WO1995033206A1 - Detection de differents genotypes de vih utilisant un immunodosage modifie par un peptide synthetique - Google Patents

Detection de differents genotypes de vih utilisant un immunodosage modifie par un peptide synthetique Download PDF

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Publication number
WO1995033206A1
WO1995033206A1 PCT/US1995/004421 US9504421W WO9533206A1 WO 1995033206 A1 WO1995033206 A1 WO 1995033206A1 US 9504421 W US9504421 W US 9504421W WO 9533206 A1 WO9533206 A1 WO 9533206A1
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WIPO (PCT)
Prior art keywords
hiv
assay
antigen
env
reagent
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PCT/US1995/004421
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English (en)
Inventor
James L. Gallarda
Barbara L. Stone
Douglas J. Liu
Susan K. Harrington
George J. Dawson
Ping Wu
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Abbott Laboratories
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Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to EP95915635A priority Critical patent/EP0763204A1/fr
Priority to JP8500836A priority patent/JPH10502165A/ja
Priority to AU22455/95A priority patent/AU2245595A/en
Publication of WO1995033206A1 publication Critical patent/WO1995033206A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to human immunodeficiency virus (HIV) and immunoassays.
  • this invention relates to an immunoassay and reagents for the simultaneous detection of more than one HIV genotypes.
  • this invention relates to an immunoassay for detecting HIV- 1 antigens and/or HIV-1 antibodies and HIV-2 antibodies in a test sample.
  • AIDS Acquired immunodeficiency syndrome
  • HIV type 1 HIV-1
  • ARC AIDS-related complex
  • HIV-1 is transmitted by sexual contact, exposure to blood and certain blood products, or from an infected mother to her fetus or child.
  • P. Piot et al. Science 239:573-579 (1988).
  • the prevalence of HIV-1 antibodies in AIDS and ARC patients and persons at risk is high, and the virus can be isolated from nearly 90% of all seropositive individuals. See, for example, M. G. Sarngadharan et al., Science 224:506-508 (1984); and D. Gallo et al., J. Clin. Micro. 25:1291-1294 (1987).
  • HIV-2 In 1986 a second human immunodeficiency virus, HIV-2, was isolated from patients with AIDS in west Africa. F. Clavel et al., Science 233:343-346 (1986). HIV-2 infections also have been identified in individuals from several countries outside of west Africa. See, for example, A. G. Saimot et al., Lancet i:688 (1987); M.A. Rey et al., Lancet i:388-389 (1986); A. Werner et al., Lancet i:868-869 (1987); G. Brucker et al., AIDS 2:141 (1988); and K Marquart et al., AIDS 2:141 (1988). Although at the present time HIV-2 appears to be endemic only in west Africa, it appears likely that, based on the experience with HIV-1, HIV-2 will spread to other parts of the world.
  • HIV-2 virus is similar to HIV-1 virus in its morphology, cell tropism, interaction with the CD4 cellular receptor, in vitro cytopathic effect on CD4 cells, overall genomic structure and its ability to cause AIDS.
  • HTV-2 differs from HIV-1 in several respects. See F. Clavel, Ibid and R.A. Weiss et al., AIDS 2:95-100 (1988).
  • HIV-1 consensus sequence, approximately 55% homolgy to HIV-2 consensus sequence, and approximately 85% homology to ANT 70 sequence.
  • recombinant antigens allows for the improved detection of anti-HIV-1 and/or anti-HIV-2 containing test samples, while minimizing non-specific reactions largely due to cross reactions with whole virus or viral lysate.
  • the use of at least one recombinant HIV protein to detect HIV antibody in a test sample with the use of labeled recombinant HIV antigens is described in the parent patent applications previously incorporated herein by reference.
  • ELISA immunosorbent assay
  • immunoassays lack sensitivity and specificity which could permit test samples, such as blood products containing virus to escape detection and thereby potentially result in the infection of those patients receiving, for example blood products.
  • the lack of specificity (i.e., false positives) in such immunoassays is often due to nonspecific binding of immunoglobulins to cellular protein in the viral lysates, or in the case of recombinant antigens, lack of specificity may be caused by shared epitopes with viruses unrelated to AIDS.
  • Gallaher, Cell, 50: 327-328 (1987) has reported that a region of HIV-1 gp41 shares an antigenic region with the respiratory syncytial virus and with the measles virus Fl glycoprotein. Thus, even highly purified
  • HIV polypeptides could potentially be responsible for false positives. In either case, such false positives could result in the misdiagnosis of AIDS.
  • HIV genomic RNA encodes (beginning at the 5' end): (i) a gag gene extending between nucleotides 310 to 1869 and encoding for the internal structure core or nucleocapsid proteins including p24, the most antigenic core protein;
  • assays which can detect HIV antigen or HIV antibody are known.
  • assays include the anti-HIV-1/HIV-2 assay described
  • the detection of more than one analyte in a test sample usually involves the separate detection of each analyte in a separate assay. Such detection methods have been preferred since hey allow for stringent quality assurance determinations to be performed for each analyte to be tested.
  • Great Britain Patent No. 2188418 teaches an assay tray assembly having reaction wells each with openings in the top surface from which a projection is extended and wherein the inner surface of each well side wall and the outer surface of each projection may be incubated simultaneously for detecting two or more specific substances present in a specimen which has been introduced into the reaction wells.
  • U.S. Patent No. 4,870,003 to Kortright et al. discloses a solid phase immunoassay for detection of an antigen and/or antibody of a single binding pair utilizing an antigen "spike" of inactivated antigen.
  • HIV-1 and/or HIV-2 antibodies or antigens could be simultaneously detected, yet each separate analyte to be detected could be individually quality controlled.
  • an assay would be an improvement over other known assays since the simultaneous determinations of the presence of either HIV antigen analyte and/or HIV antibody analyte would be performed in one well, separation of solid phase components would not be required if more than one solid phase was utilized, and the assay could be quality controlled for individual analytes which were to be detected in the simultaneous assay.
  • synthetic HIV peptide antigens having unique and highly conserved epitopes of the HIV virus.
  • Such synthetic HIV antigens are beneficial because of the relative ease and lower cost with which they can be prepared, and more importantly, because of the reduced risk of obtaining false positives due to impurities or presence of shared epitopes with viral proteins not related to AIDS.
  • synthetic peptide HIV antigens would provide increased sensitivity in detecting emerging HIV subtypes.
  • synthetic HIV peptide antigens would provide a commercially available assay employing synthetic HIV peptide antigens to consistently and reliably detect HIV genotypes, such as subtype O antibodies in test samples. The lack of such an assay presents a major challenge in worldwide detection of HIV detection in blood and other biological samples.
  • an immunoassay for detecting one or more HIV genotypes comprising the steps of contacting a test sample with recombinant antigens (hereinafter "capture reagents") of (a) HIV- 2 gp36 env , (b) HIV-1 gp41 env , and (c) HIV-1 p24 gag immobilized on one or more solid materials to form a first mixture.
  • capture reagents recombinant antigens
  • the first mixture is incubated for a time and under conditions sufficient to form HIV env antibody/ recombinant antigen complexes and HW gag antibody/recombinant antigen complexes, and the resulting complexes are contacted with (a) an improved indicator reagent comprising a synthetic antigenic site-directed cyclic HIV-2 env peptide labeled with a signal generating compound, in the presence or absence of a
  • HIV-2 env peptide labeled with a sgnal generating compound (b) an improved indicator reagent comprising a synthetic antigenic site-directed cyclic HIV-1 env antigen labeled with a signal generating compound, in the presence or absence of a recombinant HIV-1 env antigen labeled with a signal generating compound, and (c) a recombinant HIV gag antigen labeled with a signal generating compound to form a second mixture.
  • the second mixture is incubated for a time and under conditions sufficient to form HIVantibody/ capture reagent/indicator reagent complexes.
  • the presence of antibodies to one or more HIV genotypes, particularly, HIV-1 env antibodies, HIV-2 env antibodies, HIV subtype 0 antibodies, and/or HIV gag antibodies in the test sample is determined by detecting the total signal generated by the complexes.
  • HIV env peptide having an immunoreactive specificity characteristic of an immunodominant region of gp36 of HIV-2 or gp41 of HIV-1.
  • the present invention unexpectedly and surprisingly found that the inclusion of a HIV-2 gp36 synthetic peptide in the assay of the present invention significantly reduced the frequency of HIV-2 false positives as compared to HIV
  • the present invention unexpectedly and surprisingly found that the inclusion of a HIV-1 gp41 synthetic peptide in the assay of the present invention increased the sensitivity of the assay to HIV subtype O in test samples as compared to HIV immunoassays employing only recombinant antigen in the indicator reagent.
