AN AUGMENTED WESTERN BLOT FORMAT AND IMMUNOASSAY FOR DETECTION OF VIRAL ANTIBODIES

Abstract: A method for producing an improved solid phase antigenic reagent useful in an immunoassay for detecting antibodies specific for a virus, such as the human immunodeficiency virus, is disclosed which comprises the addition to a natural viral lysate a synthetic or recombinant viral protein or peptide. Also provided is an improved immunoassay utilizing the solid phase antigenic reagent.

Description: - - - AN AUGMENTED WESTERN BLOT FORMAT AND IMMUNOASSAY FOR DETECTION OF VIRAL ANTIBODIES BACKGROUND OF THE INVENTION Field of the Invention The invention relates to a test designed to detect antibodies to human retroviral infections, more specifically the Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) , the etiologic agents of Acquired Immunodeficiency Syndrome (AIDS) . Information Disclosure Statement Laboratory tests currently used to detect HIV infection are based on the observation that individuals infected with HIV will develop virus-specific antibodies within a few weeks, or months at the latest. Serologicai diagnosis of HIV infection rests on the detection of a host immune response to viral antigens encoded by the 3 major viral structural genes. The crag gene encodes the major viral core proteins, pi" (molecular weight 17 kD) , p24 (molecular weight 24 kD) and their precursor, p55 (molecular weight 55 kD) . The env gene encodes the envelope glycoprotein precursor, gplδO (molecular weight 160 kD) which is cleaved into an external envelope domain, gpl20 (molecular weight 120 kD) , and the transme brane region, gp41 (molecular weight, 41 kD) . The pol gene encodes major proteins, such as reverse transcriptase p66 and p5l molecular weights 66 and 51 kD, respectively) and a p31 endonuclease (molecular weight 31 kD) component. Several other viral proteins, mainly having regulatory functions, are encoded and expressed by the HIV genome, but none of these gene products are sufficiently immunogenic to be used as routine markers for serologicai diagnosis. Several methods for the detection of antibodies to HIV-l have been developed over the years, and, as the epidemic continues to spread, it becomes important that diagnostic methods have the capability to accurately identify infected individuals. Enzyme-linked im unoassayε (ELISAs) are widely used to screen for presence of virus-specific antibodies (Groop an, J.E. et al.. J. Infec. Pis. 153:736-742 (1986); Centers for Disease Control, "Update: Serologic testing for antibody to Human Immunodeficiency virus," MMWR 11:833-840, 845 (1988)) . Such a test format can be extremely sensitive but carries with it a potential for being less specific, leading to false positive reactions. Supplementary tests that are more specific are commonly used. A positive HIV-l serologicai diagnosis has significant psychosocial implications for the individual being diagnosed. It is all the more pressing, therefore, that test results be accurate and reliable. Furthermore, although users of HIV im unoblot confirmatory assays may appreciate the inherent limitations in assays using only purified natural viral antigens, they have become comfortable witπ the reactivity profiles obtained in such assays and may thus be hesitant to change. Supplementary tests offering greater specificity and sensitivity than a standard ELISA include the western blot test, indirect immunofluorescence and radioimmunoprecipitation assays (MMWR, 1988, suprai . Among these supplementary tests", the western blot is most informative and is the current "gold standard" for serologicai confirmation of HIV infections ("Interpretation_and use of the Western Blot Assay for serodiagnosiε of HIV-l Infections," MMWR 38, Uo . s-7 (1989) ) . In the Western Blot, HIV-l virus is harvested from tissue culture, lysed, and the individual viral proteins separated by polyacrylamide gel electrophoresiε (PAGEj . The separated proteins are then transferred to nitrocellulose- membranes by electroblotting ("immunoblot strips") . A test serum is incubated with the immunoblot εtripε and HIV- antibodies, if present, bind to the separated viral proteins. The strips are washed to remove excess unbound proteinε, and presence of the specific antibodies is visualized by incubation with enzyme-conjugated anti-human immunoglobulin antibodies and chro ogenic substrates. Thus, the presence of an antibody in sufficient concentration results in the staining of the characteristic viral protein "band." Over the past five years, laboratories and health organizations have delineated several criteria required for determining that a HIV-l western blot profile is positive. Qualitative assessment of reεultε based on the number of stained bands and the particular band patternε, haε indicated that εampleε obtained early in infection, cr infection by sor.e cross-reacting viruses, may yield "atypical" blot patterns. Such atypical patterns are generally considered "Indeterminate" with regard to diagnostic criteria of positivity. Ir. such circumstances, a follow-up evaluation of the individual is usually performed. Attempts have been made to improve the reliability o: western blot analysis by (a) removing ir. interpretation, and (b) standardization of the reage ts used ir. manufacturing. However, the configuration of the test format as currently practiced is subject to a variety of complica ing factors. For example, the relative concentrations of the viral proteinε from a particular tissue culture preparation will vary, and this variability is reflected in the