition by means of the novel coating process of this invention, of unique coating structures with smoothly varying gradations in composition and/or density properties. Since the changes in deposition parameters can be made while the coating is being continuously deposited, the gradation or changes in composition and/or density properties are very smooth. If the coating is being continuously deposited, the gradation or changes in composition and properties are very smooth. If the coating is deposited without moving the gun or torch and the substrate is stationary as well, the coating gradation will be continuous; i.e., without discrete changes as a function of thickness. In most cases, however, the coating device and substrate can be moved relative to each other and the coating is deposited in multiple layers. Using the process of this invention, each layer may be slightly different than the preceding or succeeding layer. The time between layers is only dependent on the size of the substrate and the traverse rate (the relative rate of motion between the coating device and the substrate), since coating is being deposited continuously by the coating device. The difference between layers is a function of the rate of change in deposition parameters and the traverse rate. The smoothness of the gradation is then a function of the thickness of the individual layers which can be made very thin. The total thickness of the coating and of each zone is a function of the requirements of the application. The total thickness of the coating is typically in the range of 100 to 500 microns, but may be thicker or thinner if it is necessary to satisfy the specific requirements of the application.
This invention also relates to articles with the graded coatings of this invention. Such articles include those requiring coatings with graded properties to enhance the coating""s mechanical, thermal, or electrical properties. As illustrative, but not limited, examples of these coated articles include (a) those that require a very hard, brittle surface such as an oxide or carbide graded to a tough, ductile metallic layer adjacent to the substrate to provide more impact resistance and higher bond strength of the coating to the substrate; (b) those that require a thermal barrier outer layer such as an oxide graded to a metallic layer adjacent to the substrate that provides oxidation resistance and thermal shock resistance; and (c) those that require a wear and corrosion resistant outer surface graded to an oxide layer adjacent to the substrate for electrical resistance.
As used herein, a graded coated layer shall mean at least one layer comprising a mixture of at least two coating materials in which at least one of the coating materials varies in composition to produce a heterogeneous coated layer. Also as used herein, composition of the coating material shall also include density of the coating material.
Graded coatings, as defined herein, are used extensively to enhance the mechanical impact resistance, thermal shock resistance, and corrosion resistance of a coating system, as well as for other purposes. Occasionally graded coatings are used to allow thicker coatings to be deposited than would otherwise be possible. Most commonly, the layer of coating next to the substrate is a metallic alloy, and the outermost coating layer is an oxide or cermet. The metallic layer bonds better to the substrate and the oxide or cermet than the cermet or oxide directly to the substrate. It also may improve the mechanical impact resistance and other properties of the total coating by providing a layer of intermediate mechanical properties such as elastic modulus. Other factors such as stress relief through creep of the metallic layer may also be important. An example of this type of system is the use of a nickel based alloy under a tungsten carbide cobalt coating used to repair worn machine components. The thermal shock resistance of a coated system may also be increased with a metallic intermediate layer by increasing the bond strength of the system and providing an intermittent coefficient of thermal expansion between a metallic substrate and an oxide outer coating. This type of coating is frequently used in thermal barrier coating system where the metallic alloy undercoat is also used to protect the substrate from oxidation or other forms of corrosion. A typical example of a thermal barrier coating system, such as aircraft components such as aircraft blades, uses a cobalt-nickel-chromium-aluminum-yttrium metallic alloy undercoat and a zirconia-yttria outer coating. The porosity inherent in the metallic coating is sealed by heat treatment after deposition to provide a corrosion resistant barrier to protect the substrate, frequently a superalloy turbine blade or vane in a gas turbine engine.
In accordance with this invention the properties of many graded coating systems can be improved by increasing the number of layers of coatings with increasing amounts of the material of the final outer layer. This results in a smoother transition in properties as the number of layers increases.