  • Test kits for performing the assays of the present invention also are provided.
  • Tthe detection of HIV antigen analyte and/or HIV-1 and HIV-2 antibody analytes in a test sample of the present invention can be performed according to various homogeneous and heterogenous assay formats known in the art where the various reagent additions as described above can be perforemed
  • the assay of the present invention is
  • a "specific binding member,” as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like.
  • specific binding pairs can include members that are analogs of the original specific binding member, for example, an analyte-analog.
  • Immunoreactive specific binding members include antigens, antigen fragments; antibodies and antibody fragments, both monoclonal and polyclonal; and complexes thereof, including those formed by recombinant DNA methods.
  • Analyte is the substance to be detected which may be present in the test sample.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody, and more specifically an HIV antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • analyte also includes any antigenic substances, haptens, antibodies, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein in the capture and/or indicator reagents for the determination of vitamin B 12 , or the use of a lectin in the capture and/or indicator reagents for the determination of a carbohydrate.
  • the analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.
  • the test sample can be a mammalian biological fluid such as whole blood or whole blood components including red blood cells, white blood cells including lymphocyte or lymphocyte subset preparations, platelets, serum and plasma; ascites; saliva; stools; cerebrospinal fluid; urine; sputum; trachael aspirates and other constituents of the body which may contain or be suspected of containing the analyte(s) of interest.
  • the test sample also can be a culture fluid supernatant, or a suspension of cultured cells. Mammals whose body fluids can be assayed for HIV antigen analyte or HIV antibody analyte according to the present invention include humans and primates, as well as other mammals who are suspected of containing these analytes of interest. It also is contemplated that non-biological fluid samples can be utilized.
  • the methods of the present invention are advantageously used in solid phase heterogeneous binding assays which include both sandwich and competitive assay methods.
  • Heterogeneous binding assay techniques involve the use of a solid phase material to which a member of the binding reaction becomes bound. Prior to detecting the label which indicates the presence or amount of analyte in the test sample, the immobilized reaction component is separated from excess sample and assay reagents by removing the solid phase from the reaction mixture.
  • a capture reagent as defined below typically involves a capture binding member which has been bound to a solid phase material.
  • the specific binding member can be an
  • the capture reagent is contacted to a test sample, suspected of containing the analyte, and to an indicator reagent comprising a second specific binding member that has been labeled; for example, a labeled anti-analyte antibody or labeled antigen.
  • the reagents can be mixed simultaneously or added sequentially, either singly or in
  • a binding reaction results in the formation of a capture reagent/analyte/indicator reagent complex.
  • the assay can also comprise the step of separating the resultant complex from the excess reagents and test sample.
  • the complex retained on the solid phase material is detected by examining the solid phase for the indicator reagent. If analyte is present in the sample, then label will be present on the solid phase material. The amount of label which becomes associated with the solid phase is directly proportional to the amount of analyte in the sample.
  • the assays of the present invention can be carried out using any of the sandwich assay formats, including the forward, reverse and simultaneous techniques.
  • a forward assay involves the contact of the test sample to the capture reagent followed by an incubation period which is in turn followed by the addition of the indicator reagent.
  • a reverse assay involves the addition of the indicator reagent to the test sample followed by the addition of the capture reagent after an incubation period.
  • a simultaneous assay involves a single incubation step as the capture reagent and indicator reagent are both contacted to the test sample at the same time.
  • the capture reagent again typically involves a capture binding member which has been affixed to a solid phase material and which is contacted with both test sample and an indicator reagent.
  • the indicator reagent can be formed from an analyte or analyte-analog which has been conjugated with a label.
  • a binding reaction occurs and results in the formation of complexes of (1) immobilized capture reagent/analyte complex and (2) immobilized capture
  • the immobilized specific binding member can be an analyte or analyte-analog with which the test sample analyte competes for binding to the indicator reagent.
  • the amount of label which becomes associated with the solid phase is inversely related to the amount of analyte in the sample.
  • a positive test sample will generate a decrease in signal.
  • the presence or amount of the analyte in the test sample is usually determined by detecting the presence or amount of the label which has become associated with the solid phase, although free or unbound indicator reagent may also be detected.
  • the competitive assay the more analyte present in the test sample the lower the amount of label present on the solid phase.
  • the sandwich assay the more analyte present in the sample the greater the amount of label present on the solid phase.
  • the indicator reagents of the present invention comprise a specific binding member of each analyte conjugated to a signal generating compound (label).
  • Each indicator reagent produces a detectable signal at a level relative to the amount of the analyte in the test sample.
  • each indicator reagent while comprising a specific binding member of a different analyte, is conjugated to the same signal generating compound, which is capable of generating a detectable signal.
  • the indicator reagent is detected or measured after it is captured by the capture reagent.
  • the total signal generated by the indicator reagent(s) indicates the presence of one or more of the analytes in the test sample.
  • signal generating compounds can be utilized in the practice of the present invention.
  • different fluorescent compounds could be utilized as the signal generating compounds, one for each indicator reagent, and detection could be determined by reading at different wavelengths.
  • a short-lived chemilumine scent compound such as an acridinium or
  • phenanthrindium compound and a long-lived chemiluminescent compound such as a dioxetane can be utilized to generate signals at different times for different analytes.
  • chemiluminescent compounds which are capable of generating signals at different times are the subject matter of co-pending patent application U.S. Serial No. 636,038, which enjoys common ownership and is incorporated herein by reference.
  • Acridinium and phenanthridinium compounds are described in co-pending U. S. patent application Serial No. 07/271,763 filed June 23, 1989, which enjoys common ownership and is incorporated herein by reference.
  • the specific binding member of the indicator reagent can be a member of any specific binding pair, including either biotin or avidin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor or an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to the analyte as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • an antibody can be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a recombinant antibody, a mixture thereof, or a mixture of an antibody and other specific binding members.
  • the details of the preparation of such antibodies and their suitability for use as specific binding members are well known to those in the art.
  • the HIV antigen of the indicator reagent is a synthetically- or recombinantly-produced antigen capable of binding to an immunodominant region of the core and env proteins of HIV- 1 and/or the env region of HIV-2.
  • the antigen used in the HIV-2 indicator reagent is a synthetically produced cyclic peptide corresponding to an immunodominant region of gp36 of HIV-2 and is characterized by its ability to immunoreact with antibodies induced by HIV-2.
  • the synthetic peptide is from between about 10 amino acids to about 50 amino acids in length and includes the amino acid residue sequence Cys-Ala-Phe-Arg-Gln-Val-Cys(-CAFRQVC-) in which the two cysteines have been oxidized to their disulfide cyclic form.
  • Preferred synthetic peptides include, but are not limited to:
  • the antigen used in the HIV-1 indicator reagent is a synthetically produced cyclic peptide corresponding to an immunodominant region of gp41 and is characterized by its ability to
  • the synthetic peptide is from between about 10 amino acids to about 50 amino acids in length and includes the amino acid residue sequence -Cys-Ser-Gly-Lys-Leu-Ile-Cys-(- CSGKLIC-) wherein the cysteine residues are oxidized to their disulfide cyclic form.
  • Preferred HlV-1 synthetic peptides include, but are not limited to:
  • HIV-1 peptides include but are not limited to:
  • a peptide of the present invention need not be identical to the specific amino acid sequences presented above so long as the peptide contains the sequence CAFRQVC or CSGKLIC or homologs in their oxidized form thereof and are able to bind with antibodies induced by genotypes of HIV.
  • the synthetic peptide of the present invention can be subject to various changes, such as insertions and deletions, and substitutions of one amino acid for another, either conservative or nonconservative in nature.
  • the peptides of this invention are prepared using a variety of known methods. Conventional solid phase synthesis is most preferably employed as is described in B. Erickson, et al. Solid-phase peptide synthesis, The Proteins, Vol II, 3rd ed. Academic Press, New York, New York (1976) and E. Atherton, et al. Solid Phase Peptide Synthesis : A Practical Approach. IRL Press, Oxford, (1989). However, other well known methods of peptide synthesis may also be used.
  • the resin support is any suitable resin conventionally employed in the art for solid phase preparation of peptides, preferably p-methylbenzyloxyalcohol polystyrene and p-methylbenzydrlamine resin.
  • the amino protecting group is removed by standard methods conventionally employed in the art of solid phase peptide synthesis. After removal of the amino protecting group, remaining a- amino protected and, if necessary, side chain protected amino acids are coupled, sequentially, in the desired order to obtain the product. If required, cyclization between two cysteines is accomplished by diluting the crude peptide into an oxidizing environment that promotes disulfide bond formation. The cyclized peptide is then purified and lyophilized for storage.