material used ir. manufacture of the test. These problems prompted attemptε at improvement by using viral antigens from synthetic sources (Crowl, R. et al. , 1985. Cell 41: 979-986 (1985) ; Samuel, K.P. et ai. , Gene 64: 121-134 (1988)) . Several structural and nonstructural HIV gene sequences have been cloned and their proteinε expressed. Immunogenic epitopes from such regions have also beer, identified and synthesized. Mostly, these antigens have beer, used in the ELISA format for screening test purposes. In some instances these antigens have been directly applied onto nitrocellulose sheets and evaluated for use as a replacement for the conventional western blot._Such teεt formats also have certain limitations. Molecularly cloned antigens, or synthetic peptideε may be limited in the "repertoire" of epitopes presented, thus restricting the sensitivity and range of detection of sero- reactivity of an extensive array of antibody responses. A major deficiency in the art is therefore the absence of a single confirmatory immunoblot assay method that provides not only the well-characterized natural antigen reactivity profile, but also has the capability of increaεed sensitivity and immediate qualitative differentiation of infection by viral subtypes, such as HIV-l and HIV-2. SUMMARY OF THE INVENTION It is an object of the preεent invention to overcome the deficiencies in the work discussed above. It is a further object of the invention to provide an immunoblot format and immunoassay with significantly improved senεitivity and specificity for the serologicai diagnosis of viral infection, especially with human immunodeficiency virus (HIV-l, HIV-2) . It is a further object of the invention to provide an immunoassay format with significantly improved specificity for serologically distinguishing HIV-l from HIV-2 infection. This invention is further directed to an extension of such an immunoassay format for serologically distinguishing viral infections other than HIV-l and HIV-2 , such as HTLV-I and HTLV- II, to enhance sensitivity and specificity of detecting a particular viral infection by εerodiagnosiε. Specifically, this invention relates to a new and enhanced method for the detection -of antibodies to a virus, in particular Human Immunodeficiency Virus type 1 (HIV-l) and/or type 2 (HIV-2) by use of an augmented western blot format and asεay. The invention is directed to an improved immunoassay for the detection of antibodies specific for a virus in a biological fluid comprising: (a) providing a solid phase support containing electro phoretically separated natural viral proteinε and additionally having bound thereto at leaεt one εynthetic viral protein or peptide; (b) contacting the biological fluid with the solid phase support allowing an antibody in the biological fluid to bind to the natural or εynthetic protein forming an antigen-antibody comple>T; (c) contacting the antigen-antibody complex with a detectably labelled binding partner capable of binding to the antibody; and (d detecting the detectable label on the solid phase support as a measure of the presence of the antibody in the fluid. The invention is alεo directed tc a method for pro¬ ducing an improved immunoblot format useful in an immunoassay for detecting antibodies specific for a virus, comprising the steps of: (a) applying a preparation of natural proteins of the virus derived from tisεue culture to a polyacryla ide gel; (b) electrophoretically εeparating the viral proteinε; (c) transferring the separated proteinε to a solid phase support; and (d) directly applying to the solid phase support at least one synthetic viral protein at a site not occupied by the tranεferred proteins, using the technique of stamping, imprinting, line drawing or slotting with a manifold apparatus. The above method may also include directly applying to the solid support a human anti-im unoglobulin antibody to serve as a positive control by the technique of stamping, imprinting, line drawing, or εlotting with a manifold apparatus, at a site not occupied by the transferred proteinε or the directly applied synthetic viral protein. The invention further provides a method for producing an improved immunoblot format useful in an immunoassay for detecting antibodies specific for a virus, comprising the steps of: ~~ (a) applying a mixture of a preparation of natural proteins of the virus derived from tissue culture and at least one synthetic viral protein to a polyacrylamide gel; (b) electrophoretically separating the natural and synthetic viral proteins; and (c) transferring the separated proteins tc a solid phase support. The new method and asεay of the present invention, based on augmenting the natural protein western blot with εyn¬ thetic proteins from the same virus " (or a different virus) provide a distinctly sensitive and specific detection device. Additionally use of this method of detection will provide a more complete serologicai profile. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents an immunoblot showing a typical HIV-l viral antigen profile observed with the conventional western blot asεay with HIV-l reactive serum. Figure 2 presents immunoblot profiles of the augmented immunoblot process format as described in Example 1 with sera from individuals infected with HIV-l, HIV-2, dual HIV-l/HIV-2, and non-infected individualε, aε well aε a εample of serum which is reactive only to gag proteinε: (a) Serum with HIV-l reactivity shows reactivity to the typical HIV-l viral antigen profile and to the augmented recombinant envelope band confirming HIV-l infection; (b) Serum with HIV-l reactivity but having weak to no reactivity with natural envelope proteins due to insufficient natural envelope proteins on the immunoblot strip; augmentation with recombinant envelope protein allows immediate confirmation of envelope reactivity; (c) Serum with dual HIV-l/HIV-2 reactivity iε shown aε immediately diεtinguiεhable from εerum from an individual with a single infection; (d) Serum with HIV-2 reactivity shows reactivity with the augmented HIV-2 envelope band which immediately distinguishes HIV-2 from HIV-l infection; (e) A non- reactive sera shows reactivity only to the anti-human IgG, indicating presence of a εerum sample in the assay; this serves as a positive control for the assay ensuring that reεultε are not read as a false negative; (f) Immunoblot profile of a serum which iε gag-reactive only. — Figure 3 illuεtrateε immunoblot profiles of the augmented aεεay format wherein the εtrip iε augmented with synthetic polymerase protein (a) and synthetic cere proteir. (b) . Figure 4 illustrates an immunoblot profile of the augmented assay format wherein the strip iε augmented witr. a εynthetic HTLV-I envelope protein. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention involves three basic procedureε. In the first, viral proteinε, preferably retroviral proteins of the retroviruses, HIV-l or HIV-2 , are prepared fror lysateε of virus obtained from tissue culture, and are separated by western blot, using PAGE followed by blotting onto a solid phase support, such aε nitrocellulose. In the second procedure, genetically engineered or chemically synthesized viral proteins or peptideε are directly applied to the same εolid support, in order to augment the pro¬ file of the electrophoretically tranεferred proteinε. Viral protein or peptide antigenε produced either by recombinant or εynthetic means will be referred to herein collectively as "εynthetic. " In the third procedure, an immunoassay is carried out to detect the presence of antibodies in a biological fluid, such aε the εerum, of virus-infected individuals to the transblotted as well as to the directly applied proteins. By the term "biological fluid" iε intended any fluid derived from the body of a normal or diseased subject, such aε blood, εerum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, asciteε fluid, puε and the like. Also included within the meaning of this term as used herein is a tissue extract, or the culture fluid in which any cells or tissue preparation from the subject haε been incubated. HIV-l virus particles are harvested from the supernatants of virus-infected cell lines and concentrated and purified via a sucrose density gradient using methods well- known in the art. The virions are—lysed and inactivated using a detergent, such aε NP40. The method of Popovic et al . (Science 224:497-500 (1984)) iε preferred. The antigens contained in the viral lyεate are εeparated by electrophoresis on a polyacrylamide slab gel in the preeence of sodium dodecyl sulfate. The electrophoreεed proteinε are transferred to a solid phase support using methods known in the art. (See: Towbin H, et al. , Proc Natl Acad. Sci. USA 76: 435C ( r 19"9) ; Tsang, V.C.W. et al. , Methods in Enzvmoloqv 92:377-391 (1983) , which referenceε are hereby incorporated by reference) . The εolid phaεe support uεeful for the methodε of the present invention is preferably nitrocellulose. Alternatively, other solid phase supports may be used, including, but not limited to, nylon, charge modified nylon, diazobenzyloxymethyl paper, and the like. The next phaεe of the process according to the inven¬ tion iε the application of the synthetic proteinε or peptideε directly to the solid phaεe support. The synthetic proteinε or peptides can be chemically synthesized or produced by recombinant techniques in prokaryotic cells, preferably bacteria, or in eukaryotic cells such aε yeaεt cells or mammalian cells. Synthetic protein or peptide antigens known to be antigenic in the species being tested for viral antibodies, preferably hu anε, are generally uεeful in the preεent invention. The preferred viral antigens are those known to be valuable for diagnostic purposeε. The HIV-l proteins used to augment the immunoblot of the present invention may be purified proteins from the natural virus preparation, recombinant proteinε and peptides, or func¬ tional derivatives thereof. Such purified or recombinant proteinε of HIV may be the entire, or a portion of the gpl20/160 envelope glycoprotein, gp41, p66, p31, or p2 . Better defined viral protein antigenε in the form of peptides, such aε a p24 epitope that does not cross-react with normal " human serum, are also contemplated within the scope of the invention. In one embodiment, the HIV-l recombinant envelope protein encoded by the plas id p-env S iε used (Petteway, £.R. et al. , UCLA SYMP. ON MOL. CELL. BIOL.. "Viruses and Human Cancer," 4_3: 15-28 (1987)) . In a preferred embodiment, the HIV envelope peptide having the εequence RILAYERYLKDQQLL3IV, " GCSG- " ., a variation of the UBI peptide iε uεed. In another embodiment, the peptide encoded by clone 566 (Samuel, K.F. et al . , Gene 64 . : 121-134 (19SS)) is uεed. For the HIV-2 εpecific protein, the peptide sequence LNSWGCAFRQVCHTTVPW, which is a modification cf tne peptide dis¬ closed in McCormick et al. (Science 237:1346-1349 (1987), , is preferred. For HTLV-1 εpecific proteins, a . envelope recombinant, gp21 (Samuel, K.P. et al . , Gene Anal. Techn. 