The invention relates to a novel thermal spray process in which a mixture of coating materials is fed to a thermal spray gun or torch and the rate of deposition of the individual components of the mixture varied in a controlled manner by varying the thermal parameters of the thermal spray process gases. As a simple, but not limited example, consider a coating material that consists of two components, A and B, with different properties including melting point, size, shape, heat capacity, and thermal absorption characteristics. The thermal spray deposition parameters may be initially set to optimize the deposition rate or efficiency of A and not B and then changed gradually to optimize the deposition rate or efficiency of B. Thus the deposited coating would have a gradation in composition from predominantly A to A+B to predominately B. In addition to gradations in composition, gradations in other properties such as density can also be made by changing the deposition parameters. While this invention includes coatings that are continuously graded in composition and properties it also includes coatings in which one or more layers of the coating is maintained constant for a given thickness. Any and all of these variations in gradations will be encompassed in the term xe2x80x9cgradedxe2x80x9d as used herein. The coating material is usually fed to the thermal spray device in the form of powder, although one or more of the constituents could be fed in the form of wire or rod. When two or more of the constituents of the coating material are in the form of powder they may be blended mechanically and fed from a single powder dispenser to the thermal spray device or fed individually or in partial blends from two or more powder dispensers to the thermal spray device. The coating material may be fed to the thermal spray device internally as in most detonation gun and high velocity oxy-fuel devices or externally as in many plasma spray devices. The changes in deposition parameters including gas composition and flow rates, power levels, and coating material injection rates may be changed during the deposition process either manually by the equipment operator or automatically by computer control.
This invention comprises the process for depositing a graded coating using a thermal spray coating process in which a mixture of two or more powders is fed to the thermal spray device and the deposition parameters of the device are changed continuously or discontinuously. The result is that the deposition rate or efficiency of one or the powder constituents is initially dominant and, as the operating parameters are changed, the deposition rate of another constituent more or less gradually becomes dominant. An example, not intended to be limiting, is the deposition of a mixture of two materials with differing melting points such as a metallic alloy or an oxide. If a coating with a gradation from a metal layer next to the substrate to an oxide layer on the external surface of the coating is required, the operating parameters of the device would be initially set to provide a relatively low thermal content in the gas stream, sufficient to melt or nearly melt the metallic powder, but not the oxide. Under these conditions, the deposition rate of the metal would be very high while that of the oxide would be very low or nearly nil. The deposition parameters of the thermal spray device would then be changed to increase the thermal content of the gas stream so that more and more of the oxide would be heated to near or above its melting point. This would increase the deposition rate of the oxide and the coating composition would have a higher and higher fraction of oxide. As the thermal content of the gas stream increased to a sufficiently high level, the deposition rate of the metal would decline due to vaporization of the metal or due to the superheating of the particles to such a degree that they would splatter on impact rather than adhere to the surface being coated. The resulting coating may thus have a continuous gradation in composition from almost pure metal to almost pure oxide. If it were desired to have a constant composition for some portion of the thickness of the coating, the deposition parameters of the thermal spray device could be held constant for a period of time. Thus, continuing with the example of the metal-to-oxide coating, it would be possible to have a virtually pure metallic layer of some thickness adjacent to the substrate, followed by a graded zone, followed by a virtually pure oxide layer. This example could be used to produce a superior thermal barrier coating.
In those situations in which the thermal spray device is a detonation gun, the thermal content of the gas stream in the gun, as well as the velocity of the gas stream, can be varied by changing the composition of the gas mixtures. Both the fuel gas composition and the ratio of fuel to oxidant can be varied. The oxidant is usually oxygen. In the case of detonation gun deposition, the fuel is usually acetylene. In the case of Super D-Gun deposition, the fuel is usually a mixture of acetylene and another fuel such as propylene. The thermal content can be reduced by adding a neutral gas such as nitrogen.
In those situations in which the thermal spray device is a high velocity oxy-fuel torch or gun, the thermal content and velocity of the gas stream from the torch or gun can be varied by changing the composition of the fuel and the oxidant. The fuel may be a gas or liquid as described above. The oxidant is usually oxygen gas, but may be air or another oxidant.
In those situations in which the thermal spray device is a plasma spray torch, the thermal content of the plasma gas stream can be varied by changing the electrical power level, gas flow rates, or gas composition. As described above, argon is usually the base gas, but hydrogen, nitrogen, and helium are frequently added. Some plasma spray devices use liquid water rather than a gas to create the plasma stream. The velocity of the plasma gas stream can also be varied by changing the same parameters.
Variations in gas stream velocity from the thermal spray device can result in variations in particle velocities and hence dwell time of the particle in flight. This affects the time the particle can be heated and accelerated and, hence, its maximum temperature and velocity. Dwell time is also affected by the distance the particle travels between the torch or gun and the surface to be coated.
The specific deposition parameters used with any of the thermal spray devices depend on both the characteristics of the device and the materials being deposited. The rate of change or the length of time the parameters are held constant are a function of both the required coating composition gradation, the rate of traverse of the gun or torch relative to the surface being coated, and the size of the part. Thus a relatively slow rate of change when coating a large part may be the equivalent of a relatively large rate of change when coating a small part.