  • Cyclic peptides of this invention are prepared by the direct oxidative conversion of protected or unprotected SH-groups to a disulfide bond following techniques generally known in the art of peptide synthesis.
  • the preferred method involves the direct oxidation of free SH-groups with
  • suitable coupling reagents are N,N'-diisopropylcarbodiimide or N,N'-dicyclohexylcarboiimide (DCC) either alone or preferably in the presence of 1-hydroxybenzotriazole.
  • DCC N,N'-dicyclohexylcarboiimide
  • Another useful coupling procedure makes use of performed symmetrical anhydrides of protected amino acids.
  • the label of the indicator reagent is capable of generating a measurable signal detectable by external means.
  • the labels contemplated include, but are not intended to be limited to chromogens; catalysts such as enzymes for example, horseradish peroxidase, alkaline phosphatase, and B-galactosidase; luminescent compounds such as fluorescein and rhodamine;
  • chemiluminescent compounds such as acridinium compounds
  • phenanthridinium compounds and dioxetane compounds phenanthridinium compounds and dioxetane compounds; radioactive elements; and direct visual labels.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • a variety of different indicator reagents can be formed by varying either the label or the specific binding member.
  • Peptides I - XIII of the instant invention are labeled with horseradish peroxidase (HRPO) using conventional Schiff base chemistry as described by Nakane, P., et al., J. Histochem Cytochem. 22: 1084-1091 (1974).
  • HRPO horseradish peroxidase
  • aldehyde groups on HRPO are generated by oxidation of the carboydrate residues with sodium metaperiodate. These reactive aldehyde groups are allowed to interact with amino groups on the peptide, which are preferably at the amino terminus or the the e-amino group of lysine.
  • the Schiffs base that forms is subsequently stabilized upon reduction with sodium borohydride and the resulting conjugate is stored until use.
  • the capture reagents of the present invention comprise a specific binding member for each of the analytes of interest which are attached to at least one solid phase and which are unlabeled.
  • the capture reagent is specific for the analyte as in a sandwich assay, it can be specific for indicator reagent or analyte in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay.
  • the capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample. This attachment can be achieved, for example, by coating the specific binding member onto the solid phases by absorption or covalent coupling.
  • Coating methods and other known means of attachment, are known to those in the art.
  • the specific binding member of the capture reagent can be any molecule capable of specifically binding with another molecule.
  • the specific binding member of the capture reagent can be an immunoreactive compound such as an antibody, antigen, or antibody/antigen complex. If an antibody is used, it can be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a recombinant antibody, a mixture thereof, or a mixture of an antibody and other specific binding members.
  • the "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads and microparticles, membranes, plastic tubes, walls of wells of reaction trays, glass or silicon chips and tanned sheep red blood cells are all suitable
  • Suitable methods for immobilizing capture reagents on solid phases include ionic, hydrophobic, covalent interactions, and the like.
  • 60-well polystyrene reaction trays and 1/4 inch polystyrene beads are utilized, while in another example, a 96-well reaction tray is the only solid phase utilized. It is contemplated that all solid phases be present during the quantitation of signal, thus eliminating the need to separate solid phases for detection of signal.
  • a “solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, and other configurations known to those of ordinary skill in the art.
  • the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens.
  • Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well.
  • Such useful solid supports include but are not intended to be limited to natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic
  • inert carriers such as paper, glass, plastic films, or fabrics.
  • the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents including monoclonal antibodies.
  • Nylon also possesses similar characteristics and also is suitable.
  • porous solid supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm.
  • the pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.
  • the surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support. The irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
  • Preferred solid phase materials for flow-through assay devices include filter paper such as a porous fiberglass material or other fiber matrix
  • the thickness of such material is not critical and will be a matter of choice, largely based upon the properties of the sample or analyte being assayed, such as the fluidity of the test sample.
  • a charged substance can be coated directly to the material or onto microparticles which then are retained by a solid phase support material.
  • microparticles can serve as the solid phase, by being retained in a column or being suspended in the mixture of soluble reagents and test sample, or the particles themselves can be retained and immobilized by a solid phase support material.
  • retained and immobilized is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within the support material.
  • the particles can be selected by one skilled in the art from any suitable type of particulate material and include those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials.
  • the size of the particles is not critical, although it is preferred that the average diameter of the particles be smaller than the average pore size of the support material being used.
  • embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U. S.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi- automated systems wherein the solid phase comprises a microparticle.
  • Such systems include those described in pending U. S. Patent Application 425,651 and U. S. Patent No. 5,089,424, which correspond to published EPO applications Nos. EP 0 425 633 and EP 0424 634, respectively, and U.S. Patent No. 5,006,309 all of which enjoy common ownership and are incorporated herein by
  • a test sample suspected of containing any of the HIV antigen analyte or HIV antibody analytes of interest is simultaneously contacted with a solid phase to which a first specific binding member of a first analyte is attached, and a solid phase to which a first specific binding member of a second analyte has been attached, thereby forming a mixture.
  • the specific binding members serve as capture reagents to bind the analyte(s) to the solid phases. If the specific binding member is an immunoreactant, it can be an antibody, antigen, or complex thereof, specific for each analyte of interest.
  • the specific binding member is an antibody
  • it can be a monoclonal or polyclonal antibody, an antibody fragment, a recombinant antibody, as well as a mixture thereof, or a mixture of an antibody and other specific binding members.
  • This mixture is incubated for a time and under conditions sufficient for a binding reaction to occur and which incubation results in the formation of capture reagent/first analyte complexes of the first analyte if it is present in the test sample, and/or the formation of capture reagent/second analyte complexes of the second analyte if it is present in the test sample.
  • an indicator reagent for each analyte is contacted with the complexes.
  • the indicator reagent for the first analyte comprises a specific binding member of the first analyte of interest which has been labeled with a signal generating compound.
  • the indicator reagent for the second analyte comprises a specific binding member of the second analyte of interest which has been labeled with the same signal generating compound as the indicator reagent for the first analyte, thereby forming a second mixture.
  • This second mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator reagent complexes and/or capture
  • reagent/second analyte/indicator reagent complexes The presence of either analyte is determined by detecting the signal generated in connection with the complexes formed on the solid phase as an indication of the presence of one or more analytes in the test sample. If the indicator employs an enzyme as the signal generating compound (label), then the signal can be detected visually or measured spectrophotometrically. Or, the label can be detected by the measurement of fluorescence, chemiluminescence, radioactive energy emission, etc., depending on the label used to generate the signal.
  • the capture reagents can be attached to the same solid phase, or can be attached to different solid phases. It is contemplated that all capture reagents could be attached to the same solid phase, or that each capture reagent could be attached to a separate solid phase, or that combination of capture reagents could be attached to separate solid phases. For example, if microparticles were the solid phase of choice, then separate microparticles could have at least one capture reagent(s) attached to it. A mixture of microparticles (solid phases) could be used to capture the various analytes which may be present in the test sample by using the mixture of microparticles. It is contemplated that different ratios of capture reagents attached to solid phases could be utilized in such an assay, to optimize analyte(s) detection.
  • the specific binding member used as a capture reagent for the HIV-1 antibody analyte be HIV-1 gp41 antigen
  • the specific binding member used as the capture reagent for the HIV-1 antigen analyte be anti-HIV-1 gp24 antibody.
  • the HIV-1 gp41 used as capture reagent be a recombinantly prepared antigen (protein).
  • the specific binding member for the antibody analyte indicator reagent is a HIV-1 gp41
  • HIV-1 p41 antigen be synthetically produced, and that the enzyme be horseradish peroxidase (HRPO).
  • HRPO horseradish peroxidase
  • a test sample suspected of containing any of the analytes of interest is simultaneously contacted with a first solid phase to which a first specific binding member of a first analyte and a first specific binding member of a second analyte have been attached, an indicator reagent for the first analyte comprising a specific binding member for the first analyte labeled with a signal generating compound and an indicator reagent for the second analyte comprising a specific binding member for the second analyte labeled with a signal generating compound, to form a mixture.
  • the specific binding members serve as capture reagents to bind the analyte(s) to the solid phases.
  • the specific binding member is an immunoreactant, it can be an antibody, antigen, or complex thereof, specific for each analyte of interest. If the specific binding member is an antibody, it can be a monoclonal or polyclonal antibody, an antibody fragment, a recombinant antibody, as well as a mixture thereof, or a mixture of an antibody and other specific binding members.
  • the indicator reagents comprise specific binding members of the first and second analytes of interest which have been labeled with a signal generating compound.
