JP2001504572A - Assays for detecting HIV antigens and HIV antibodies - Google Patents

Abstract

  (57) [Summary] An assay for simultaneously detecting the presence or absence of an HIV antigen analyte and / or an HIV-1 antibody and / or an HIV-2 antibody in a test sample. The analyte is captured on the same or a different solid phase and the presence or absence of the analyte is determined by detecting a signal generated by an indicator reagent specific for the analyte.

Description

DETAILED DESCRIPTION OF THE INVENTION Assays for Detecting HIV Antigens and HIV Antibodies Background of the Invention The present invention relates generally to immunoassays, and more particularly to HIV-1 antigen analytes and HIV-1 antibody analytes and / or Alternatively, the present invention relates to an immunoassay and a substance for simultaneously detecting an analyte of an HIV-2 antibody. Acquired immunodeficiency syndrome (AIDS) is a disease of the immune system associated with opportunistic infections and / or tumors that has reached epidemic proportions in the United States, Europe and Central Africa. Epidemiological data suggest that AIDS is caused by at least two types of human immunodeficiency virus (HIV). HIV type 1 (HIV-1) has been isolated from patients with AIDS and AIDS-related complex disease (ARC) and from healthy humans at high risk of AIDS. For example, F. Barre-Sinoussi et al., Science 220: 868-871 (1983); Popovic et al., Science 224: 497-500 (1984); C. See Gallo et al., Science 224: 500-503 (1984). HIV-1 is transmitted to the fetus or child from sexual intercourse and contact with blood or certain blood products, or from an infected mother. P. Science 239: 573-579 (1988) by Piot et al. AIDS and ARC patients and at-risk humans have high HIV-1 antibody prevalence, and can isolate virus from almost 90% of all seropositive individuals. For example, MG. Science 224: 506-508 (1984); Sarngad haran et al .; J. Gallo et al. Clin. Micro. 25:12 91-1294 (1987). In 1986, a second human immunodeficiency virus (HIV-2) was isolated from an AIDS patient in West Africa. F. Science 233: 343-346 (1986), Clavel et al. HIV-2 infection has also been identified in individuals from several countries outside West Africa. For example, AG. Saimot et al. Lancet i: 688 (1987); MA. Rey et al. Lancet i: 388-389 (1986); Werner et al. Lancet i: 868-869 (1987); Brucker et al. AIDS 2: 141 (1988); See Marquart et al. AIDS 2: 141 (1988). At present, HIV-2 seems to be prevalent only in West Africa, but based on experience with HIV-1, it is clear that HIV-2 will spread to other parts of the world. The HIV-2 virus is similar to the HIV-1 virus in its morphology, cell tropism, interaction with the CD4 cell receptor, in vitro cytopathic effect on CD4 cells, whole genome structure and AIDS pathogenicity. is there. F. Clavel, AIDS 1: 135-140 (1987). However, HIV-2 differs from HIV-1 in several ways. F. Clavel, ibid. A. See Weiss et al. AIDS 2: 95-100 (1988). According to serum tests, the cross-reactivity of the envelope glycoproteins of HIV-1 and HIV-2 is extremely low, but HIV-1 and HIV-2 share many common epitopes within their core antigens. F. Clavel, see above. This limitation of envelope antigen cross-reactivity means that currently available serum assays for HIV-1 cannot react with sera from individuals carrying HIV-2 antibodies. Can be explained. F. J. Denis et al. Clin. Micro. 26: 1000-1004 (1988). An assay for an HIV-1 / HIV-2 antibody, recently marketed by the applicant, called Abbott HIVAB® HIV-1 / HIV-2 (rDNA) EIA, is a test for these two viral proteins, namely HIV- Recombinant antigens corresponding to 1 envelope and HIV-2 envelope are used. The use of these recombinant antigens allows for the detection of test samples containing anti-HIV-1 and / or anti-HIV-2 while minimizing non-specific reactions, mainly caused by cross-reaction with whole virus or virus lysate. Can be improved. The use of at least one recombinant HIV protein to detect HIV antibodies in a test sample using a labeled recombinant HIV antigen has been disclosed by the present applicant in JP-A-3-28766, JP-A-62-168052. And so on. Based on nucleotide analysis of the viral genome, the HIV genomic RNA encodes (sequentially from the 5 'end): (i) Corresponds to nucleotides 310 to 1869 and has a core or nucleocapsid protein of internal structure (eg, p24, A gag gene encoding the most antigenic core protein); (ii) a pol gene corresponding to nucleotides 1,629 to 4,673; encoding a enzyme or reverse transcriptase; and (iii) an envelope glycoprotein corresponding to nucleotides 5,781 to 8,369. The env gene encoding (eg, gp41, the most antigenic envelope protein). Ratner et al., Nature 313: 277-284 (1985). One of the challenges facing the medical community today is the prevention of HIV contamination by blood products, which have been detected in blood products (and other human body fluids) and reported to have been transmitted by blood transfusion. Today, several assays are available, such as those described in US Patent No. 4,520,113 to Gallo et al. Other assays that can detect HIV antigens or HIV antibodies are also known. Such assays include the anti-HIV-1 / HIV-2 assays disclosed in the previously cited applications, and D.I. Paul, U.S. Pat. No. 4,748,110; Includes the assays disclosed in Stewart et al., US Pat. No. 4,983,529 and US Pat. No. 5,173,399. However, all known assays for the detection of HIV antigen or HIV antibody analytes that have received federal approval detect either HIV antigen or HIV antibody analytes in test samples. You can only do it. Known federally-approved commercial assays are not available for detecting both HIV antigen and / or HIV antibody analytes using test samples in a single assay. Detection of two or more analytes in a test sample usually consists of separately detecting each analyte in a separate assay. Such a detection method was preferred because rigorous measurements of guaranteed quality could be performed for each analyte to be tested. Medical advances have led to the recognition of new markers for many diseases and clinical conditions, and the need for clinical testing of these markers. Inspection laboratories are facing the problem of timely handling increasing inspection volumes while seeking cost reduction. For example, human T-leukemia virus type 1 (HT LV-1), HIV and hepatitis C virus (HCV) have been added to a panel of donor blood test factors at these institutions, and the presence or presence of these factors. Tests for blood banks have surged as exposures to blood have been tested. A possible solution, especially for reducing the labor of laboratories caused by blood bank testing demands, is to find a way to combine the assays. However, it is difficult to combine assays without lowering individual performance criteria, and more importantly, to overcome manufacturing and quality control issues. Some researchers