This invention also comprises graded coatings made by the process of this invention. These coatings have two or more constituents. The coatings of this invention include, but are not limited to, coatings that are virtually continuously graded from the composition of one constituent in the powder mixture to that of another constituent and also coatings graded from one constituent to another with zones of virtually constant composition for a given thickness. The gradation may have a very sharp transition zone such that the coating""s composition changes from A to B with little discernable intermediate mixed zone. Alternatively, the transition may be very gradual from A to A+B to B with the A+B zone gradually changing from predominately A to predominantly B. More than two constituents may be included in the gradations. Thus, the gradation may be from A to B to C or may be from A to B+C, or from A to A+B to B+C, etc. In addition to gradations in composition, gradations in properties such as density can be made by changing the deposition parameters, either simultaneously while changing composition or independently while holding the composition constant for a given thickness of the coating.
The coatings of this invention may consist of metallic, ceramic, cermet, or polymeric materials, alloys thereof, or any combination of these. The coatings of this invention may be used for a wide variety of purposes including, but not limited to, wear resistance, corrosion resistance, thermal characteristics, mechanical characteristics, optical characteristics, or electrical characteristics or for any combination of these. A few examples, not intended to be limited in any way, include their use in the following ways: (a) as wear resistant surfaces where the gradation in properties may be from a metallic zone adjacent to a metallic substrate to hard wear resistant cermet composition on the exterior surface with the metallic zone and gradation serving to increase bond strength and ameliorate the mechanical stresses imposed in the coating in service; (b) as thermal barriers where the coating is graded from a zone of metallic coatings adjacent to the metallic substrate to an oxide with low thermal conductivity where the metallic layer provides corrosion protection and better bond strength and the gradation provides better thermal shock resistance; or (c) as electrically insulated and wear resistant coatings where the composition of the coating adjacent to a metallic substrate is an electrical insulator and the gradation is to a more wear resistant cermet surface where the gradation provides better bond strength and ameliorates thermal stresses placed on the coating in service.
The coated articles of this invention comprise those articles having a graded coating produced using the process of this invention. Said articles may include a very wide variety of components of manufacturing equipment or end products of production. A few examples intended to illustrate such articles, but not to be limiting in any way, include the following: (a) components requiring wear resistance in service that includes high mechanical loading where the coating may include a gradation from a relative tough, but ductile, metallic zone adjacent to a metallic substrate to a wear resistant cermet exterior zone with the gradation providing better bond strength and dissipation of the mechanical stresses placed on the coating""s surface; (b) components requiring a thermal barrier to protect them from excessive heat or thermal shock or to increase the thermal efficiency of a process where the coating is graded from a metallic to an oxide as noticed above; or (c) components requiring electrical insulation with either the insulating layer being adjacent to the substrate with gradation to a more wear resistant surface as noted above or where the coating is graded from a metallic adjacent to substrate with gradation to an insulating oxide on the exterior surface with the gradation providing better bond strength and amelioration of imposed thermal or mechanical stresses.

Process for producing graded coated articles


The invention relates to seroreactive regions on proteins E1 and E2 of human papillomavirus (HPV) 16. The application also relates to a vaccine which contains such peptides which contain the seroreactive regions. The invention likewise embraces compositions for diagnostic purposes which contain peptides with the seroreactive regions.

The invention relates to seroreactive regions on proteins E1 and E2 of human papillomavirus (HPV) 16.
The application also relates to a vaccine which contains such peptides which contain the seroreactive regions.
The invention likewise embraces compositions for diagnostic purposes which contain peptides with the seroreactive regions.
HPV 16 is one of the human papillomaviruses (Proc. Natl. Acad. Sci., USA 80, 3813-3815 (1983). The organization of the genome of HPV 16 has been described in Virology, 145,181-185 (1985).
Genomic sequences of HPV can be detected in most cases of preinvasive and invasive cervical tumors. HPV 16 has been identified world-wide as the virus type predominating in these tumors. The HPV 16 genome is detectable in more than 50% of cervical tumors, in which case it is often present integrated into the cellular DNA. Little is known about the immune response after infections with HPV 16 or other papillomaviruses.
Initial data: patients suffering from cervical tumors were compared with healthy individuals with regard to the presence of antibodies against viral proteins. These viral proteins were then linked as fusion products with various prokaryotic peptides at their N terminus and then used as antigens in Western blots.
The object of the present invention is the further identification of HPV 16 viral structures which can be used as tool in the prophylaxis, diagnosis and therapy of HPV 16-dependent tumorous diseases in humans. The identification of such structures is a prerequisite for the development of ELISAs which make it possible to test a large quantity of human sera for the presence of HPV 16.