2 . : 60- 66 (1985)) is preferred. By "functional derivative" iε meant a "fragment," "variant," "analog," or "chemical derivative" of a viral protein, which termε are defined below. A functional derivative retainε at leaεt a portion of the function of the viral protein which permitε itε utility in accordance with tne preεent invention, that iε, itε recognition by and binding to an antibody. A "fragment" of a viral protein refers to any subset of the molecule, that iε, a shorter peptide. A "variant" of a viral protein referε to a molecule εubstantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methodε well- known in the art. Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutionε of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may alεo be made to arrive at the final conetruct, provided that the final conεtruct poεεeeεes the desired activity. An "analog" of a viral_ protein referε to a non- natural molecule εubεtantially similar to either the entire molecule or a fragment thereof. A "chemical derivative" of a viral protein contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that iε capable of reacting with εelected εide chainε or terminal residues. The viral protein or peptide is directly applied onto the solid phase support to augment the serum reactivity profile provided by antigens of the natural viral lysate. For reproducible sensitive western blot profileε, it is desirable that all individual viral proteins be preεent in approximately equal concentrationε. It iε rare that viral antigens in lysates of virions from tissue culture provide such uniformity in lot to lot harvestε. Alεo, the electrophoretic tranεfer of the natural viral proteinε separated by SDS-PAGE may result in non- random selective retention of different viral proteins baεed on molecular weight verεuε transblot time. Lower molecular weight proteinε tend to tranεfer more rapidly from the gel to the nitrocelluloεe. Commonly, viral glycoproteins are transferred inefficiently due both to their chemical nature as well aε molecular weight. Tnerefore augmentation of the immunoblot profile of natural viral antigens with synthetic viral protein or peptide antigens provides several advantages. The antigens can be made available reproducibly and uniformly, decreasing the lot-to-lot variability of the immuno- blotε. Immunogenic viral proteinε which are inherently preεent in lower concentration, such as envelope glycoproteins or gag proteinε, like p24, can be applied directly onto the solid phase εupport to improve the εenεitivity and εpecificity of the teεt. Viral proteins or peptides specific to viruses other than HIV-l, such as, for example, HIV-2, HTLV-I , HTLV-II , hepa¬ titis virus, Epstein-Barr Virus, Herpes Simplex Virus 1 and I, and the like, can also be applied to the solid εupport. Presence of such viral antigens will allow immediate serologicai detection and distinction of viral infections. The εynthetic proteins or peptides may be applied to the εolid phaεe support by any one of several known procedures. If the molecular weight of the synthetic viral protein or peptide iε εuch that it iε within the range of molecular weights of the natural viral proteins, and further, if it will be electrophoretically distinct, such a protein or peptide can be mixed with the natural viral lysate prior to electrophoresiε and transblotting (Towbin H, et al. , supra; Tsang, V.C.W. et al . , supra) . However, genetically engineered proteins may occur aε aggregateε or in more than one form dependent on the nucleotide sequences inserted into the expression vectors. In εuch caεeε, aε well aε in the caεe of low molecular weight synthetic pep¬ tides, it iε preferable that theεe proteinε be directly applied via a slot or dot blotting procedure which are well known in the art. Other procedureε like stamping, or line drawing, alεo known in the art, can be uεed. (See, for example, Linnecke, U.S. Patent No. 4, 748, 042 j . In a preferred embodiment of the preεent invention, three proteins are uεed to augment the western blot: anti human IgG antibody or human IgG, a genetically engineered HIV-l enve¬ lope protein and synthetic peptide corresponding to a portion of a HIV-2 envelope protein. More preferably, an HIV-l envelope peptide, RILAVERYLKDQQLLGIWGCSGK, iε uεed in place of the envelope protein. A preferred means of applying the augmenting proteins or peptides to the immunoblot is by slotting on to the nitrocellulose after the natural viral proteins have been electrophoresed and tranεblotted. Anti-human IgG or human IgG is applied directly onto the membrane to provide within-aεεay quality control. Thus the addition of sera and conjugate, respectively, will be indicated by color development of this band—on the nitrocellulose after the performance of the immunoaεεay. Thiε will obviate the possibility of a false negative result due to the poeεibility that a test serum sample was inadvertently left off. Whenever a human εerum εample iε added, the anti-human IgG will react with it and the band in the appropriate location will be detected. A εample lacking any anti-viral antibody (seronegative) will have no reactivity to any viral bands but will be positive for the anti-human IgG band. Abεence of a colored band where the anti-human IgG band iε located iε an indication that a εerum sample was not added in the immunoassay. In the preferred slotting procedure, the tranεblotted nitrocellulose containing the electrophoresed natural viral antigens iε placed onto a slotting manifold. The manifold is constructed such that each slot provideε a band preferably not more than 1.5 mm wide and 13 cm in length. Slotting iε preferably performed