  • This mixture is incubated for a time and under conditions sufficient for a binding reaction to occur and which incubation results in the formation of capture reagent/first analyte/indicator reagent complexes of the first analyte and/or capture reagent/second analyte/indicator reagent complexes of the second analyte, if either or both the first or second analyte are present in the test sample.
  • the presence of either analyte is determined by detecting the signal generated in connection with the complexes formed on either or both solid phases as an indication of the presence of the first analyte and/or the second analyte in the test sample.
  • the indicator employs an enzyme as the signal generating compound (label)
  • the signal can be detected visually or measured spectrophotometrically.
  • the label can be detected by the measurement of fluorescence, chemiluminescence, radioactive energy emission, etc., depending on the label used.
  • the assay can include the use of a hapten-anti-hapten system, in which case the indicator reagent can further comprise a hapten such as biotin.
  • the use of a biotin/anti-biotin system for assays is the subject matter of cop-pending U.S. Patent Application Serial No. 687,785 which corresponds to published European Patent Application No. 0160900 (published November 13, 1985), which enjoys common ownership and is incorporated herein by reference.
  • the specific binding member used as a capture reagent for the HIV-1 antibody analyte be HIV-1 gp41 antigen, and that the specific binding member used as the capture reagent for the HIV-1 antigen analyte be anti-HIV-1 p24 antibody. It is most preferred that the HIV-1 gp41 used be a recombinantly prepared antigen
  • the specific binding member for the antibody analyte indicator reagent is HIV-1 p41 antigen, labeled with an enzyme. It is most preferred this HIV-1 p41 antigen be recombinantly or synthetically produced, and that the enzyme be horseradish peroxidase (HRPO). It is most prefered that this HIV-1 gp41 antigen be snthetically produced.
  • Solid phases preferred include a magnetic or non-magnetic bead, a well of a reaction tray, and microparticles, either alone or in any combination.
  • Positive and negative controls can be included in the assay of the present invention to ensure reliable results.
  • a blank solid phase(s), to which no capture reagent has been attached, can be utilized as the negative reagent control.
  • Positive controls can include a positive control for each analyte which control is tested separately, and a combined positive control wherein the presence of all analytes to be detected in the assay are determined.
  • recombinantly-prepared antigens be used as capture reagents on the solid phase and that synthetic HIV peptides conjugated to exnzme be used as indicator reagents in the assay.
  • the present invention is not limited to the combination of recombinant proteins reagents and synthetic peptide reagents as described above, but that other combinations are contemplated.
  • one or more or all of the capture reagent proteins can be synthetically produced as can the protein of the labeled reagents.
  • Recombinant HIV-1 and HIV-2 may labeled with ezyme may also be used in combination with synthetic peptides as indicator reagents.
  • viral lysates or isolates of the specific analyte can be employed, provided that a labeled HIV-2 synthetic peptide reagent of the present invention is employed.
  • rDNA recombinant DNA
  • the present invention utilizes recombinantly-produced HIV envelope proteins as assay reagents.
  • the cloning of the HIV genome and expression of HIV envelope and core protein in E. coli, the purification and characterization of gp41 and p24, and various assay formats which utilize these recombinant proteins are described in U.S. Patent Application Serial No. 07/020,282 filed February 27, 1987 and previously incorporated herein by reference, from which this present invention claims priority. Briefly, HIV-infected HT-9 cells were harvested and total cellular DNA was isolated an subjected to digestion. The DNA segments encoding for the core protein and for the envelope glycoprotein were further subcloned into bacterial expression vectors using well-known recombinant technology.
  • U.S.Patent Application Serial No. 07/020,282 also teaches that, in the detection of HIV-1 antibody, the use of recombinant antigens as the capture reagent and the indicator reagent allows for the detection of anti-HIV-1 antibodies of different immunoglobulin classes. These immunoglobulin classes include IgG, IgA, IgE and IgM. The detection of anti- HIV-1 IgG, IgM and IgA using the Abbott HIVAB® HIV-1/HIV-2 (rDNA) EIA assay has been described in an abstract by J. L. Gallarda et al., 5th Annual Forum on AIDS. Hepatitis and Other Blood-Borne Diseases. Atlanta, Georgia, March 29-April 1, 1992.
  • recombinant antigens produced in heterologous sources or synthetic HIV peptides can be utilized in the assay and will contribute an even greater lessening of false positive results. For example, if an E. coli prepared
  • recombinant antigen such as gp41 is used as the capture reagent, then a recombinant antigen gp41 produced in any suitable source different than E. coli, such as in a suitable yeast host or other suitable host such as B.
  • HIV peptides of varying length, as described above, can be used.
  • the present invention also utilizes antibodies which specifically bind to HIV antigen analytes.
  • anti-HIV p24 antibody is used.
  • a mixture of monoclonal antibodies, both specific for HIV p24 antigen is used. In this mixture, one monoclonal antibody which specifically binds to an epitope on HIV-1 p24 to which epitope human anti-HIV-1 p24 IgG does not competitively bind is used with another monoclonal antibody which specifically binds to a different epitope of HIV-1 p24 to which different epitope human anti-HIV-1 p24 IgG does competitively bind.
  • the monoclonal antibody which does not competitively bind human anti-HIV-1 p24 IgG also specifically binds to HIV-2 p24 antigen.
  • These monoclonal antibodies and their use in HIV antigen assays are the subject matter of co-pending U. S. Patent Application Serial No. 07/204,798, which enjoys common ownership and is incorporated herein by reference.
  • These monoclonal antibodies are designated as 31-42-19 and 31-90-25.
  • Hybridoma cell line 31-42-19 producing monoclonal antibody 31-42-19 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville,
  • Hybridoma cell line 31-90-25 producing monoclonal antibody 31-90-25 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 on May 26, 1988 and has been accorded ATCC Deposit No. HB 9725.
  • the use of these monoclonal antibodies as antibody fragments in HIV antigen assays also has been described in U.S. Patent No.
  • HIV-2 p41 would be attached to a solid support as the HIV-2 antigen capture reagent, in addition to the previously-described HIV-1 p41 antigen capture reagent and HIV-1 antibody capture reagents.
  • the solid support can be the same solid support to which all other capture reagents are attached, it can be the same solid support to which HIV-1 recombinant antigens have been attached, or it can be attached to a solid phase to which no other capture reagent (except for HIV-2 p41) has been attached.
  • the assay procedure would be the same as described hereinabove for the various embodiments of the invention.
  • the HIV-2 antibody analyte indicator reagent would comprise recombinant and/or synthetically-prepared HIV-2 gp36 antigen attached to a detectable label.
  • the recombinantly prepared HIV-2 p41 is utilized.
  • the sequence for the HIV-2 virus (including p41 antigen) is described in EP 0 347,365, published December 20, 1989 to Diagen Corp, which is incorporated herein by reference.
  • a most preferred HIV-2 recombinant antigen encodes the first 104 amino acids of the HIV-2 p41 antigen.
  • the resulting plasmid designated as pJC104 expresses the HIV-2 env protein as a fusion with CKS protein. This plasmid encodes a recombinant protein
  • HIV-2 peptides of varying lengths, as described above, conjugated to enzyme are used as indicator reagents.
  • HIV-1 peptide I conjugated to HRPO is the indicator reagent.
  • This procedure utilized 1/4 inch polystyrene beads (available from Abbott Laboratories, Abbott Park, IL 60064) and a 60-well polystyrene reaction tray (available from Abbott Laboratories, Abbott Park, IL 60064).
  • Two different anti- HIV-1 p24 monoclonal antibodies were coated on the beads, as follows. The beads were coated at a concentration of 8 ⁇ g/ml (approximately 1.6 ⁇ g/ml/bead) in a 0.25 M sodium citrate buffer (pH 7.2) for two hours at 45°C. The beads then were washed in the 0.25 M sodium citrate buffer (pH 7.2), and then they were reacted with a detergent solution containing 0.1% Triton X-100 ®
  • Hybridoma cell line 31-42-19 producing monoclonal antibody 31-42-19 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville,
  • Hybridoma cell line 31-90-25 producing monoclonal antibody 31-90-25 was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 on May 26, 1988 and has been accorded ATCC
  • the wells of the 60-well reaction tray were coated with HIV antigen, as follows.
  • the recombinant protein HIV-1 p41 env protein designated as pTB319 was added to each well at a concentration of 1 ⁇ g/ml in 0.1 M 3-
  • pTB319 plasmid producing recombinant protein pTB319 is the subject matter of a patent application to Boiling and Mandecki, "CKS Method of Protein Synthesis," U.S.Patent Application Serial No. 167,067, filed March 11, 1988, previously incorporated herein by reference.
  • pTB319 was produced by inserting a synthetically-produced DNA fragment which encoded the carboxy terminal 42 amino acids of HIV-1 pl20 into the plasmid pTB315, as described in Boiling and Mandecki, Ibid.