The present invention therefore embraces seroreactive 5 regions of the E1 protein of HPV 16, which have one of the following amino-acid sequences:
seroreactive regions of the E2 protein of HPV 16, which have one of the following amino-acid sequences;
The invention furthermore embraces peptides with one or more of the seroreactive regions identified above, a vaccine which contains one or more of the peptides identified above, a composition for diagnostic purposes for the identification of specific antibodies against HPV E1 and/or E2 protein, which likewise contains the peptides identified above, monoclonal antibodies which have an affinity for one or more of the seroreactive regions of the E1 or E2 protein of HPV 16, and a composition for diagnostic purposes which contains these monoclonal antibodies.
In order to identify seroreactive regions in proteins E1 and E2 of HPV, the experimental route described in Science 228, 1315-1317 (1985) was followed. Subgenomic HPV 16 DNA fragments which had been randomly generated by ultrasound treatment and partial DNAse I treatment were cloned into the phage vector fusE1 and then expressed as part of a phage coat protein. Seroreactive phage recombinants were identified using sera prepared against E1 and E2, and purified, and the seroreactive regions were characterized by sequencing the HPV 16 portion. Polyclonal rabbit sera against an HPV 16 E1 MS2 polymerase fusion protein and against the amino- and carboxyl-terminal part of HPV 16 E2 (separate, likewise ) MS2 fusion proteins) were prepared.
The filamentous phages embrace the three groups f1, fd, and M13. It is common to them all that binding and uptake of the phages takes place via F pili of the bacteria, i.e. that only F+ strains can be infected. The fd wild-type phage, from which the vector system used is derived, forms particles which are about 900xc3x976 nm in size and which are composed in particular of about 2700 subunits of the main coat protein. In addition, in each case, 5 molecules of the minor coat proteins pIII, pVI, pVII, and pIX are located at both ends of the virions. The single-stranded, circular phage genome which, in the case of the fd wild-type, is 6408 bp in size, carries the information for a total of 10 different proteins.
In the fd derivatives fuse1, fuse2 (Parmley and Smith, Gene, 7, 305-318 (1988)), and fusemm, a tetracycline-resistance gene is integrated, by insertion of a part of the Tn10 transposon, in the phage genome, which has been enlarged to about 9.2 kbp in this way. This means that the replicative DNA double-stranded phage genomes behave in the bacteria like selectable plasmids and can accordingly be prepared and used for clonings. Another modification from the wild-type is the presence of a reading frame mutation in the gene for the minor coat protein pIII in conjunction with an inserted restriction site for cloning expressable DNA fragments. The gene for pIII is composed of two almost completely independent domains (Crissmann and Smith, 1984): an N-terminal domain which mediates the binding of the phages to the bacterial cell receptor (F pili), and a C-terminal protein domain which is responsible for phage morphogenesis. The reading frame mutation, which is located directly behind the signal sequence of the protein, thus leads to inactivation of the gene and accordingly also prevents the formation of infectious particles. This is of importance for the replication of these phage mutants as plasmids because the fd genomes inactivated in the morphogenesis do not damage the host bacteria (Smith, in: Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Butterworth Publishers, Stoneham, Mass. 61-85, 1987).
Insertion of suitable DNA fragments and restoration of gene III functions lead to the formation of infectious phage particles which carry additional amino-acid sequences on their coats. These sequences are accessible to various ligands, for example antibodies, in the natural state of the phages.
The fd expression system used in this invention is essentially based on setting up phage banks by cloning DNA foreign sequences into the gene III, and examining the latter with the aid of monoclonal or polyclonal sera for seroreactive recombinants. An amplification normally takes place in preparation of these expression banks. The extent of this replication of individual clones in turn depends on the nature and size of the inserted DNA sequence. This means that different clones differ in frequency, which may differ by up to several powers of ten. It is therefore possible to derive from the stated properties the following two features of the fd expression banks:
Amplification of the banks, which leads to repeated cloning of identical phage clones isolated by immunoscreening;
Possibility of enriching seroreactive phages by affinity chromatography (columns) because phages in the active state can be bound and eluted again.
The repeated isolation of identical recombinants was avoided by using, separately set up banks, there being an extremely low probability of cloning a DNA fragment prepared identically and in parallel, or of the phage recombinant derived therefrom.