have developed or attempted to develop assays for simultaneously testing two or more analytes in a test sample. Such an assay would be advantageous because the time required to detect two or more analytes in a test sample is greatly reduced. Also, performing such assays requires less processing time, reagents and equipment than existing assays, so the cost of each assay will also be lower. For example, US Pat. No. 4,315,907 to Fridlender et al. Teaches a heterogeneous specific binding assay system that separates bound species from labeling reagent in free species form. U.S. Pat. No. 4,378,344 to Zahradnik et al. And EP 027008 teach solid phase devices for determining the presence of each analyte consisting of a container and an insert. In this device, the presence of each analyte is determined by the assay method described in the claims. UK Patent No. 2188418 teaches an assay tray assembly with reaction wells, each well having an opening in the top surface overhanging the protrusion, and the inner surface of each well sidewall and the outer surface of each protrusion at the same time. After incubation, two or more specific substances present in the sample introduced into the reaction well are detected. European Patent Publication No. 0 351 248 filed by IDEXX Corporation discloses a co-immunoassay for feline virus or HIV that is capable of detecting antigen and / or antibody components of a single binding pair. Further, Papsidero U.S. Pat.No. 5,039,604 teaches an immunoassay in which two different antigens are added, followed by a single labeled antibody reactive with these antigens to simultaneously detect two HTLV or HIV antibodies. ing. Further, U.S. Pat. No. 4,870,003 to Kortright et al. Discloses a solid-phase immunoassay for detecting antigens and / or antibodies of a single binding pair using the antigen "spike" of the inactivated antigen. Further, detecting one or more analytes using two or more solid phases is described in co-pending U.S. Patent Application No. 574,821 (European Patent Publication 473 065 published March 4, 1992). ). An important factor identified in the development of a simultaneous assay is that two assays to be performed simultaneously must be performed with the same sample volume, the same incubation time and the same cut-off calculation. Such a simultaneous assay further requires that each analyte can be individually quality controlled by both the manufacturer and the laboratory using the assay to ensure the sensitivity, specificity and reproducibility of the immunoassay. Accordingly, it would be advantageous to provide an assay that can simultaneously detect the presence of two or more HIV analytes and that can individually control the quality of each analyte to be detected. The presence of the HIV antigen analyte and / or HIV antibody analyte is measured simultaneously in one well, and the use of two or more solid phases does not require separation of the solid phase components, and the assay Such assays are superior to other known assays because the quality of the individual analytes to be detected in a simultaneous assay can be controlled. SUMMARY OF THE INVENTION The present invention provides an assay for simultaneously detecting the presence or absence of an HIV antigen analyte and / or an HIV antibody analyte that may be present in a test sample. The assay comprises the steps of: (i) at least one HIV antigen analyte capture reagent attached to a solid phase that is specific for the HIV antigen analyte; and (ii) a HIV antibody analyte specific. At least one HIV antibody analyte capture reagent attached to a solid phase to form a first mixture; an HIV antigen capture reagent / HIV antigen analyte complex and / or an HIV antibody capture reagent Incubating the first mixture for a time and under conditions sufficient to produce an HIV antibody analyte complex; the complex is (i) a signal-generating compound specific for the HIV antigen analyte. An HIV antigen analyte indicating reagent consisting of one member of a labeled binding pair, and (ii) an HIV antibody analyte indicating one member of a binding pair that is specific to the HIV antibody analyte and labeled with a signal generating compound. Contact with reagents to form a second mixture; HIV antigen capture Incubate the second mixture for a time and under conditions sufficient to produce a drug / HIV antigen analyte / indicating reagent complex and / or HIV antibody capture reagent / HIV antibody analyte / indicating reagent complex. By detecting the generated signal as an indication of the presence of one or both analytes in the test sample, the HIV antigen analyte and / or the HIV antibody analyte in the test sample; To determine the presence or absence of The present invention further provides an assay for simultaneously detecting the presence of an HIV antigen analyte and / or an HIV antibody analyte in a test sample. The assay comprises the steps of: (i) at least one HIV antigen capture reagent attached to a solid phase, which is specific for the HIV antigen analyte; and (ii) a specific, solid At least one HIV antibody capture reagent attached to the phase, and (iii) at the same time as the HIV antigen analyte indicator reagent, which is specific for said HIV antigen analyte and comprises one member of a binding pair labeled with a signal generating compound. Contacting to produce a first mixture; sufficient time to produce HIV antigen capture reagent / HIV antigen analyte / HIV antigen indicator reagent complex and HIV antibody capture reagent / HIV antibody analyte complex. Incubating the first mixture under conditions; contacting the complex with an HIV antibody analyte-indicating reagent that is specific for the HIV antibody analyte and that is one member of a binding pair labeled with a signal-generating compound. To produce a second mixture; HIV antibody capture reagent / HIV Incubating said second mixture for a time and under conditions sufficient to produce an analyte / HIV antibody analyte-indicating reagent complex; detecting said signal to produce said HIV antigen-binding in a test sample. Determining the presence of the analyte and / or the HIV antibody analyte. The HIV antibody capture reagent and the antigen capture reagent may be attached (bound) to the same or different solid phases. Preferred solid phases include magnetic beads, non-magnetic beads, reaction tray wells, microparticles, nylon strips and nitrocellulose strips. The HIV antigen analyte is the HIV 24 pgag antigen, and the HIV antigen analyte capture reagent is a monoclonal, polyclonal or recombinantly derived anti-HIV 24p antibody or fragment thereof. The HIV antigen capture reagent is a mixture of monoclonal antibodies secreted by the hybridoma cell lines of ATCC Accession Numbers HB9725 and HB9726, and the HIV antibody capture reagent is HIV-1 p41 protein, or HIV-1 p41 protein and HIV- 2 It is preferably a