on areaε on the transblotted nitrocellulose not occupied by the transblotted viral proteinε. In a preferred embodiment, the slotted bands are 1.5 mm apart and the proteinε are slotted 1 to 1.5 mm from the lower end of the zone occupied by the transblotted natural viral proteinε. Slotting can be performed immediately after the tranεblot of the natural viral antigenε. Alternatively, the tranεblotted nitrocelluloεe can be dried, kept in a dry container until ready for εlotting at another time. The amountε of synthetic proteinε slotted are predetermined via a εerieε of εenεitivity and specificity titrationε, known to one of skill in the art. The proteinε are diluted preferably in 0.1 M carbonate buffer, pH 9.6, applied into the slots using a micro- pipettor or syringe. A 1 ml syringe iε preferred. The slotting apparatus iε a modification of a εlotting dot manifold equipment available commercially from companieε like BIO-RAD (Richmond, CA) or Life Scienceε, Inc. (Gaitherεburg, MD) . The εolid phaεe support, preferably nitrocellulose, to be slotted iε held in place via clamps or screwε. After addition of the protein or peptide, exceεε material iε removed from the slots- via aspiration through the slotε attached to a vacuum pump. In addition to εlotting, the proteins can be applied by stamping, line drawing, or dot blotting. The εolid phaεe εupport iε removed and blocked ir. a buffer containing a detergent and blotting proteins. Nonfat dry milk iε a preferred blocking agent althougn any prctem- containing blocking agent known in the art can be uεed. Tne εolid phaεe εupport iε dried and cut into 3 or 4 mm-wide εtrips and stored in desiccated dry containers until ready for use. The presence or absence of antibodies tc the viral antigens is detected via an immunoaεεay procedure. This can be performed following previously establiεhed procedureε well known in the art. The serum εample to be teεted iε diluted in the presence of a Tris-based buffer containing blocking proteinε, such as non-fat dry miIT;. The diluted εerur sample iε incubated with the augmented viral antigen εtrip prepared aε described above. Antibodies to HIV-l will bind to antigens located on the strip aε discrete bands. The strips are then washed to remove unbound material. Visualization cf the antibodies specifically bound to the viral antigens iε accomplished in situ using a εpecieε-εpecific εecond antibody εpecific for the immunoglobulin compriεing the anti-HIV antibody, εuch aε goat anti-human IgG. Alternatively, a ligand which will bind specifically to the human antibody, such aε Staphylococcal protein A, is used. Once the secondary binding reagent iε choεen, the appropriate detection reagent can be selected. Useful detection reagents for the present invention include enzymes, preferably alkaline phosphataεe (AP) , or horεeradiεh peroxidaεe (HRP) . Other enzymeε which can be uεed include, but are not limited to, malate dehydrogenaεe, εtaphylococcal nucleaεe, delta-V-εteroid iso erase, yeast alcohol dehydrogenaεe, alpha- glycerophosphate dehydrogenase, trioεe phoephate iεomeraee, aεparaginaεe, glucoεe oxidaεe, beta-galactoεidaεe, ribo- nucleaεe, ureaεe, catalaεe, glucoεe-6-phoεphate dehydrogenaεe, glucoamylaεe and acetylcholineεteraεe. In another embodiment, colloidal gold εphereε rather than enzymeε are uεed as the detectable label. The enzyme or colloidal gold εphereε are preferably conjugated to the secondary binding reagent (εuch aε goat anti- human IgG or protein A) . For detection of the enzyme-conjugated reagent, a chromogenic εubεtrate iε uεed. For alkaline phoεphataεe, the chemical εubεtrateε, 5 bromo-4 chloro-3-indoyl phoεphate (BCIP; and nitroblue tetrazolium (NBT) are uεed. For horεe radish peroxidase, 3,3'.- diaminobenzidine (DAB) , 4 chlorol-naphthol (4CN) and 3- amino-9-ethyl carbazole iε uεed. One of εkill in the art will readily determine which substrate to use. The uεe of the colloidal gold εphereε provideε a one- step detection εyεtem aε the gold provideε a roεe/red color and no further incubation steps are required. If virus-specific antibodies are present in the test sera, colored bands corresponding to the position of— one or more of the viral antigens will be seen on the nitrocellulose εtrip. In a preferred embodiment, goat anti-human IgG con¬ jugated with alkaline phoεphataεe is uεed aε the enzyme conjugated εecond reagent. In another embodiment, the εecondary reagent (εuch aε the goat anti-human IgG antibody may be conjugated to a firεt enzyme which iε "coupled" to a second εyεte which reεultε ultimately in generation and depoεition of a chromophore where the antigen-antibody reaction occurred on the εolid phase. Thus, for example, the conjugated firεt enzyme may act on a firεt substrate which yields a product which when coupled with a second enzyme εubεtrate reaction, or with a second coupled' dye (such aε a diazo dye) , yieldε a product which iε detectable and reflectε the preεence of the first enzyme. The second substance may be a co-enzyme precursor plus a chromogenic precursor, wherein the firεt enzyme reactε with the co-enzyme precurεor, εuch as, for example nicotinamide adenine dinucleotide phosphate (NADP) , under suitable conditions to produce NAD, which iε then employed in a εerieε of cyclic chemical reactions forming a generation catalyst which acts to produce a detectable product from a precurεor (εuch aε a chromogenic precurεor) . The generation catalyst car. be another enzyme, e.g. diaphorase, aε a reduction catalyst. The presence of a chromophore so generated can be visually detected cr