  • Example 2 Simultaneous Assay for HIV antigen and HIV antibody
  • the two solid phases prepared as described in Example 1 were used in an assay for detection of HIV antigen and/or HIV antibody in a test sample, as follows.
  • An HIV-1 seroconversion panel which contained 65 serum samples derived from nine HIV-1 infected individuals undergoing seroconversion, was used in the assay.
  • Each serum sample was diluted in a separate well of the 60- well tray previously prepared in Example 1 by adding 150 ⁇ l of the serum sample to 50 ⁇ l of specimen diluent, which contained 2% Tween 20®
  • Example 2 polyoxyethylenesorbitan, available from Sigma Chemical Co., St. Louis, MO. Then, a bead previously coated with anti-HIV p24 antibodies as described in Example 1 was placed in each well containing a serum sample. The wells of each tray were incubated for 60 minutes at 40°C under continuous rotation. Following incubation, each well of the 60-well reaction tray was washed with 15 ml of deionized water (dH 2 O) in the Abbott Parallel Processing CenterTM (PPC, available from Abbott Laboratories, Abbott Park, IL).
  • dH 2 O deionized water
  • an HIV p24 antibody probe reagent (rabbit polyclonal F[ab']2 anti-HIV-1 [active ingredient: anti-p24 antibody at a concentration of 2 to 6 ⁇ g/ml] in an antibody diluent (2.25% BSA, 7.5% calf serum, 7.5% goat serum, 25% human recalcified plasma, 0.1% sodium azide) was added to each well/bead and then the resulting mixture was incubated for 60 minutes at 40°C without rotation. Each bead/well in the reaction tray was washed with 15 ml of dH2 ⁇ .
  • conjugate diluent 0.18% Tris, 1.19 % Tris-HCl, 0.38% NaCl, 9.0% calf serum, 0.9% goat serum, 10.0% human calcified plasma, 4.5% Triton X- 100 ® , 0.013% gentamicin sulfate, 0.009% thimerosal
  • conjugate diluent 0.18% Tris, 1.19 % Tris-HCl, 0.38% NaCl, 9.0% calf serum, 0.9% goat serum, 10.0% human calcified plasma, 4.5% Triton X- 100 ® , 0.013% gentamicin sulfate, 0.009% thimerosal
  • conjugate diluent 0.18% Tris, 1.19 % Tris-HCl, 0.38% NaCl, 9.0% calf serum, 0.9% goat serum, 10.0% human calcified plasma, 4.5% Triton X- 100 ® , 0.013% gentamicin sulfate, 0.009%
  • Each bead/well of the 60-well reaction tray was washed with 15 ml of dH 2 O as previously described. Then, 300 ⁇ l of o- phenylenediamine-2HCl (OPD) was added to each well/bead and then was incubated for 30 minutes at room temperature in the dark. The reaction then was stopped by adding 300 ⁇ l of a stopping reagent (1 N H 2 SO 4 ) to each well/bead. The reaction was read using the Abbott PPC which measured the optical density of the reaction at 492 nm using a 630 nm reference. The cutoff value was established as 0.1 OD + mean OD of the negative control. Thus, serum samples were considered reactive (positive) if the sample to cutoff value was greater than 1.
  • OPD o- phenylenediamine-2HCl
  • HIV antigen assay HIV antigen assay
  • HIV antibody assay Human Immunodeficiency Virus Types 1 and 2: E. coli and B. megaterium. recombinant antigen, Abbott HIVAB ® HIV-1/ ⁇ IV-2 (rDNA) ELA; available from Abbott Laboratories, Abbott Park, IL
  • the data from Table 1 indicates that the method of the present invention had greater sensitivity than either the HIV- 1/2 Ab or the HIV-1 Ag test when the results from the three individual tests were compared separately to each other. It is expected that the assay can be optimized even further to detect both HIV p24 antigen which is present early in the course of infection and also in the final stages of HIV infections, as well as HIV antibodies which appear later in infection at the time of seroconversion.
  • blocking reagent comprising 5% non-fat dry milk, 10 mM Tris [pH 8.0] 150 mM NaCl and 0.05% Tween-20 ® ) for one hour at room temperature.
  • the solid phase prepared as described in Example 3 was used in an assay to detect the presence of HIV antigen and/or HIV antibody in a test sample, as follows.
  • Each serum sample of a 12-member seroconversion panel (Panel G available from Boston Biomedica Inc., Boston MA) as well as positive and negative controls were tested.
  • 150 ⁇ l of each serum sample or positive or negative control was diluted in a separate well of the microtiter plate with 50 ⁇ l of specimen diluent (containing 15 ⁇ l of Triton X-100® and 35 ⁇ l of blocking reagent, as described in Example 3) and incubated for 60 minutes at room temperature without rotation.
  • washing buffer 0.05% non-fat dry milk, 10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween 20®
  • HIV p24 antibody probe reagent goat polyclonal F[ab']2 anti-HIV-1 [active ingredient: anti-p24 antibody at a concentration of 2 to 6 ⁇ g/ml) in an antibody diluent (2.25% BSA, 7.5% calf serum, 7.5% goat serum, 25% human recalcified plasma, 0.1% sodium azide) were added to each well and incubated for 60 minutes at room temperature without rotation. After incubation the wells were washed with eight cycles of wash buffer as previosuly described.
  • conjugate diluent (as described in Example 2) which contained a mixture of recombinant HIV-1 p41 protein (pTB 319) labelled with HRPO and HRPO-labelled goat anti-rabbit IgG (previously described in Example 2) were added to each well and incubated for 60 minutes at room temperature without rotation. The wells were washed with eight cycles of wash buffer (described previously herein) and then rinsed with dH 2 O. Then, 125 ⁇ l of OPD substrate was added to each well and the wells were incubated at room temperature for 10 minutes in the dark. The reaction was stopped by adding 125 ⁇ l of stopping reagent (previously described in Example 2).
  • the assay of the invention was capable of detecting the presence of HIV antibody and/or HIV antigen in the
  • HIV Antibody and HIV Antigen Capture Reagents Both monoclonal anti-HIV p24 antibodies previously described (31-42-19 and 31-90-25) and the recombinant HIV-1 p41 antigens previously described (HIV-1 p41 recombinant protein pTB319, and HIV-2 p41 recombinant protein pJC104) are together and simultaneously coated onto a uniform 0.5%
  • microparticles After blocking the microparticles with 10 mg/ml casein in 0.01 M Tris (pH 8.0), 0.15 M NaCl at 56°C for 18-24 hours, the microparticles again are washed as described herein and diluted to 0.015% suspension (wt./volume) in 0.05 M Tris (pH 8.0), 0.15 M NaCl, 1% BSA, 15% sucrose and 0.1% sodium azide.
  • the Abbott IM x ® Microparticle Enzyme Immunoassay (MELA) system is used, although any system which employs microparticles can be used.
  • the Abbott IM x ® MELA system is thoroughly described in the Abbott LM x ®
  • Example 5 100 ⁇ l of the 0.15% suspension prepared in Example 5 is mixed together with 100 ⁇ l of test sample suspected of containing HIV-1 and/or HIV-2 antibody and/or HIV-1 antigen, and incubated at 40°C for ten minutes in an Abbott LM x ® reaction cell to form a reaction mixture. HIV antibodies and/or HIV antigens bind to the microparticles in an antibody/antigen/microparticle complex. 150 ⁇ l of the reaction mixture is transferred onto a glass fiber matrix to which the
  • microparticles are retained in an irreversible binding.
  • the antibody/ antigen/microparticle complexes then are reacted with 50 ⁇ l of a probe consisting of biotinylated recombinant HIV-1 and HIV-2 recombinant p41 antigens (pTB319 and pJC104, previously described) and biotinylated F(Ab')2 anti-HIV-1 p24 in 0.05M Tris 9pH8.0), 2% BSA, 0.25% saponin and 0.1% sodium azide at 40°C for ten minutes.
  • an antibody conjugate consisting of goat anti-biotin alkaline phosphatase in 0.1 M Tris (pH 8.0), 0.5M NaCl, 0.9% Brij- 35®, 1.0% BSA and 0.1% sodium azide then is allowed to react with the biotin probe/antibody/antigen/microparticle complexes for ten minutes at 40°C.
  • microparticle complexes are washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCl and 0.1 % sodium azide, the biotin probe/antibody/antigen microparticle complex is reacted with 50 ⁇ l of the substrate methylumbelliferyl phosphase (MUP, Abbott Laboratories, Abbott Park, IL), and the fluorescence of the product , methylumbelliferon, (MU) is measured.