mixture with the p41 env protein. Most preferably, these antigens are produced recombinantly. The signal generating compound of the HIV antigen analyte indicating reagent and the signal generating compound of the HIV antibody analyte indicating reagent are selected from the group consisting of enzymes, luminescent compounds, chemiluminescent compounds and radioactive elements. Moreover, one or both indicator reagents may further comprise a hapten. The foregoing assays may be suitable for separately detecting both HIV-1 antigen analyte and HIV-1 / HIV-2 antibody analyte. This assay step comprises the steps of: (a) combining a test sample with a solid phase having at least one anti-HIV p24 antibody, at least one recombinant HIV-1 p41 antigen and at least one HIV-2 p41 antigen attached thereto. Contacting to form a mixture; (b) anti-HIV p24 antibody / HIV-1 p24 antigen complex and / or HIV-1 p41 antigen / HIV-1 antibody and / or HIV-2 p41 antigen / anti-HIV-2 p41 antigen Incubating the mixture for a time and under conditions sufficient to produce a complex; (c) (i) a time sufficient to produce an anti-HIV p24 antibody / HIV p24 gag antigen / anti-HIV p24 antibody complex And contacting the anti-HIV p24 antibody capable of specifically binding to the HIV p24 antigen under the conditions with the anti-HIV p24 antibody / HIV p24 antigen complex, and (ii) an anti-species antibody labeled with a signal-generating compound. The indicator reagent is brought into contact with the anti-HIV p24 antibody / HIV p24 antigen / anti-HIV p24 antibody complex, and (iii) the generated signal is detected in the presence of the HIV p24 antigen in the test sample. (D) (i) detecting the presence of the HIV-1 p41 antigen / anti-HIV-1 p41 antibody complex and / or HIV-2. The p41 antigen / anti-HIV-2 antibody complex is contacted with an indicator reagent consisting of an HIV-1 p41 antigen labeled with a signal generating compound and an HIV-2 p41 antigen labeled with a signal generating compound, and (ii) generating the generated signal. By detecting the presence of anti-HIV-1 p41 antibody and / or HIV-2 p41 antibody in the test sample, the anti-HIV-1 p41 antibody and / or HIV-2 p41 antibody in the test sample are detected. To determine the presence or absence of This assay can also be performed using two (or more) solid phases. Furthermore, a test kit for performing the assay of the present invention is provided. DETAILED DESCRIPTION OF THE INVENTION An assay is provided for detecting an HIV antigen analyte and / or an HIV antibody analyte in a test sample. Although the invention is not limited to immunoreactivity assays, it is preferred that the assays be performed as immunoassays. Any assay using a specific binding member can be performed. As used herein, a "specific binding member" is a member of a specific binding pair, i.e., two different molecules, where one molecule specifically binds to a second molecule by chemical or physical means. Therefore, in addition to the specific binding element of antigen / antibody in a normal immunoassay, specific binding pairs include biotin and avidin, carbohydrate and lectin, complementary nucleotide sequences, effector molecules and receptor molecules, coenzymes and enzymes, enzyme inhibitors And enzymes and the like. Furthermore, a specific binding pair can include a member that is an analog of the original specific binding member, eg, an analyte analog. Immunoreactive specific binding members include antigens, antigen fragments; monoclonal and polyclonal antibodies and antibody fragments; and complexes thereof (eg, complexes produced by recombinant DNA methods). As used herein, "analyte" is a substance to be tested that may be present in a test sample. The analyte can be any substance for which a natural specific binding member (eg, an antibody) is present or capable of producing a specific binding member. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substance, hapten, antibody and combinations thereof. Because the analyte is a member of a specific binding pair, the natural specific binding partner (pair) (e.g., the use of intrinsic factor proteins in the capture and / or indicator reagents ₁₂ (Or by measuring carbohydrates using lectins in the capture and / or indicator reagents). Analytes include proteins, peptides, amino acids, hormones, steroids, vitamins, drugs (including drugs administered for treatment and drugs for unauthorized purposes), bacteria, viruses, and any of the foregoing. Metabolites of such substances or antibodies may be included. The test sample is a mammalian biological fluid [eg, whole blood or whole blood components (white blood cells such as red blood cells, lymphocytes or lymphocyte preparations, platelets, serum and plasma); ascites; saliva; stool; Fluid; urine; sputum; tracheal aspirate as well as other components of the body that contain or may contain the analyte of interest]. The test sample may be a culture supernatant or a cell suspension. Examples of mammals capable of assaying a body fluid for an HIV antigen analyte or an HIV antibody analyte based on the present invention include humans, primates, and other mammals that are thought to contain the analyte of interest. included. Non-biological fluid samples can also be used. The indicator reagent includes a label bound to a specific binding member of each analyte. Each indicator reagent produces a detectable signal at a level commensurate with the amount of the analyte in the test sample. In a preferred embodiment, each indicator reagent consists of a specific binding element for a different analyte, but is conjugated to the same signal producing compound (label) capable of producing a detectable signal. Generally, the indicator reagent is detected or measured after being captured on the solid phase material. In the present invention, the total signal generated by the indicator reagent indicates that one or more analytes are present in the test sample. It is contemplated that different signal producing compounds may be used in practicing the present invention. Thus, for example, different fluorescent compounds can be used as signal-generating compounds, one for each indicator reagent, and can be read and detected at different wavelengths. Alternatively, short-lived chemiluminescent compounds (eg, acridinium or phenanthridinium compounds) and long-lived chemiluminescent compounds (eg, dioxetane) can be used to generate different numbers of signals for different analytes. A method detailing the use of two or more chemiluminescent compounds capable of producing different numbers of signals is described in the subject of a co-pending PCT application filed December 23, 1991 (Tokuhei 6-504374). It is. Acridinium and phenanthridinium compounds are described in co-pending US patent application Ser. No. 07 / 271,763, filed Jun. 23, 1989. All are by the present applicant and are incorporated