meaεured by colorimetric techniqueε known in tne art. For example, a cyclic NAD/NADH reaction with different oxidation- reduction catalyεtε for phoεphatase determination is discioεe:: in EPO Publication 0058539. In an additional example, tne enzyme lactic dehydrogenaεe (LDH) , in the preεence of lactate, cauεeε reduction of NAD to NADH, which can reduce a tetrazolium εalt to a reduced for azan, which can be detected viεually or colorimetrically at the appropriate wavelength. The amount of formazan formed iε a eaεure of LDH activity. Chromophore precurεorε uεeful in thiε embodiment include tetrazolium εaltε such aε 2- (p-iodophenyl) , 3- (p-nitrophenyl) -5- phenyltetrazolium chloride (INT) ; 3- (4 , 5-dimethylthiazol-2-yl) - 2,5-diphenyl tetrazolium bromide (MTT) ; 2 , 2 ' 5, 5 '-tetra- (p-nitrcpnenyl) -3 , 3 ' - (3,3' -dimethoxy-4 , 4 ' -diphenylene) diphenyltetrazoliu chloride (TNBT) ; 2,2 ' -di (p-nitrophenyl) -5, 5 '-diphenyl-3 , 3 '-(3, 3 '- dimethoxy-4 ,4 '-diphenylene) diphenyl tetrazolium chloride (NBT) ; 2 ,2-diphenylene-3 , 3 ' 5, 5 ' -tetraphenyl ditetrazolium chloride (neotetrazolium chloride or NT) ; 2,3-5- triphenyltetrazolium chloride (TT) ; and the like. Alternatively, alkaline phosphatase activity results in generation of NAD from NADP. The NAD can couple to the LDH reaction described above and result in similar formazan formation, which in thiε case would reflect the preεence of alkaline phoεphataεe bound to the solid support. As will be evident to one of skill in the art, the immunoassayε and methodε of the preεent invention can be uεed for improved detection of many typeε of viruεes from any of a number of εpecieε. The invention iε particularly uεeful for aεεay of retroviruε-specific antibodies in humans. However, it is also intended for other viruses, for example, retroviruseε of other mammalian εpecieε εuch aε εimian retroviruεeε, feline retroviruses, and the like, and iε therefore uεeful in veterinary aε well aε human medicine. Having now generally deεcribed the invention, the sane will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unlesε εpecified. EXAMPLE 1 Immunoblot Capable of Diεtinquiεhing HIV-l from HIV-2. Because of the extensive i munological cross- reactivity of HIV-l and HIV-2 , it is often not posεible to distinguish an "indeterminate" HIV-l immunoblot from a true HIV-2 seropoεitive εample. The immunoblot configuration deεcribed herein allowε thiε diεtinction. A. PREPARATION OF IMMUNOBLOT STRIPS The polyacrylamide gel was polymerized between glass plates measuring 18 cm by 16 cm comprising of a 11% reεolving lower gel and a 6% upper εtacking—gel. Approximately 6C μg of HIV-l viral antigen lysate was mixed in a volume of 800 ul with εample buffer containing glycerol, 2-mercaptoethanol, and bromophenol blue and pyronin Y aε tracking dyee. Thiε mixture was layered onto the upper stacking gel and electrophoresed at 15mA/gel, for the upper gel then at 25mA/gel for the lower gel until the pyronin Y marker had migrated 10.5 cm into the lower gel. The gel was removed from thre εandwiching glaεε plateε, and the upper gel and 0.5cm from the edge of the lower gel marker were trimmed off. The proteins were then electrophoretically transferred onto nitrocelluloεe, using modifications of an established methodology (Towbin et al. , supra) . A piece of nitrocellulose measuring 12.8 cm by 10.5 cm, preεoaked in a Tris-baεed buffer, was layered onto a piece of wet blotting paper, meaεuring 14 cm by 11.5 cm. The trimmed gel was gently layered onto the nitrocellulose paper. A uniform physical contact between the gel and nitrocelluloεe was maintained by removing exceεε air bubbles using a pair of smooth rollers. Another piece of wet blotting paper was placed on top, thereby sandwiching the gel. The blotting paper-gel-nitrcceilulose- blotting paper sandwich was further sandwiched by I pieces o: Scotch-Brite pads. Thiε multilayer preparation was placed within the tranεblot cell holder, i merεed into the tranεblct tank with the nitrocelluloεe side of the εandwich directed toward the poεitive electrode (anode) . The proteinε were tranεblotted by applying a voltage of 30V overnight (16 hours) followed by 100V for 1 hour. The nitrocellulose sheet was removed and kept moist in a tray containing a phosphate buffer. Commercially available anti-human IgG was diluted, to a concentration of 10 μg/ml in O.IM carbonate buffer, pH9.6. A genetically engineered HIV- 1 envelope protein, at 7 μg/ml, and a HIV-2 εpecific εynthetic peptide from the envelope εequence, at 15 μg/ml, all diluted in carbonate buffer, were applied to the tranεblotted nitrocelluloεe uεing a εlctting apparatuε. The anti-human IgG waε slotted at approximately 1.6cm from the lower end of the electrophoreεed, transblotted protein zone, followed by the HIV-l envelope protein at 1.3cm and synthetic - IS - HIV-2 peptide at 1.0 cm respectively from the lower end. The lower end of the nitrocellulose iε the area beyond which the lower molecular weight viral proteinε had migrated. The protein εolutionε were removed by aεpiration with a vacuum pump. The nitrocelluloεe waε removed from the εlotting apparatuε and blocked in PBS buffer containing 5% non-fat dry milk for 45 minutes at room temperature, (25§C) , on a rocker platform. The nitrocellulose was rinsed for 15 minutes at room temperature with PBS-Tween buffer. Nitrocellulose sheetε were removed, air dried on paper towelε, then left in a 37@C oven for overnight drying. The εheet waε cut into 3mm or 4mm wide strips and kept in dry containers till ready for the asεay. B. IMMUNOASSAY Serum εamples that had been previously identified aε εeropoεitive for HIV-l, εeropoεitive for HIV-2 and indeterminate for HIV-l with conventional immunoblot assays were aεεayed by incubation with the augmented western blot stripε. Theεe particular εampleε were εelected for purpoεee of illustration. In normal use, an unknown εerum εample from a patient would be tested. The strips v/ere placed, right side up, onto each well of commercially available trayε uεed for conventional HIV-l weεtern blot aεεays. The εtripε were prewetted by incubating in 2 ml each of a waεh buffer (Tris- based, containing Tween- 20 detergent) . After 15-30 minutes, the buffer was aspirated, and 2 ml of blotting buffer, containing Tris, inactivated goat serum and 5% non fat dry milk were added. 