  • the rate of MU methylumbelliferyl phosphase
  • HIV Antibody and HIV Antigen Capture Reagents are coated separately onto polystyrene microparticles (available from Seradyne Inc.,
  • each of the reagents may be coated separately from each other or in various combinations with each other. After each of the analyte capture reagents is coated on their respective microparticles, the various coated microparticles are pooled together and used in the assay.
  • the microparticles again are washed as described in Example 5, pooled together, and diluted to a 0.015% suspension (wt/volume) in 0.05 M Tris 9pH 8.0), 0.15 M NaCl, 1% BSA and 15% sucrose.
  • the microparticles may be pooled at various ratios to affect sensitivity and specificity of the assay in order to optimize their use.
  • the Abbott IM x ® Microparticle Enzyme Immunoassay (MELA) system is used, although any system which employs microparticles can be used.
  • the Abbott IM x ® MELA system is thoroughly described in the Abbott LM x ®
  • Example 5 100 ⁇ l of the 0.15% suspension prepared in Example 5 is mixed together with 100 ⁇ l of test sample suspected of containing HIV-1 and/or
  • HIV-2 antibody and/or HIV-1 antigen incubated at 40°C for ten minutes in an Abbott LM x ® reaction cell to form a reaction mixture. HIV antibodies and/or HIV antigens bind to the microparticles in an Abbott LM x ® reaction cell to form a reaction mixture. HIV antibodies and/or HIV antigens bind to the microparticles in an Abbott LM x ® reaction cell to form a reaction mixture. HIV antibodies and/or HIV antigens bind to the microparticles in an
  • antibody/antigen/microparticle complex 150 ⁇ l of the reaction mixture is transferred onto a glass fiber matrix to which the microparticles are retained in an irreversible binding.
  • the antibody/antigen/microparticle complexes then are reacted with 50 ⁇ l of a probe consisting of biotinylated recombinant HIV-1 and HIV-2 recombinant p41 antigens (pTB319 and pJC104, previously
  • 50 ⁇ l of an antibody conjugate consisting of goat anti-biotin alkaline phosphatase in 0.1 M Tris (pH 8.0), 0.5M NaCl, 0.9% Brij-35®, 1.0% BSA and 0.1% sodium azide then is allowed to react with the biotin probe/antibody/antigen/microparticle complexes for ten minutes at 40°C.
  • microparticle complexes are washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCl and 0.1 % sodium azide, the biotin probe/antibody/antigen microparticle complex is reacted with 50 ⁇ l of the substrate methylumbelliferyl phosphase (MUP, Abbott Laboratories, Abbott Park, IL), and the fluorescence of the product , methylumbelliferon, (MU) is measured.
  • the rate of MU production is proportional to the concentration of analyte(s) in the test sample.
  • HIV-1 genomic library was prepared by ligating a partial EcoRI digestion of genomic DNA derived from HIV-1 infected HT-9 cells (obtained from Dr. Robert Gallo, National Cancer Institute, Laboratories of Tumor Cell Biology, Lot No. P3-21) with bacteriophage lambda Charon 4A EcoRI arms and transfecting into E. coli C600.
  • the library was screened by hybridization with cDNA made from HIV-1 viral RNA, and a single phage (designated Phage 4B) was obtained containing the entire HIV-1 genome.
  • Phage 4B DNA was digested with Kpnl and ligated into the Kpnl site of pUC18 (Bethesda Research Laboratories). A clone (designated pcK2) containing the entire p41 region of the HIV-1 env gene was identified and mapped.
  • Phage 4B DNA was digested with EcoRI and ligated into the EcoRI site of pBR322.
  • a clone (designated pcR23) containing the entire HIV-1 gag gene was identified and mapped.
  • a DNA fragment containing the/env gene from HIV-2 prophage isolate D1945 was identified within a lambda genomic library of prophage DNA. This fragment was subcloned into an EcoRI site of an E. coli expression vector (lambda PL vector pKH20). The resulting plasmid was named pEHa.
  • the construction of the envelope expression vector was a two step process.
  • the first step involved the construction of an E. coli plasmid
  • the second step involved the construction of an expression vector with the ability to survive in both Escherichia Sp. and Bacillus sp., and the introduction of the env fragment into this plasmid (designated pOM10).
  • An 854 base pair (bp) BglII/BamHI DNA fragment obtained from plasmid pcK2 was ligated into the BamHI site of pUC9 (Pharmacia).
  • a clone containing a part of the env gene in the same orientation as the lacZ gene was identified, mapped, and designated p41A.
  • a 557 base pair bp BamHI DNA fragment obtained from plasmid pcK2 was ligated into the BamHI site of plasmid p41A.
  • a plasmid containing the complete p41 sequence of the env gene in the same orientation as the lacZ gene was identified, mapped, and
  • plasmid pE194 which had previously been restricted with XbaI ⁇ md blunt ends were formed using E. coli DNA polymerase 1 (Klenow fragment) to fill in the "sticky" DNA ends (blunt-end treatment).
  • a plasmid (designated pAS5) was isolated, mapped and shown to have the ability to survive in both E. coli and B. subtilis. The env gene was then inserted into pAS5.
  • a DNA fragment from the plasmid p41C containing the env gene was generated via EcoRI/Sall digestion and subsequent blunt-end treatment. This DNA fragment was ligated to plasmid pAS5 which had been linearized with Sail and blunt-end treated.
  • One isolated clone (designated pAS14) was determined to have the env gene fused to the scoVG promoter in the proper orientation.
  • erythromycin resistance gene in pAS14 was replaced by the chloramphenicol resistance gene from a related Bacillus plasmid pC194 as follows.
  • a 1107 bp DNA fragment containing the chloramphenicol acetyl transferase (CAT) gene from a Clal/Dral digest of the plasmid pC194 was isolated. This DNA fragment was ligated to the 6407 bp DNA fragment isolated from a Clal/Smal digestion of pAS14 (a treatment which removes all of the original erythromycin resistance gene).
  • the final plasmid obtained was designated pOM10.
  • the promoter region, transcriptional start, and ribosomal binding site span bases 4840-4971.
  • the coding region (bases 4972-6183) consists of sequences derived from the spoVG region of the parent plasmid pVGl (bases 4972-5004), sequences derived during DNA ligations (bases 5005-5010) and sequences derived from the HIV-1 env gene gpl20 (bases 5011-5145) (Ratner, L., et. al. Nature 313:277-284, 1985).
  • the p41 sequences are from bases 5146-6180.
  • the translation is terminated at the native termination codon of the env gene (bases 6181-6183).
  • the DNA sequence coding for the recombinant protein was confirmed by sequencing of the plasmid harvested after fermentation for three lots. The sequenced lots were fermented from the same cell bank used to produce antigen for clinical master lots.
  • Plasmid pOM10 expresses the HIV-1 envelope protein as a fusion protein containing 11 amino acids derived from the amino-terminus of the spoVG protein, 2 amino acids derived as a result of DNA manipulations during ligations, followed by the final 45 amino acids from the p120 envelope protein and the entire p41 protein sequence. This protein is referred to as recombinant p41 (rp41). Preparation of recombinant HIV-1 gp41 fusion protein
  • This recombinant E. coli clone expressing the HIV-1 CKS- 120/41 fusion antigen was carried out in several steps. First the gene for the gp41 portion of the HIV-1 antigen was synthesized and inserted into a modified pUC18 giving the plasmid pTB315. Next, the DNA sequence coding for the carboxyl 42 amino acids of the gpl20 protein was synthesized and inserted into pTB315 resulting in plasmid pTB316. Finally, the gp120/41 gene was transferred to an expression plasmid (pTB210) which allowed efficient expression of the antigen as a fusion protein. The resulting plasmid, pTB319, was isolated and mapped.
  • a gene encoding the amino acids 519-673, and 712-863 of the HIV-1 gp160 envelope protein was designed to be constructed from a series of synthetic DNA fragments in a pUC18 plasmid derivative.
  • a 129 base pair double stranded DNA fragment representing the carboxy-terminus of gp120 was synthesized (311.3 and 311.4) and inserted into the remaining Narl site of pTB315. This fragment was inserted into plasmid pTB315 which was digested with Narl. A plasmid designated pTB316 was isolated and screened such that the orientation of the inserted fragment was in the same orientation as the gp41 gene.
  • This plasmid derived from plasmid pBR322, contains a modified lac promoter fused to a kdsB gene fragment (encoding the first 239 of the entire 248 amino acids of the E. coli CMP-KDO Synthetase or CKS protein), and a synthetic linker fused to the end of the kdsB gene fragment.
  • the synthetic linker includes multiple restriction sites for insertion of genes, translational stop signals and the trPA rho-independent trans criptional terminator.