herein by reference. The specific binding element of the indicator reagent may be any specific binding pair element (e.g., biotin or avidin, carbohydrate or lectin, complementary nucleotide sequence, effector or receptor molecule, complement Enzymes or enzymes, enzyme inhibitors or enzymes). The immunoreactive specific binding member can be an antibody, antigen or antibody / antigen complex that can bind the analyte in a sandwich assay, the capture reagent in a competition assay, and the auxiliary specific binding member in an indirect assay. When an antibody is used, it may be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a recombinant antibody, a mixture thereof, or a mixture of the antibody and another specific binding member. Details of how to produce such an antibody and its suitability for use as a specific binding member are well known to those skilled in the art. The signal producing compound (label) of the indicator reagent can generate a measurable signal that can be detected by external means. Various possible signal producing compounds (labels) include chromogens; catalysts (eg, enzymes such as horseradish peroxidase, alkaline phosphatase and β-galactosidase); luminescent compounds (eg, fluorescein and rhodamine); chemiluminescent compounds (eg, acridinium) Compounds, phenanthridinium compounds and dioxetane compounds); radioactive elements; and direct visible labels. The choice of a particular label is not critical, but the label may generate a signal by itself or in combination with one or more additional substances. A variety of different indicator reagents can be produced by changing the label or specific binding member. The capture reagent of the invention consists of a specific binding element for each analyte of interest, is attached to at least one solid phase and is unlabeled. The capture reagent is specific for the analyte in a sandwich assay, but is specific for the indicator reagent or analyte in a competitive assay, or for an auxiliary specific binding element that is itself specific for the analyte in an indirect assay. obtain. The capture reagent can bind directly or indirectly to the solid phase material before or during the performance of the assay to separate the immobilized complex from the test sample. Such attachment can be performed, for example, by coating the specific binding member on a solid phase by absorption or covalent bonding. Coating methods and other known means of attachment are well known to those skilled in the art. The specific binding member of the capture reagent can be any molecule that can specifically bind to another molecule. The specific binding member of the capture reagent can be an immunoreactive compound (eg, an antibody, antigen or antibody / antigen complex). When an antibody is used, it may be a monoclonal antibody, a polyclonal antibody, an antibody fragment, a recombinant antibody, a mixture thereof, or a mixture of the antibody and another specific binding member. 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, membranes, plastic tubes, reaction tray well walls, glass or silicon chips and brown sheep red blood cells are all suitable examples. Suitable methods for immobilizing the capture reagent on the solid phase include ionic interactions, hydrophobic interactions, covalent interactions, and the like. In one embodiment of the present invention, a 60-well polystyrene reaction tray and 1/4 inch polystyrene beads are used; in another embodiment, a 96-well reaction tray is the only solid phase used. Since all the solid phase is present during signal quantification, it is not necessary to separate the solid phase for signal detection. As used herein, "solid phase" means any material that is insoluble or may become insoluble in subsequent reactions. The solid phase can be selected for its inherent ability to attract and immobilize the capture reagent. Alternatively, the solid phase may carry additional receptors that can attract and immobilize the capture reagent. The additional receptor may consist of a charged material having a charge opposite to the capture reagent itself or a charged material associated with the capture reagent. As another alternative, the receptor molecule can be any specific binding member that can be immobilized (attached) to a solid phase and immobilize the capture reagent by a specific binding reaction. The receptor molecule can indirectly bind the capture reagent to the solid phase material before or during the assay. Thus, the solid phase is plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter wells, sheets, beads, microparticles, chips, and other shapes known to those skilled in the art. obtain. It is contemplated that the solid phase may also include a suitable porous material having sufficient porosity to be able to bind the antigen with the detection antibody and appropriate surface affinity, and is within the scope of the present invention. Although a microporous structure is generally preferred, a hydrated gel structure material may be used. Such useful solid phases are described below: Natural polymer carbohydrates and synthetically modified, crosslinked or substituted derivatives thereof (eg agar, agarose, crosslinked alginic acid, substituted and crosslinked guar gums, especially with nitric acid and carboxylic acids) Cellulose esters, mixed cellulose esters, cellulose ethers); Natural polymers containing nitrogen [eg proteins and derivatives (crosslinked or denatured gelatin)]; Natural hydrocarbon polymers (eg latex and rubber); Synthetic polymers which can be prepared with suitable porous structures [For example, vinyl polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and its partially hydrolyzed derivatives, polyacrylamide, polymethacrylate), the copolymers and terpolymers, condensation polymers (e.g., polyester, poly Amides) and other polymers (eg, polyurethanes or polyepoxides)]; porous inorganic materials [eg, alkaline earth metals and magnesium sulfates or carbonates (eg, barium sulfate, calcium sulfate, calcium carbonate), alkali / alkaline earth metals] Silicates of aluminum and magnesium]; oxides or hydrates of aluminum or silicon [eg clay, alumina, talc, kaolin, zeolite, silica gel or glass (these materials can be used as filters with the aforementioned polymeric materials) Mixtures or copolymers of the above class (eg, graft copolymers obtained by initiating polymerization of a synthetic polymer on an existing natural polymer). All of these materials can be used in a suitable form (e.g., a membrane, sheet or plate) or coated, bonded or bonded to a suitable inert carrier (e.g., paper, glass, plastic film or cloth), or It can be laminated. The porous structure of nitrocellulose shows excellent absorption and adsorption properties for various reagents (for example, monoclonal antibodies). Nylon has similar properties and is suitable. The porous solid support as described above preferably has a thickness of about 0. 