In this invention, a total of 11 different expression banks for HPV 16 DNA in fusE1 were set up. The number of primary, tetracycline-resistant, and insert-harboring recombinants was, in this case, between 2,000 and 90,000 per bank. Since complete plasmids composed of about 4 kb vector portion and 8 kb HPV portion in sheared form were always used for the cloning, the HPV-containing fd recombinants are reduced by about 30%. The fragments cloned in were then expressed, as already mentioned, as a fusion protein of the gene III coat protein. The cloning site in gene III is in this case directly behind the translated signal sequence for protein export. In order to restore the function of the gene, it is necessary for an insert to have a defined size (3n+2; n=0, 1, 2, 3 . . . ). In order to express a defined protein sequence as fusion protein of the gene III product, it is necessary in addition for both the 3xe2x80x2 and the 5xe2x80x2 junction to be in the correct reading frame, and for the corresponding insert to be present in the correct orientation. This therefore applies overall to only about every 18th (3xc3x973xc3x972) HPV DNA-containing recombinant. A small portion thereof is in turn inactivated by translation stop codons present in the insert, or by proteins which are not functional because of their folding. Because of the stated parameters it is difficult to estimate what is the minimum number of recombinants necessary to express with great probability any required part of an HPV genome as fd fusion protein in the phage bank. In papillomaviruses, about 10 kb of the genome (partly by overlapping open reading frames) code for proteins. Of 2,000 tetracycline-resistant insert-harboring recombinants, about 100 ({fraction (1/18)}) clones express HPV sequences in a suitable manner. With average HPV fragment sizes of 50-150 bp, the expressed HPV sequence amounts to about 5,000-15,000 bp. In fact, fd banks with about 2,000 recombinants prove to be sufficiently representative.
In order to ensure the specificity of the immuno-screening, either several different recombinants of a seroreactive region or at least several identical but independently isolated phage recombinants were always isolated.
The amino-acid position indicators in FIGS. 2 and 5 relate to the E1 and E2 proteins, and not to the positions of the open reading frames. The first methionine was given position 1.
Preparation of Polyclonal Antisera Against HPV 16 E1
In order to isolate seroreactive phage recombinants from the HPV 16 fd expression bank, initially polyclonal rabbit sera against HPV 16 E1 MS2 fusion proteins were prepared. For this, the Pst I A fragment of HPV 16 (bp 875-3693) was cloned into the Pst I cleavage site of the expression vector pEX12mer (Seedorf et al., EMBO J. 6, 139-144, 1987), by which amino acids 5-649 of HPV 16 E1 ORF are expressed (FIG. 1). This vector is a derivative of the plasmid pPLC24 (Remaut et al., Gene 15, 81-93,1981) which has been modified by insertion of the pUC8 polylinker behind the MS2 polymerase portion. The fusion protein is transcribed in the pEX12mer by the temperature-inducible lambda pL promoter. The N-terminal fusion portion of the MS2 protein amounts to 100 amino acids.
Since the original HPV 16 isolate (Seedorf et al., Virology, 145, 181-185, 1985) has a reading frame mutation in the region of the E1 open reading frame (nucleotide position 1138), recourse was had to an HPV 16 isolate from a cervical carcinoma with a complete E1 ORF. Because of the selected restriction cleavages, the HPV 16 E1 open reading frame (bp 865-2811) is thus completely expressed apart from three N-terminal amino acids.
The clonings and plasmid analyses were initially carried out using the E. coli strain W6 in which there is constitutive expression of the repressor for the lambda promoter. This prevented expression of the fusion proteins, in order to prevent counterselection after the transformations. After examination of the cloning by restriction analysis, and Southern blot hybridization with radioactive labelled HPV 16 DNA (Pst I A fragment), the plasmid DNA of the construct was used for transformation in E. coli N6045. This strain is able, because of its temperature-sensitive repressor of the lambda promoter, to express the MS2 fusion proteins.
It was then possible in a Western blot to examine, with the aid of a monoclonal antibody directed against the MS2 portion of the fusion protein, by comparison of extracts from induced and non-induced bacteria the size and the expression rate of the fusion protein. Since the band of the MS2 E1 fusion protein corresponded to the expected size of about 90 kD, no examination of the cloning junctions by sequencing was carried out. In the two other reading frames of the HPV 16 Pst I A fragment, expression of larger proteins is impossible because of the presence of translation stop codons. In addition, both Pst I cleavage sites of the vector-insert junctions were retained. Correct expression of the E1 open reading frame was confirmed by the results of the inununoscreening of the HPV 16 fd expression banks, which are described in the following section.
The MS2-E1 fusion protein was then purified from induced E. coli cultures by differential extraction and by electroelution from SDS polyacrylamide gels, and was then used to immunize two rabbits.