  • This plasmid encodes 239 amino acids of CKS and 22 amino acids coded for by the synthetic linker.
  • Plasmid pTB316 was digested with BamHI and KpnI and a 1073 bp fragment was isolated. This fragment consisted of the original synthetic gp41 gene with the carboxyl 42 amino acids of the gpl20 gene inserted in the proper location. This fragment was inserted into pTB210 which was previously digested with Bglll and KpnI. The resulting plasmid, designated pTB319, was isolated and mapped.
  • the promoter region, transcriptional start, and ribosomal binding site span bases 45-125.
  • the coding region is comprised of sequences derived from the 239 amino acids of the CKS protein (bases 126-842) and the 11 amino acids from the synthetic polylinker (bases 843-875). This is followed by 42 residues of the p120 HIV-1 env (bases 876-1001) and 185 residues of the HIV-1 p41 env (bases 1002-1556).
  • the 38 amino acid deletion of the transmembrane region is between base pairs 1466 and 1467.
  • the construction of the p24 gag expression vector was a multi-step process.
  • the first step involved the construction of an E. coli plasmid, pB1, with a smaller gag containing DNA fragment.
  • the second step involved the construction of an expression vector, designated pKRR951, with the proper molecular signals to allow efficient expression.
  • a 949 bp PvuII/Bglll DNA fragment obtained from plasmid pcR23 was ligated into the plasmid pUC9 (Pharmacia) previously digested with Hindll and BamHI.
  • a clone containing a part of the gag gene (including the p24 coding region) in the same orientation as the lacZ gene was identified, mapped, and designated pB1.
  • gag gene DNA fragment was then introduced into an expression vector pKRR810 which placed the gag gene expression under the control of the
  • E. coli la mbda phage P L promoter while allowing efficient termination of protein synthesis.
  • a 963 bp DNA fragment containing most of the gag gene was obtained by an EcoRI (complete)/Pstl (partial) digestion of plasmid pB1.
  • a synthetic oligonucleotide DNA fragment of 36 bp was added to the gag gene fragment to reconstruct the amino-terminus of the encoded protein and to place an EcoRI site immediately upstream of the initiation codon. This modified fragment was inserted into the EcoRI site of the expression vector pKRR810.
  • a clone (pKRR950) with the gag gene in the same orientation as the phage P L promoter was identified, isolated and mapped. The size of this clone was reduced by 106 bp by Apal digestion and religation of the pKRR950 plasmid resulting in a plasmid designated pKRR951.
  • the lambda cI ts regulatory gene and the E. coli lambda phage P R promoter were included within the construct.
  • the addition of this temperature sensitive gene allows control of the lambda promoters and subsequently of the gag gene expression.
  • a 2392 bp DNA fragment containing the lambda cI ts regulatory gene and the E. coli lambda phage P R promoter was obtained via Bglll digestion of a plasmid called pRK248.cI ts . This fragment was inserted into the Bglll site of plasmid pKRR951 resulting in plasmid pKRR955.
  • the promoter region, transcriptional start, and ribosomal binding site span bases 7757-271. This region is derived from two different lambda phage mutants and a synthetic region.
  • the coding region is comprised of a synthetic sequence which duplicates the NH 2 end of the lacZ' gene from pUC9 (bases 272-
  • sequences coding for a portion of the HIV-1 gag gene including the entire p24 sequence (bases 344-1036), followed by a short sequence from the synthetic three frame translation terminator of the vector pKRR810 (bases 1169-1180). Translation is terminated at the third termination codon in this segment (bases 1181-1183).
  • the sequence shows the rrnBt 1 transcription terminator (bases 1184-1241).
  • the plasmid pKRR955 produces a fusion protein comprised of 12 amino acids derived from the lacZ protein and the pUC9 polylinker region, followed by a portion of the gag protein (including the final 12 amino acids of the p17 protein, the entire 231 amino acids of the p24 protein and the first 44 amino acids of the pl5 protein), followed by 4 amino acids derived from the terminator portion of the pKRR810 vector.
  • This protein is referred to as recombinant p24 (rp24).
  • rp24 recombinant p24
  • This recombinant E. coli clone expressing the rp41 HIV-2 antigen was carried out in two steps. First a fragment of the HIV-2 env gene was isolated from a HIV-2 prophage and subcloned into an E. coli expression vector designated pEHa. Second, a HIV-2 env gene fragment was subcloned from plasmid pEHa into an alternative expression vector, pTB210N, resulting in the plasmid pJC104.
  • a DNA fragment containing the env gene from HIV-2 was identified within a lambda genomic library of prophage DNA. This fragment was subcloned into an EcoRI site of an E. coli expression vector (lambda PL vector pKH20). The resulting plasmid was named pEHa. This work was done by DIAGEN GmbH, Neiderheider Strasse 3, 4000 Dusseldorf (Kuhnel et. al. Proc. Natl. Acad. Sci. USA 86:2383-2387, 1989).
  • the cloning vector pTB210 allows the fusion of recombinant genes to the
  • This plasmid consists of the plasmid pBR322 with a modified lac promoter fused to a kdsB gene fragment (encoding the first 239 of the entire 248 amino acids of the E. coli CMP-KD0 Synthetase or CKS protein), and a synthetic linker fused to the end of the kdsB gene fragment.
  • the synthetic linker includes: multiple restriction sites for insertion of genes, translational stop signals, and the trpA rho-independent transcriptional terminator.
  • the plasmid pTB210N contains a Ncol site in the synthetic linker and is derived from the plasmid pTB210.
  • Plasmid pEHa was digested with Ncol and a 314 base pair fragment encoding the first 104 amino acids of the HIV-2 p41 protein was isolated and inserted into the Ncol site of plasmid pTB210N. This plasmid, designated pJC104, expresses the HIV-2 env protein as a fusion with the CKS protein.
  • the promoter region, transcriptional start, and ribosomal binding site span bases 45-125.
  • the coding region is comprised of sequences derived from the 239 amino acids of the CKS protein (bases 126-842) and the 13 amino acids from the synthetic polylinker (bases 843-881). This is followed by 104 residues of the amino end of the HIV-2 env (bases 882-1193) and 15 amino acids of the remainder of the polylinker (bases 1194-1238).
  • the translation is terminated at the termination codon at bases 1239-1241.
  • the plasmid pJC104 encodes a recombinant protein containing the first
  • This protein is referred to as recombinant p41 HIV-2 (rp41 HIV-2.
  • the plasmid pOM10 prepared as described above, was transformed into protoplasts of B. megaterium strain PY361 (a prototrophic derivative of strain QMB1551 cured of native plasmids) and viable chloramphenicol resistant cells were allowed to regenerate.
  • Expression of rp41 antigen was under the control of the spoVG promoter ⁇ md was observed when the cells entered the sporulation growth phase.
  • This plasmid replicated as an independent element, was non- mobilizable, and was maintained at approximately 10 to 30 copies per cell.
  • E. coli K-12 strain XL-1 recA1, endA1, qyrA96, thi-1, hsdR17, supE44, relA1, lac-/F', proAB, lacIqZdeltaM15, TN10
  • the expression of the rpCKS-41 protein is under the control of the lac promoter.
  • Recombinant pCKS-41 expression was induced by the addition of LPTG to 100 ⁇ g/ml. This plasmid replicated as an independent element, was non-mobilizable and was maintained at
  • the expression of rp24 protein was under the control of both the lambdaPL and lambdaPR promoters and the cI ts repressor expressed from the cI ts gene present on the plasmid. Recombin ⁇ mt p24 expression was induced by temperature shift from 30oC to 42°C. This plasmid replicated as an independent element, was non- mobilizable and was maintained at approximately 10 to 30 copies per cell.
  • the plasmid pJC104 prepared as described above, was transformed into E. coli K-12 strain XL-1 (recA1, endA1, qyrA96, thl-1, hsdR17, suDEi4, relA1, lac-/F', proAB, lacIqZdeltaM15, TN10) cells made competent by the calcium chloride method.
  • E. coli K-12 strain XL-1 recA1, endA1, qyrA96, thl-1, hsdR17, suDEi4, relA1, lac-/F', proAB, lacIqZdeltaM15, TN10
  • the expression of the rp41 HIV-2 fusion protein was under the control of the lac promoter.
  • Recombinant p41 HIV-2 expression was induced by the addition of IPTG to 100 ⁇ g/ml. This plasmid replicated as an independent element, wzs non-mobilizable and was
  • the plasmid pTB210 prepared as described above, was transformed into E. coli K-12 strain XL-1 (recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac-/F', proAB, laciqZdeltaM15, TN10) cells made competent by the calcium chloride method.