01-0. 5 mm, more preferably about 0. It is considered to be a 1 mm sheet shape. The pore size can vary over a wide range, and is preferably about 0. 025-15 microns, especially about 0. 15 to 15 microns. The surface of such a carrier can be activated by a chemical treatment that allows covalent attachment of the antigen or antibody to the carrier. However, in general, irreversible binding of antigens or antibodies occurs by adsorption on porous materials with poorly understood hydrophobic forces. Preferred solid phase materials for the flow-through assay include filter paper (eg, a porous glass fiber material or other fiber matrix material). The thickness of such materials is not critical and is selected primarily based on the properties of the sample or analyte to be assayed (eg, the flow rate of the test sample). To change or increase the intrinsic charge of the solid phase, the charged substance can be directly coated on the microparticles or material carried by the solid support material. Alternatively, the microparticles can be used as a solid phase by being retained on a column or suspended in a mixture of a soluble reagent and a test sample. Alternatively, the particles themselves can be retained / immobilized on a solid support material. "Retained / immobilized" means that the particles on or within the carrier material cannot substantially migrate to other locations within the carrier material. The particles may be selected by those skilled in the art from any suitable type of particulate material. The particles include materials comprising polystyrene, polymethyl acrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate or similar materials. It is preferred that the average diameter of the particles be smaller than the average pore size of the carrier material used, but the particle size is not critical. Thus, embodiments using various other solid phases are also contemplated and are within the scope of the present invention. For example, the fixing described in the applicant's JP 1-227061 (corresponding to EP-A-0326100) and JP-A 3-44399 (corresponding to EP-A-0464773), incorporated herein by reference. An ion capture procedure for immobilizing the labile reaction complex with a negatively charged polymer can be used in the present invention to generate a rapid solution phase immunochemical reaction. The immobilizable immune complex is separated from the rest of the reaction mixture by ionic interaction between the negatively charged polyanion / immune complex and the pretreated positively charged porous matrix, and the various signal generation systems described above [eg, System described in Chemiluminescence signal measurement described in JP-A-63-112564 (corresponding to EP-A-0 273,115). Said patent is assigned by the present applicant and is incorporated herein by reference. Furthermore, the method of the present invention may be suitable for use in systems using microparticle technology (eg, automated / semi-automated systems in which the solid phase consists of microparticles). Such systems include those described in JP-A-3-60981 (corresponding to European Patent Publication No. 0 425 633), U.S. Patent No. 5,089,424 (corresponding to European Patent Publication No. 0 424 634) and U.S. Patent No. 5,006,309. The system is included. All of the foregoing patent applications are by the applicant and are incorporated herein by reference. In practicing one embodiment of the present invention, a test sample suspected of containing the HIV antigen analyte or HIV antibody analyte of interest is attached to the first specific binding element of the first analyte. The mixture is contacted at the same time as the solid phase being applied and the solid phase to which the first specific binding member of the second analyte is attached. The specific binding element acts as a capture reagent, binding the analyte to the solid phase. If the specific binding member is an immunoreactant, it can be an antibody, antigen or complex thereof specific for each analyte of interest. When the specific binding member is an antibody, it can be a monoclonal or polyclonal antibody, an antibody fragment, a recombinant antibody, and mixtures thereof, or a mixture of an antibody and another specific binding member. The mixture is incubated for a time and under conditions sufficient for the binding reaction to take place. This incubation results in the formation of a first analyte capture reagent / first analyte complex if the first analyte is present in the test sample and / or a second analyte in the test sample. When present, a capture reagent of the second analyte / second analyte complex is generated. Next, the indicator reagent of each analyte is brought into contact with the complex. The first analyte indicator reagent comprises a specific binding element of the first analyte of interest labeled with a signal generating compound. The second analyte indicator reagent comprises a specific binding element of the second analyte of interest labeled with the same signal generating compound as the first analyte indicator reagent to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to produce a capture reagent / first analyte / indicating reagent complex and / or a capture reagent / second analyte / indicating reagent complex. The presence of each analyte is determined by detecting the signal generated in association with the complex formed on the solid phase as an indication of the presence of one or more analytes in the test sample. When the indicator reagent utilizes an enzyme as the signal-generating compound (label), the signal can be detected visually or measured by spectroscopy. Alternatively, the label used for signal generation can be detected by measuring fluorescence, chemiluminescence, radioactive energy emission and the like. The capture reagents can be attached to the same solid phase or different solid phases. All capture reagents may be attached to the same solid phase, each capture reagent may be attached to a separate solid phase, or a combination of capture reagents may be attached to individual solid phases. it is conceivable that. For example, if the selected solid phase is a microparticle, each microparticle may have at least one capture reagent attached. The particulate mixture (solid phase) can be used to capture various analytes that may be present in the test sample. In such assays, it is believed that capture reagents can be attached to the solid phase in various ratios to optimize analyte detection. In the above embodiment, the specific binding element used as the capture reagent for the HIV-1 antibody analyte is HIV-1 p41 antigen, and the specific binding element used as the capture reagent for the HIV-1 antigen analyte is anti-HIV. Preferably, it is a -1 p24 antibody. Most preferably, the HIV-1 p41 used is a recombinantly