Identification of Seroreactive Regions on the HPV 16 E1 Protein
Both of the polyclonal rabbit sera prepared against HPV 16 E1 were used to examine five different HPV 16 fd expression banks for reactive recombinants. It was possible in this way to identify a total of at least two different antibody binding sites represented by non-overlapping phage clones. In total, 19 independent phage clones which contain seven different classes of HPV 16 inserts were isolated (FIG. 2). Six classes have a common overlapping region which codes for the HPV 16 E1 specific peptide EDLVDFIVND (SEQ ID NO: 23). The second identified epitope on the E1 protein is represented by a recombinant phage (clone 1059) which codes for the E1 peptide GSPLSDIS (SEQ ID NO: 7).
The original HPV 16 isolate has a reading frame mutation in the E1 open reading frame (nucleotide position 1138). The DNA of this HPV 16 isolate was used to prepare the fd expression banks. Two of the isolated seroreactive fd recombinants contain this region and therefore also have the reading frame mutation. In clone 1145 this leads to a change of reading frame, and this results in C-terminal attachment of three HPV 16-E2 non-specific amino acids ( . . . ValValHis). Clone 1059 starts in the wrong frame and is converted into the correct HPV 16 E1 reading frame by the reading frame mutation of the HPV 16 isolate used. The clone codes for the peptide STGSKTKVFGSPLSDIS (SEQ ID NO: 24), of which only the C-terminal amino acids . . . GSPLSDIS (SEQ ID NO: 7) derive from the actual HPV 16 E1 protein and must form the epitope.
Both clones which contain the reading frame mutation have the correct insert size (3n+2 base pairs) to restore the reading frame of gene III of the phage vector.
Preparation of Polyclonal Antisera Against HPV 16 E2
Like the case of the HPV 16 E1 open reading frame, no suitable antisera were available for the HPV 16 E2 protein either. For this reason, the HPV 16 E2 open reading frame (nucleotide position 2756-3850; AA 1-365) was expressed in the vector pEX12mer as already described for the E1 protein.
Firstly the HPV 16 DNA fragment was cloned via the Hinf I cleavage site at position 2761 into the pEX12mer vector. In this case the starting material was an already sub-cloned HPV 16 fragment (bp 2367-4467). This fragment was cut out of the vector again, via the additionally inserted non-HPV 16-specific restriction sites Xba I (5xe2x80x2 end) and Bam HI (3xe2x80x2 end), and prepared. This DNA fragment, which is 2.1 kb in size (Xba II/Bam HI), was then partially cut with Hinf I. This results, among other things, in a fragment which is 1700 hp in size between the 3xe2x80x2-terminal Bam HI cleavage site and Hinf I site at bp 2761. The internal Hinf I cleavage site (bp 3539) in this fragment is uncleaved, and the HPV 16 E2 ORF is completely present apart from three amino-terminal amino acids. After preparation, the Hinf/Bam fragment was cloned into the pEX12mer expression vector which had been cleaved with Bam HI. This resulted, via the compatible Bam HI sites, in linear products of vector and insert. The free ends of these products were filled in with Klenow polymerase and then closed by ligation. This results in an MS2-E2 junction at the filled-in cleavage sites Bam HI (vector) and Hinf I (E2 insert) with loss of the two restriction sites. Using Eco RI/Bam HI double restriction cleavages, it was possible to identify recombinants which harbored the HPV 16 E2 fragment in the correct orientation.
After transformation into the E. coli expression strain 6045 it was not possible, using a monoclonal antibody directed against the MS2 polymerase, to find any production whatever of the MS2 fusion protein. In order to rule out a displacement of the reading frame at the MS2-E2 junction, the plasmid DNA of a total of 16 different MS2-E2 recombinants was hybridized in a Southern blot with an oligonucleotide derived from the correct Bam HI/Hinf I junction. Since an unambiguous hybridization signal was identifiable with 15 clones, it was assumed that the cloning had taken place in the correct reading frame, and expression of the complete E2 ORF is not possible in pEX vectors. As a substitute, the HPV 16 E2 protein was then expressed in two halves in the pEX12mer vector.
Expression of the Amino-terminal Region of HPV 16 E2
The amino-terminal region of the E2 open reading frame between nucleotide position 2761 and 3209 was cloned into the pEX12mer vector and expressed. Since the E2 open reading frame starts at nucleotide position 2756, the MS2-E2 fusion protein lacks the first two amino acids (Met-Glu) of the E2 protein (FIG. 4).