  • E. coli K-12 strain XL-1 recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac-/F', proAB, laciqZdeltaM15, TN10
  • CKS protein was under the control of the lac promoter.
  • CKS expression was induced by the addition of IPTG to 100 ⁇ g/ml. This plasmid replicated as an independent element, was non-mobilizable and was maintained at approximately 10 to 30 copies per cell.
  • HIV-1/ ⁇ LV-2 Antibody Assay Using One Solid Phase One solid phase was coated with the recombinant antigens rp24, rp41, and rp36 as described in Example 3 and used in an assay to detect the presence of HIV-1/HIV-2 antibody in a test sample.
  • the assay conditions were
  • Example 3 essentially as described in Example 3, except that sample volume was decreased to 50 ⁇ L, from 150 ⁇ L used in the licensed assay of Example 3.
  • HIV antibody assays using synthetic HIV-2 peptide I were compared to the results obtained for the same serum sample when using an HIV antibody assay (Human Immunodeficiency Virus Types 1 and 2: E. coli and B. megaterium. recombinant antigen, Abbott HIVAB ® HIV-1/HIV-2
  • Immunodeficiency Virus Types 1 and 2 E. coli and B. megaterium
  • HIV-1 Peptide XIII -modified ELAs on samples identified as Subtype 0 are shown below. The results indicate an increased sensitivity in the assay to subtype O antibody when the synthetic peptide is present.
  • the assay of the invention can be optimized even further by varying assay conditions and/or incubation times, using various combinations of antigen or antibody capture or probe reagents, and other methods, reagents and conditions known to those skilled in the art.
  • various other antibody capture reagents can be used, including HIV p24, gp120, gp160, p17,, and others.
  • the variance of the antibody capture reagent may then require the use of a different antigen capture reagent. All these variations are contemplated to be within the scope of this invention.
  • manual methods or other automated analyzers can be used or adapted to the assay of the present invention.

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Abstract

L'invention concerne un dosage destiné à détecter simultanément la présence d'un ou de plusieurs sous-types d'anticorps anti VIH-1 et/ou d'anticorps anti VIH-2 du VIH, dans un échantillon de test. Les analytes sont capturés soit sur les mêmes phases solides soit sur des phases solides différentes, et on détermine la présence d'analytes par détection d'un signal généré à l'aide d'un cocktail de réactifs indicateurs contenant un antigène synthétiques et recombinés. On peut citer à titre de réactif indicateur préféré un peptide cyclique synthétique à 19 acides aminés homologue à une région immunodominante de gp 41 du VIH-1, et un peptide cyclique synthétique à 19 acides aminés homologue à une région immunodominante de la gp 36 du VIH-2.
PCT/US1995/004421 1994-05-31 1995-04-10 Detection de differents genotypes de vih utilisant un immunodosage modifie par un peptide synthetique WO1995033206A1 (fr)

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EP95915635A EP0763204A1 (fr) 1994-05-31 1995-04-10 Detection de differents genotypes de vih utilisant un immunodosage modifie par un peptide synthetique
JP8500836A JPH10502165A (ja) 1994-05-31 1995-04-10 合成ペプチド−改良イムノアッセイを使用する異なるｈｉｖ遺伝子型の検出
AU22455/95A AU2245595A (en) 1994-05-31 1995-04-10 Detection of different hiv genotypes utilizing a synthetic peptide-modified immunoassay

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041179A1 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Systeme d'expression pour genes non secreteurs
WO2000001719A2 (fr) * 1998-07-02 2000-01-13 Peptide Solutions Peptides du virus varicelle-zona et autres virus permettant d'assurer un diagnostic et une therapie dans le cas du vih-2
WO2003095968A3 (fr) * 2002-05-10 2004-03-25 Bio Rad Pasteur Procede de detection simultanee d'un antigene et d'un anticorps d'un microorganisme infectueux
WO2004027066A2 (fr) * 2002-09-17 2004-04-01 Biomerieux Protéine recombinante chimérique et diagnostic in vitro du hiv
EP2309269A1 (fr) * 2004-09-08 2011-04-13 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Compositions et procédés pour la détection d'une infection par VIH-1/VIH-2
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US8524461B2 (en) 2003-04-11 2013-09-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Multiple antigenic peptide assay for detection of HIV or SIV type retroviruses
WO2017018939A1 (fr) * 2015-07-27 2017-02-02 Mp Biomedicals Asia Pacific Pte Ltd Procédés et trousse pour la différenciation d'anticorps vih-1 et vih-2
US11437141B2 (en) 2009-04-30 2022-09-06 Dexcom, Inc. Performance reports associated with continuous sensor data from multiple analysis time periods

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101322033B (zh) * 2005-09-26 2012-10-10 乌尔里希·洛斯 用于检测针对tsh受体的自身免疫抗体的方法和新型tsh受体嵌合体
KR101619862B1 (ko) 2014-09-30 2016-05-12 주식회사 녹십자 간접효소면역측정법을 이용하는 인간 Fc 포함 단백질의 역가 측정키트 및 이를 이용한 Fc 포함 단백질의 역가 측정방법

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003376A1 (fr) * 1991-07-29 1993-02-18 Biochem Pharma Inc. Dosage immunologique destine a la detection simultanee d'anticorps diriges contre le virus hiv-1, hiv-2, htlv-i et htlv-ii
WO1993021346A1 (fr) * 1992-04-09 1993-10-28 Abbott Laboratories Dosage pour la detection de l'antigene du vih et de l'anticorps du vih

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003376A1 (fr) * 1991-07-29 1993-02-18 Biochem Pharma Inc. Dosage immunologique destine a la detection simultanee d'anticorps diriges contre le virus hiv-1, hiv-2, htlv-i et htlv-ii
WO1993021346A1 (fr) * 1992-04-09 1993-10-28 Abbott Laboratories Dosage pour la detection de l'antigene du vih et de l'anticorps du vih

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
F. DATI ET AL.: "A synthetic peptide based enzyme immunoassay for the simultaneous detection of antobodies to HIV-1 and HIV-2.", CLINICAL CHEMISTRY, vol. 36, no. 6, 25 July 1990 (1990-07-25), WINSTON-SALEM NC USA, pages 1090 - 1091 *
J.S. WEBBER ET AL.: "Automated simultaneous detection of antibody to HIV-1 and HIV-2 using the Abbott IMx analyzer.", IXTH INTERNATIONAL CONFERENCE ON AIDS. JUNE 1993, 1 June 1993 (1993-06-01), BERLIN FRG, pages 263 *
K. FRANSEN ET AL.: "Evaluation of a line immunoassay for the simultaneous confirmation of antibodies to HIV-1 and HIV-2.", EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASES, vol. 10, no. 11, MUNICH FRG, pages 939 - 946 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041179A1 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Systeme d'expression pour genes non secreteurs
WO2000001719A2 (fr) * 1998-07-02 2000-01-13 Peptide Solutions Peptides du virus varicelle-zona et autres virus permettant d'assurer un diagnostic et une therapie dans le cas du vih-2
WO2000001719A3 (fr) * 1998-07-02 2000-02-24 Peptide Solutions Peptides du virus varicelle-zona et autres virus permettant d'assurer un diagnostic et une therapie dans le cas du vih-2
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
WO2003095968A3 (fr) * 2002-05-10 2004-03-25 Bio Rad Pasteur Procede de detection simultanee d'un antigene et d'un anticorps d'un microorganisme infectueux
WO2004027066A2 (fr) * 2002-09-17 2004-04-01 Biomerieux Protéine recombinante chimérique et diagnostic in vitro du hiv
WO2004027066A3 (fr) * 2002-09-17 2004-05-21 Biomerieux Sa Protéine recombinante chimérique et diagnostic in vitro du hiv
US8524461B2 (en) 2003-04-11 2013-09-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Multiple antigenic peptide assay for detection of HIV or SIV type retroviruses
US8722324B2 (en) 2004-09-08 2014-05-13 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Methods for the detection of HIV-1-specific antibodies employing GP41 polypeptides
US9121855B2 (en) 2004-09-08 2015-09-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for the detection of HIV-1 antibodies utilizing a peptide containing a novel gp41 epitope
EP2309269A1 (fr) * 2004-09-08 2011-04-13 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Compositions et procédés pour la détection d'une infection par VIH-1/VIH-2
US11437141B2 (en) 2009-04-30 2022-09-06 Dexcom, Inc. Performance reports associated with continuous sensor data from multiple analysis time periods
WO2017018939A1 (fr) * 2015-07-27 2017-02-02 Mp Biomedicals Asia Pacific Pte Ltd Procédés et trousse pour la différenciation d'anticorps vih-1 et vih-2

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