produced antigen (protein). Further, the specific binding element of the antibody analyte indicator reagent is an HIV-1 p41 antigen labeled with an enzyme, and the specific binding element of the antigen analyte indicator reagent is an anti-HIV-1 p24 antibody labeled with an enzyme. Preferably, there is. Most preferably, the HIV-1 p41 antigen is produced recombinantly and the enzyme is horseradish peroxidase (HRPO). In another embodiment of the present invention, a test sample suspected of containing any analyte of interest is labeled with a first specific binding member of a first analyte and a first specific binding member of a second analyte. A first solid phase to which a binding element is attached, a first analyte-specific indicator reagent comprising a specific binding element of a first analyte labeled with a signal generating compound, and a specificity of a second analyte labeled with a signal generating compound The mixture is produced simultaneously with the second analyte-indicating reagent comprising the binding element. The analyte is bound to the solid phase using the specific binding member as a capture reagent. Where the specific binding member is an immunoreactant, it may be an antibody, antigen or complex thereof specific for each analyte of interest. When the specific binding member is an antibody, it may be a monoclonal or polyclonal antibody, an antibody fragment, a recombinant antibody and a mixture thereof, or a mixture of the antibody and another specific binding member. The indicator reagent consists of a specific binding element of the first and second analytes of interest, labeled with a signal producing compound. The mixture is incubated for a time and under conditions sufficient for the binding reaction to take place. By this incubation, if one or both of the first and second analytes are present in the test sample, the first analyte capture reagent / first analyte / indicating reagent complex and / or the second analyte An analyte capture reagent / second analyte / indicating reagent complex is formed. By detecting a signal generated in association with the complex formed on one or both solid phases as an indication of the presence of the first analyte and / or the second analyte in the test sample. The presence or absence of each analyte is determined. When the indicator reagent utilizes an enzyme as the signal-generating compound (label), the signal can be detected visually or measured by spectroscopy. Alternatively, depending on the label used, the label can be detected by measuring fluorescence, chemiluminescence, radioactive energy emission, and the like. It is further contemplated that this assay may involve the use of a hapten anti-hapten system. In this case, the indicator reagent may further include a hapten (eg, biotin). The use of a biotin / anti-biotin system in the assay is the subject of JP 60-250257 (corresponding to EP-A-0160900 published November 13, 1985). This is by the applicant and is hereby incorporated by reference. In the above embodiment, the specific binding element used as the capture reagent for the HIV-1 antibody analyte is HIV-1 p41 antigen, and the specific binding element used as the capture reagent for the HIV-1 antigen analyte is anti-HIV. Preferably, it is a -1 p24 antibody. Most preferably, the HIV-1 p41 used is a recombinantly produced antigen (protein). Further, it is preferable that the specific binding element of the antibody-analyte indicating reagent is an HIV-1 p41 antigen labeled with an enzyme. Most preferably, the HIV-1 p41 antigen is produced recombinantly and the enzyme is horseradish peroxidase (HRPO). Preferred solid phases include magnetic or non-magnetic beads, reaction tray wells and microparticles used alone or in any combination. For reliable results, positive and negative controls can be included in the assays of the present invention. A blank solid phase with no capture reagent attached can be used as a negative reagent control. Positive controls can include a positive control for each analyte that is tested separately and a combined positive control that determines the presence of all analytes to be detected in the assay. As mentioned above, it is preferred that the recombinantly produced antigen be used in the assay. It is known to use inactivated whole virus cultured in cell lines capable of virus replication as reagents for assays for HIV antibodies, but false positive results may be obtained from uninfected humans. The detection of false positive (reaction) results in blood product screening assays is one of the greatest concerns with many known HIV detection methods. These incorrect results are often associated with non-specific binding of immunoglobulins to cellular proteins in the virus isolate. These reagents also have the disadvantage of being a potentially dangerous manufacturing method, as workers can be exposed to the HIV virus during in vitro culture, isolation and inactivation of live virus. Isolation of the HIV virus remains the definitive test for HIV infection, but isolation of HIV in culture is difficult, time consuming, and impractical for large-scale use. Is a typical method. The use of recombinantly produced HIV protein (antigen) as an antigen reagent in the assay method can solve the above-mentioned problems. Recombinant proteins are non-infectious, and thus production / isolation of such proteins is safer than culturing whole viruses. Furthermore, the use of recombinant pure viral proteins should eliminate some false positive reactions due to non-specific reactions with contaminating proteins. Furthermore, standardization of the reagents should improve the specificity and reliability of the assay. Expression of HIV gp41 or a portion of HIV gp41 has demonstrated the utility of recombinant DNA (rDNA) -derived HIV envelope sequences in diagnostic assays. Cold et al., Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold Spring Harbor, New York, May 22-26 (1985); Chang et al., Biotechnology 3: 905-909 (1985); Cowl et al., Cell 41: 979. -986 (1985); Cabradilla et al., Biotechnology 3: 128-133 (1986). Although it is generally known that viral proteins expressed in E. coli or other microorganisms are potentially useful in diagnostic assays, these reagents also exhibit specificity and sensitivity over live viral proteins from cell cultures Deploying immunoassays using was a difficult task. Furthermore, expression of HIV gag protein in E. coli indicated that HIV gag protein produced by rDNA technology could have potential diagnostic value. Wood et al., Cold Spring Harbor Symposium on RNA Tumor Viruses, Cold Spring Harbor, New York, May 22-26 (1985); Dowbenko et al., PNAS USA 82: 7748-7752 (1985); Ghrayeb et al., DNA 5: 93-99. (1986); Steimer et al., Virology, 150: 283-290 (1986). The present invention uses recombinantly produced HIV envelope proteins as assay reagents. The cloning of the