Plasmid DNA composed of pEX12mer and HPV 16 E2, which were obtained from the cloning described above, was truncated at the carboxyl terminus (i.e., 3xe2x80x2 end) by deletion of a Hinc II (HPV 16 bp 3209)/Bam HI fragment and religation (blunt/flush from Hinc II and Bam HI). This results in expression of the N-terminal part of HPV 16 E2 between nucleotide position 2761 (Hinf I) and 3209 (Hinc II). A fusion protein about 30 kD in size was detectable in induced bacteria in a Western blot with an anti-MS2 molecule antibody.
The fusion protein was purified by differential extraction of the induced bacterial lysate and by electroelution of the protein band from SDS polyacrylamide gels stained with Coomassie blue, and used for immunizing rabbits.
Expression of the Carboxyl-terminal Region of HPV 16 E2
The C-terminal region of the HPV 16 E2 open reading frame between nucleotide position 3209 and 3850 was expressed in the pEX12mer vector (FIG. 3). The region is thus directly connected to the expressed amino-terminal part, described above, of the HPV16 E2 open reading frame.
For this, recourse was had to the Xba/Bam fragment which has been described above and which contains the complete HPV 16 E2 reading frame. After restriction cleavage, a Hinc II/Bam HI fragment (nucleotide position 3209-4467) which contains the carboxyl-terminal half of HPV 16 E2 was isolated. This fragment was inserted into the Bam HI cleavage site of the pEX12mer expression vector (5xe2x80x2Bam HI/Hinc II-Bam HI/Bam HI 3xe2x80x2). It was possible with the aid of the anti-MS2 monoclonal antibody to identify in extracts of induced bacteria a fusion protein of about 30 kD, which was purified by differential extraction and electroelution from SDS polyacrylamide gels, and was used to immunize rabbits.
Identification of Seroreactive Regions on the HPV 16 E2 Protein
Available for the immunoscreening of the fd HPV 16 expression banks was a total of four different anti-HPV 16 E2 antisera: in each case two sera against the amino-terminal part (bp 2761-3209; AA 3-152) and two against the carboxyl-terminal part of the E2 (bp 3209-3850; AA 153-365) open reading frame. These sera were used to examine five different expression banks for seroreactive recombinants. This resulted in isolation of a total of 32 clones, of which 26 contain amino-terminal sequences of the E2 protein. These 26 clones form a total of 11 different classes which represent four different non-overlapping regions (FIG. 5).
All the epitopes are located in a restricted region comprising 88 amino acids of the amino terminus of the E2 open reading frame which is located between nucleotide position 2792 (AspLyslle . . . ) and 3055 ( . . . SerLeuGlu).
It was possible to locate in the carboxyl-terminal region at least two independent non-overlapping epitopes (TSVFSSNEVSSPEII (SEQ ID NO: 19) and TEETQTTIQRPRISEPDTGN (SEQ ID NO: 22), FIG. 5). These are represented by a total of four classes of recombinants with six independent isolates. The region of the E2 open reading frame which is covered by the clones is located between nucleotide position 3343 (ThrSerVal . . . ) and 3502 ( . . . ThrGlyAsn) and comprises 52 amino acids.
Five classes of recombinants (12 isolates) extend over nucleotide position 2926. All the clones have a point mutation (Axe2x86x92G transition) here, but this does not lead to a change in the corresponding amino acid (glutamine).
Immunoscreening of fd Phase Expression Banks
1. Phage Affinity Concentration with Protein A-Sepharose Columns
The phage banks prepared in the fd phage expression system used unavoidably underwent amplification on cloning. The extent of this replication of the original clones is in turn greatly influenced by the nature of the individual recombinants, for example by different sizes of inserts or conformation of the coat proteins, inhibition of physiological processes in the infected bacteria and many others, and it was therefore not to be expected that uniform amplification of all phages takes place. In order to isolate underrepresented phage recombinants or clones from large libraries, seroreactive phage recombinants were concentrated. For this, use was made of the circumstance that the foreign sequences expressed in each case appear as part of an fd gene III fusion protein on the coat of natural phage particles. Large amounts of phages (109-1012 particles) were for this purpose bound to protein A antibody columns and eluted again.