20 μl of each test εerum v/ere added to each well containing buffer and nitrocell loεe εtrip, and incubated overnight at room temperature on a rocker platform. The fluid waε then removed by aspiration and the εtripε waεhed 3 timeε with 2 ml of waεh buffer, with 5 minute εoakε on the rocking platform between each waεh. A commercially available anti- human IgG-alkaline phoεphataεe conjugate diluted with blotting buffer at recommended dilutione waε added to the εtripε, incubated 30 minuteε at room temperature and waεhed 3 tiraeε aε deεcribed in the previouε εtep. The viral bande were visualized after incubation of the εtripε with a commercially available εolutionε of nitroblue tetrazolium and 5 bromo-4- chloro-3 indolyl phosphate. Purplish- blue viral εpecific bandε were observed indicating the presence of specific antibodies the test sera. The enzymatic reactions were stopped by rinsing the εtrips with distilled water. C. RESULTS Representative profiles of teεt sera are diagrammed in Figure 1. Sera that contained εpecific anti-HIV-1 antibodieε reacted with all natural viral proteinε on the immunoblot εtrip, aε well aε the anti-human IgG band and the HIV-l recombinant antigen band. A serum containing anti-HIV- 2 antibodies tested on conventional HIV-l immunoblot strips would normally react with HIV-l proteins v/hich are products of gag and pol genes, including p55, p24, pl7, and p31, but would not react v/ith HIV-l specific en gene products. Such a reaction pattern v/ould be claεεified aε "indeterminate." Uεing the augmented immunoblot εtripε, the HIV-2 εera reacted with the HIV-l p24 and p31 bandε, the anti-nu a IgG band (poεitive control) and the HIV-2 envelope-specific peptide band. This allowed immediate distinction of the HIV- 2 seropositive sample from a HIV-l seropositive sample. EXAMPLE 2 The HIV-l viral lysate profile could be further aug¬ mented by the direct application of synthetic viral proteinε from other viruεeε εuch aε HTLV-I. HIV-l viral lysate antigens were electrophoreεed and tranεblotted as deεcribed in Example 1. The tranεblotted nitrocellulose waε placed onto the εlotting apparatuε, and anti human IgG was slotted on to the nitrocellulose 1.6 cm from the lower end. A genetically-engineered HTLV-I envelope protein at 7 μg/ml waε slotted into the next slot 1.3 cm from the lower end of the nitrocellulose. A HIV-l poεitive εerur fror a patient co-infected with HTLV-I will react with the HIV-l specific viral antigens aε well aε with the HTLV-I recombinant protein. An immunoblot εtrip formatted in thiε way allowed for the simultaneous serologicai distinction of HIV-l infection and probable HTLV-I infection. EXAMPLE 3 " This augmented immunoblot format was further extended by using electrophoresed HTLV-I lysate antigens and HTLV-I recombinant antigens or synthetic peptideε to augment the HTLV- I viral lysate profile. HTLV-I viral lyεate generally compriεed i munologically "weak" envelope glycoproteinε, which reεult in weakly stained immunoblot profiles with HTLV-I seropoεitive serum. To a εtandard HTLV-I viral lyεate waε added 3 μg/cm gel of a HTLV-I recombinant envelope protein, and the mixture was electrophoreεed and tranεblotted aε deεcribed in Example 1. Electrophoresiε was performed on 12% polyacrylamide gelε. The recombinant protein migrated aε a band diεtinct fror that of the other viral lyεate proteinε. The addition of the recombinant envelope protein therefore enhanced the detection of antibodies to the HTLV-I envelope proteins. EXAMPLE 4 The immunoblot format aε deεcribed in Example 3 ie further configured to diεtinguiεh HTLV-I and HTLV-II εpecific infectionε. A HTLV-II έpecific recombinant protein iε alεo added to the HTLV-I viral lysate for electrophoreεiε. Alternately, HTLV-I and HTLV-II εynthetic proteinε are applied in the εlot format aε in example 1 creating a configuration for the differentiation of HTLV-I from HTLV-II. An i munoaεεay performed aε above using this format allows the detection of co-existing antibodies to HTLV-I and II. Having now fully deεcribed thiε invention, it will be appreciated by thoεe εkilled in the art that the εame can be performed within a wide range of equivalent parameters, concen¬ trations, and conditions v/ithout departing from the εpirit and εcope of the invention and without undue experimentation. While thiε invention haε been deεcribed in connection with εpecific embodiments thereof, it will be understood that it iε capable of further modifications. Thiε application iε intended to cover any variationε, uεeε, or adaptations of the inventionε following, in general, the principles of the invention and including εuch departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the eεεential featureε hereinbefore set forth aε follov/s in the εcope of the appended claims.