HIV genome, expression of the HIV envelope and core proteins in E. coli, purification and characterization of gp41 and p24, and various assay formats using these recombinant proteins are described in European Patent Publication, published September 12, 1990. No. 386 713 (JP-A-3-28766). Briefly, HIV infected HT-9 cells are harvested and total cellular DNA is isolated and digested. DNA fragments encoding the core protein and the envelope glycoprotein were further subcloned into bacterial expression vectors using well-known recombinant techniques. The prior application also teaches that the use of recombinant antigens as capture and indicator reagents in the detection of HIV-1 antibodies can detect anti-HIV-1 antibodies of different immunoglobulin types. These immunoglobulin types include IgG, IgA, IgE and IgM. A method for detecting anti-HIV-1 IgG, IgM and IgA using the Abbott HIVAB® HIV-1 / HIV-2 (rDNA) EIA assay is described in L. Gallarda et al. Describe in the summary of the 5th Annual Forum on AIDS, Hepatitis and Other Blood-Borne Diseases, Atlanta, Georgia, March 29-April 1, 1992. Recombinant antigens produced from heterologous sources can be used in the assays, and it is believed that these antigens will significantly reduce false positive results, which is within the scope of the invention. For example, if a recombinant antigen (eg, p41) produced in E. coli is used as a capture reagent, a suitable source other than E. coli (eg, a suitable yeast host or B. Other suitable hosts such as megaterium) can be used. Furthermore, although the present invention preferably uses recombinantly produced antigens, it is well within the scope of the present invention to use synthetic proteins in place of recombinantly produced antigens. Thus, various synthetically prepared HIV peptides of different lengths can be used. The invention further uses antibodies that specifically bind to an HIV antigen analyte. In a preferred embodiment, an anti-HIV p24 antibody is used. In a most preferred embodiment, a mixture of monoclonal antibodies, both specific for the HIV p24 antigen, is used. In this mixture, a monoclonal antibody that specifically binds to an epitope on HIV-1 p24 to which the epitope human anti-HIV-1 p24 IgG does not competitively bind, a different epitope human anti-HIV-1 p24 IgG competitively binds For use with other monoclonal antibodies that specifically bind to different epitopes of HIV-1 p24. Furthermore, monoclonal antibodies that do not competitively bind to human anti-HIV-1 p24 IgG also bind specifically to the HIV-2 p24 antigen. These monoclonal antibodies and their use in HIV antigen assays are the subject of US Pat. No. 5,173,399, issued Dec. 22, 1992. These monoclonal antibodies are designated 31-42-19 and 31-90-25. Hybridoma cell line 31-42-19 producing monoclonal antibody 31-4 2-19 was deposited on May 26, 1988 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 under ATCC accession number HB 9726. Was done. The hybridoma cell line 31-90-25 producing the monoclonal antibody 31-90-25 was deposited on May 26, 1988 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 under ATCC Accession No. HB 9725. Was. The use of these monoclonal antibodies as antibody fragments in HIV antigen assays is also described in US Pat. No. 5,173,399. It is believed that the assay of the present invention is capable of detecting the HIV-2 antigen, which is also within the scope of the present invention. In this assay format, HIV-2 p41 is attached to a solid phase as an HIV-2 antigen capture reagent in addition to the above-mentioned HIV-1 p41 antigen capture reagent and HIV-1 antibody capture reagent. The solid phase may be the same solid phase to which all other capture reagents are attached, the same antigen to which the HIV-1 recombinant antigen is attached, or other than HIV-2 p41 May be attached to a solid phase to which no capture reagent is attached. The assay procedure is a procedure as described in the various embodiments of the present invention. The HIV-2 antibody analyte indicator reagent comprises the HIV-2 p41 antigen attached to a detectable label. In a preferred embodiment, it is preferred to use recombinantly produced HIV-4 p41. The sequence of the HIV-2 virus (eg, the p41 antigen) is described in Diagen Corporation, European Patent Publication No. 347,365, published Dec. 20, 1989. This patent is incorporated herein by reference. The most preferred HIV-2 recombinant antigen encodes the first 104 amino acids of the HIV-2 p41 antigen. The resulting plasmid, pJC104, expresses the HIV-2 env protein as a fusion with the CKS protein. This plasmid is disclosed in Bolling and Mandecki European Patent Publication No. 475 182, published March 18, 1992 (U.S. Patent Application No. 167,067, filed March 11, 1988, filed by the applicant and incorporated herein by reference. According to the method disclosed in the "CKS Method of Protein Synthesis" (incorporated herein), the first 239 amino acids of the CKS protein, 13 amino acids from the pTB210N multiple restriction site linker, and 104 amino acids from the HIV-2 env protein ( It encodes a recombinant protein consisting of amino acids 506-609) and an additional 15 amino acids from the pTB210N multiple restriction site linker. EXAMPLES Example 1 Coating Procedure Using Two Solid Phases This procedure is based on 1/4 inch polystyrene beads (available from the applicant) and 60-well polystyrene reaction (available from the applicant). A tray was used. Two different anti-HIV-1 p24 monoclonal antibodies were coated on the beads in the following manner. 0. 25M sodium citrate buffer (pH 7. In 2), 8 μg / ml (about 1. Beads were coated at a concentration of 6 μg / ml / bead) at 45 ° C. for 2 hours. The beads are then 25M sodium citrate buffer (pH 7. 2) Wash in, 0. 1% Triton X-100® (Sigma Chemical Co. , St. The reaction was carried out at 45 ° C. for 1 hour with a detergent solution containing Louis, MO polyoxyethylene ether). The beads were then washed with 1% bovine serum albumin (BSA) at 0. 25M sodium citrate buffer (pH 7. Block at 45 ° C for 30 minutes in 2), then 0. It was overcoated with 2% sucrose, 1% phosphate glass in 25M sodium citrate buffer at 15-30 ° C for 15 minutes and dehydrated. The two monoclonal antibodies used are 31-42-19 and 31-90-25. These antibodies are the subject of parent application US patent application Ser. No. 07 / 204,798, which describes the production and use of the antibodies previously incorporated herein by reference. The use of these antibodies is also described in Applicant's US Pat. No. 5,204,798, which is incorporated herein by reference. The hybridoma cell line 31-42-19 producing the monoclonal antibody 31-42-19 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 on May 26, 1988 under ATCC accession number HB 9726. Was done. The hybridoma cell line 31-90-25 producing the monoclonal antibody 31-90-25 was deposited on May 26, 1988 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 under ATCC accession number HB 9725. Was done. The wells of the 60-well reaction tray were then coated with HIV antigen by the following method. The recombinant protein HIV-1 p41 env protein pTB319 was added at a concentration of 1 μg / ml to 0. 