For this, initially protein A-Sepharose was swollen with PBS for 30 min and was washed with PBS. Subsequently the protein A-Sepharose was incubated with about 1 to 2 ml of suitable polyclonal sera (rabbit or human) or with corresponding protein A-binding monoclonal antibodies in Eppendorf reaction tubes on a rotary shaker at 4xc2x0 C. for 1 to 2 days. Subsequently, the protein A-Sepharose was washed 10 times by the Sepharose, being alternately resuspended in 10 ml of PBS and pelleted again by centrifugation (2 min, 6,000 rpm). The protein A-Sepharose-IgG complexes formed were then incubated with an appropriate amount of phages as above. Then the Sepharose was washed with PBS several times as before and packed into a Pasteur pipette closed with a glass bead and washed with several liters (2-15 I) flowing through. The column material was removed and then incubated in the same volume of elution buffer (1 mg/ml BSA, 0.1 M HCl, glycine, pH 2.2) for 15 min. After brief centrifugation, the supernatant, which now contains free phages and antibodies, was neutralized with ⅕ of the volume of tris base (0.5 M). Antibodies which recognize the re-combinant gene are able to inhibit binding of the phage to the bacterial cells and thus the cycle of infection. For this reason the phages were added in 100-200 xcexcl aliquots of the eluates immediately after neutralization to exponentially growing E. coli K91 and plated out on complete medium plates. It emerged during the work that replica filters of these phage platings were unsuitable for immunoblotting, probably because of contaminants in the eluate. For this reason the resulting plaques were again rinsed off the plates with complete medium and subsequently plated out from the phage suspensions obtained in this way, which had undergone renewed amplification, and the immunoblotting was carried out on minimal agar plates.
2. Phage Platings and Preparation of Nitrocellulose Replica Filters for the Immunoblotting
All the fd phage derivatives were plated out on a lawn formed by E. coli K91 (Lyons and Zinder, Virology, 49, 45-60, 1972). This strain is distinguished by a large number of F pili (5 per cell, compared with about 0.5 per cell in most F+ strains) which are responsible for uptake of filamentous phages. This is particularly important for the fd expression system used in this study because the recombinant fuse phages have, owing to the uptake of a part of the Tn10 transposon (tetracycline resistance), a genome which is distinctly enlarged compared with the wild-type, and for this reason form particularly small plaques.
To plate out the phages, a R91 overnight culture was diluted 1:100 in complete medium (2xc3x97YT) and incubated at 37xc2x0 C. for 3 to 4 h. After a density of E600=0.8-1.2 was reached, 200 xcexcl of the bacteria were plated out with an appropriate amount of phages, together with 3.5 ml of agarose (0.6% agarose, 10 mM MgSO4, 50xc2x0 C.) on prewarmed bacteria plates. Minimal agar plates were always used for every plating intended to be used for nitrocellulose replicas for the immunoscreening. Platings out for determination of the phage titer or for DNA hybridization were carried out on complete medium plates.
Use of complete medium plates for the immunoblotting always lead to very high non-specific reactivity of the filters with the sera used.
The plates were incubated at 37xc2x0 C. overnight. After about 16 hours, a nitrocellulose filter was placed on for 10-15 min, marked with four asymmetric pricks with a needle, and removed again using flat-ended forceps. The filters were labelled and then inverted onto a fresh minimal agar plate and incubated further at 37xc2x0 C. for 5-6 hours. This increased the amount of phage particles (proteins) on the filter since the reincubation makes it possible for the bacteria and phages bound to the filters to grow further via the nutrients diffusing from the plate. Subsequently the filters were removed and saturated in 10% milk (skim milk powder in PBS) for 30-60 min. The filters were then incubated with suitable dilutions of appropriate sera in 5% milk at 4xc2x0 C. overnight.
3. Immunostaining of Replica Filters and Cloning of Reactive Recombinants
After removal of the replica filters, blocking in 10% milk (in PBS) for 60 min and overnight incubation with antisera, the nitrocellulose filters were washed in PBS, 0.05% Tween 20 (5 changes of washing buffer) for 30 min. The filters were then incubated with 1:1000 dilutions of appropriate second antibodies (peroxidase-coupled goat anti-human, anti-rabbit, or anti-mouse) in 5% milk at RT for 2 h. This was followed by renewed washing (see above) and incubation in the following staining mixture:
After sufficient color had developed, the filters were removed from the solution, placed in water for 30 min, and then dried on 3 MM paper.
The prick holes and signals on the filters were then copied onto a sheet or the lid of a bacteria dish. This made it possible to assign a position or, if the phage dilution was sufficiently large (round 2 or higher), a plaque to a signal. A sterile toothpick was gently stabbed into the position or the plaque, and the toothpick was placed in 500 xcexcl of complete medium for 10-15 min. This phage suspension then contained generally about 106-107 infectious particles, which comprises about 0.1-1% of the phages in a plaque. The phage suspensions were then incubated at 65xc2x0 C. for 15-20 min in order to kill bacteria which had been carried over, and were then 5 stored at 4xc2x0 C.

Antibodies specific for seroreactive regions on HPV 16 protein E1