Claims: WHAT IS CLAIMED IS: ~~ 1. An immunoaεsay for the detection of antibodies specific for a retrovirus in a biological fluid compriεing: (a) providing a solid phase support containing electro phoretically separated and transferred natural viral proteins and additionally having bound thereto, at a site not occupied by said transferred proteinε, at leaεt one εynthetic viral protein or peptide; (b) contacting said biological fluid v/ith said solid phaεe εupport allowing an antibody in said biological fluid to bind to said natural or synthetic protein forming an antigen-antibody complex; (c) contacting said antigen-antibody complex with a detectably labelled binding partner capable of binding to said antibody; and (d) detecting said detectable label on said εolid phaεe εupport aε a meaεure of the preεence of said antibody in said fluid. 2. The immunoassay of claim 1 wherein said binding partner iε an antibody εpecific for a human immunoglobulin molecule. 3. The immunoassay of claim 1 wherein said binding partner iε an immunoglobulin-binding bacterial protein. 4. The immunoassay of claim 1 wherein said solid phase εupport iε nitrocelluloεe or nylon. 5. The immunoaεεay of claim 1 wherein said virus iε a strain of human immunodeficiency virus (HIV) . 6. The immunoassay of claim 1 wherein εaid virus iε human T lymphotropic virus, type I (HTLV-I) . 7. The immunoaεεay of claim 1 wherein εaid natural viral proteinε and said synthetic viral protein are derived from HIV-l. 8. The immunoaεεay of claim 5 wherein εaid natural viral proteinε are derived from HIV-l and said εynthetic viral proteinε are derived from HIV-l and HIV-2. 9. The immunoaεsay of claim 7 wherein εaid εynthetic viral protein has the amino acid sequence RILAVERYLKDQQLLGIWGCSGK. 10. The immunoassay of claim ε wherein εaid εynthetic viral protein derived from HIV-l has the amino acid εequence RILAVERYLKDQQLLGIWGCSGK and εaid εynthetic viral protein derived from HIV-2 haε the amino acid εequence LNSWGCAFRQVCHTTVP . 11. The immunoaεεay of claim 1 wherein εaid natural viral proteinε are derived from HIV-l and εaid εynthetic viral protein iε derived from HTLV-I. 12. The immunoaεεay of claim 1 wherein εaid natural viral proteinε and εaid εynthetic viral protein are derived from HTLV-I. 13. The immunoaεεay of claim 1 wherein εaid natural viral proteinε are derived from HTLV-I and εaid εynthetic viral protein is derived from HIV-l. 14. The immunoaεεay of claim 1 wherein εaid natural viral proteinε are derived from HTLV-I and εaid εynthetic viral proteinε are derived from HTLV-I and HTLV-II. 15. A method for producing a solid phase antigenic reagent useful in an immunoassay for detecting antibodies εpecific for a retroviruε, comprising the steps of: (a) applying a preparation of natural proteinε of said virus derived from tissue culture to a polyacrylamide gel; (b) electrophoretically separating εaid viral proteins; (c) transferring said separated proteinε to a εolid phase support; and (d) directly applying to said εolid phase support at least one synthetic viral protein at a site not occupied by said transferred proteinε, thereby producing said solid phase antigenic reagent. 16. The method of claim 15 wherein said direct applying of step (d) is performed by the technique of stamping, imprinting, line drawing or εlotting with a manifold apparatuε. 17. The method of claim 15 additionally comprising: (e) directly applying to said εolid εupport a human anti-immunoglobulin antibody by the technique of εtamping, imprinting, line drawing, or εlotting with a manifold apparatuε, at a εite not occupied by said transferred proteins or εaid directly applied εynthetic viral protein. 18. The method of claim 15 wherein said εolid phaεe support is nitrocellulose or nylon ^ 19. The method of claim 15 wherein said virus iε a strain of human immunodeficiency virus (HIV) . 20. The method of claim 15 wherein said virus iε human T ly photropic viruε, type I (HTLV-I) . 21. The method of claim 15 wherein εaid natural viral proteinε and said εynthetic viral protein are derived from HIV-l. 22. The method of claim 21 wherein εaid synthetic viral protein has the amino acid εequence RILAVERYLKDQQLLGIWGCSGK. 23. The method of claim 15 wherein εaid natural viral proteinε are derived from HIV-l and said synthetic viral proteins are derived from HIV-l and HIV-2. 24. The method of claim 23 wherein εaid εynthetic viral protein derived from HIV-l haε the amino acid εequence RILAVERYLKDQQLLGIWGCSGK and εaid εynthetic viral protein derived from HIV-2 haε the amino acid εequence LNSWGCAFRQVCHTTVP . 25. A method for producing a εolid phaεe antigenic reagent uεeful in an immunoaεεay for detecting antibodiee specific for a retrovirus, comprising the εtepε of: (a) applying a mixture of a preparation of natural proteinε of εaid virus derived from tissue culture and at leaεt one εynthetic viral protein to a poly¬ acrylamide gel; - (b) electrophoretically separating εaid natural and εynthetic viral proteinε; and (c) tranεferring εaid separated proteins to a εolid phase support.

Inventors: CHAN LILY, LIM LEE FANG, SUM YOKE WAH, YIN MAY FONG, CHAN, LILY, LIM, LEE, FANG, SUM, YOKE, WAH, YIN, MAY, FONG
Applicants: DIAGNOSTIC BIOTECH INC, DIAGNOSTIC BIOTECHNOLOGY, INC.
Application year: 1991
Main classification: C12Q1
IPC codes: C07K14, C07K16, G01N33