1M 3- [cyclohexylamino] -1-propanesulfonic acid (CAPS buffer, pH 11) was added to each well and incubated at 40 ° C. for 2 hours. The wells were then filled with 400 μl of phosphate buffered saline (PBS, pH 7. Wash twice with 5), then use 0. The reaction was carried out with 1% Tween-20 (registered trademark) at 40 ° C. for 1 hour, and then blocked with PBS containing 3% BSA at 40 ° C. for 1 hour. The wells were then overcoated with 5% sucrose PBS for 20 minutes at room temperature and dehydrated. The pTB319 plasmid that produces the recombinant protein pTB319 is described in the subject of U.S. Patent Application No. 167,067 to Bolling and Mandecki, filed March 11, 1988, previously incorporated herein by reference, entitled "CKS Method of Protein Synthesis". is there. pTB319 was produced by inserting a synthetically produced DNA fragment encoding the carboxy-terminal 42 amino acids of HIV-1 P120 into plasmid pTB315, as described in Bolling and Mandecki (ibid). Example 2 Simultaneous Assay for HIV Antigen and HIV Antibody The two solid phases prepared by the method described in Example 1 are used in a detection assay for HIV antigen and / or HIV antibody in a test sample as follows. did. An HIV-1 seroconversion panel consisting of 65 serum samples from 9 HIV-1 infected patients undergoing seroconversion was used in the assay. In each well of the 60-well tray previously prepared in Example 1, 2% Tween 20® (Sigma Chemical Co. , St. Each serum sample was diluted by adding 150 μl of serum sample to 50 μl of sample diluent containing Louis, MO polyoxyethylene sorbitan). Beads previously coated with anti-HIV p24 antibody as described in Example 1 were then placed in each well containing serum samples. The wells of each tray were incubated at 40 ° C. for 60 minutes under continuous rotation. After incubation, Abbott Parallel Processing Center ^TM Each well of a 60-well reaction tray in 15 mL of deionized water (dH _Two Washed with O). 200 μl of HIV p24 antibody probe reagent (rabbit polyclonal F [ab '] _Two Antibody dilution containing anti-HIV-1 [active ingredient: anti-p24 antibody at a concentration of 2-6 μg / ml] (2.25% BSA, 7.5% calf serum, 7.5% goat serum, 25% human heavy mineralized plasma, 0.1% Sodium azide)) was added to each well / bead, and the resulting mixture was incubated at 40 ° C. for 60 minutes without rotation. Fill each bead / well of the reaction tray with 15 ml dH _Two Washed with O. A 200 μl conjugate dilution (0.18% Tris, 1.19% Tris) containing a mixture of recombinant HIV-1 p41 antigen labeled with horseradish peroxidase (pTB319 bound to HRPO) and HRPO-labeled goat anti-rabbit IgG antibody was then added. -HCl, 0.38% NaCl, 9.0% calf serum, 0.9% goat serum, 10.0% human heavy mineralized plasma, 4.5% Triton X-100®, 0.013% gentamicin sulfate, 0.009% thimerosal) in a reaction tray. Added to each bead / well and incubated at 40 ° C. for 60 minutes without rotation. Transfer each bead / well of the 60-well reaction tray to 15 ml dH as described above. _Two Washed with O. Then, 300 μl of o-phenylenediamine-2HCl (OPD) was added to each well / bead and incubated for 30 minutes at room temperature in the dark. Then, 300 μl of stop reagent (1N H _Two SO _Four ) Was added to each well / bead to stop the reaction. The reaction was read using an Abbott PPC measuring the optical density of the reaction at 492 nm with reference to 630 nm. The cut-off value was determined as 0.1 OD + mean OD of the negative control. Therefore, a serum sample was considered reactive (positive) if the sample / cutoff value was greater than 1. All 65 serum samples from the aforementioned nine patients were tested according to this procedure. The results obtained were compared with the HIV antigen assay (HIVAG®, available from the applicant) and the HIV antibody assay (human immunodeficiency virus types 1 and 2: E. coli and B. megaterium, recombinant antigen, Abbott HIVAB Results obtained from the same serum sample using HIV-1 / HIV-2 (rDNA) EIA (available from the applicant) according to the manufacturer's instructions specified for each product insert And compared. The data is shown in Table 1. In the table, “OD” means optical density reading, “S / CO” means sample / cutoff ratio, “Result” means test decision, “HIV-1 / 2 Ab HIV “-1 Ag Comb” indicates the assay of the present invention, “HIV-1 / 2 Ab” indicates the HIVAB® HIV-1 / HIV-2 (rDNA) EIA assay, and “HIV-1 Ag” indicates Abbott. 1 shows the HIVAG® assay. * nt: This sample from the seroconversion panel could not be used for testing. Comparing the results from three individual tests individually, the data in Table 1 show that the method of the invention is more sensitive than the HIV-1 / 2 Ab test or the HIV-1 Ag test. It is believed that the assay can be further optimized to detect HIV p24 antigen, which is present early in infection and late in HIV infection, and HIV antibodies that appear during seroconversion late in infection. Example 3 Single Solid Phase Coating Procedure In this procedure, a single solid phase was coated with HIV antigen and HIV antibody by the following method. Each well of a 96-well microtiter plate (Immulon 4®, DynateCh, Alexandria VA) contains monoclonal antibodies 31-42-19 (as described in Example 1), monoclonal antibodies 31-90-25 and Recombinant HIV-1 p41 antigen, designated pTB319, was coated with 0.1 M carbonate buffer (pH 9.5) at a concentration of 1 μg / ml for 2 hours at room temperature. The wells were then blocked with 300 μl of blocking reagent (consisting of 5% nonfat dry milk, 10 mM Tris [pH 8.0], 150 mM NaCl and 0.05% Tween-20®) for 1 hour at room temperature. Example 4 HIV antigen / antibody assay using a single solid phase The solid phase produced by the method described in Example 3 was used in an assay for detecting the presence of HIV antigen and / or HIV antibody in a test sample as follows. Used for Twelve seroconversion panels (Boston Biomedica Inc., Panel G, Boston MA) were tested for each serum sample and positive / negative controls. In individual wells of the microtiter plate, dilute 150 μl of each serum sample or positive or negative control with 50 μl of sample diluent (consisting of 15 μl Triton X-100® and 35 μl blocking reagent as described in Example 3). And incubated for 60 minutes at room temperature without rotation. After incubation, 300 μl of wash buffer (0.05% nonfat dry milk, 10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween) was used using Nunc 8-channel “immunowash” manifold (Nunc, Denmark). -20 (registered trademark)) eight times. Then, HIV p24 antibody probe reagent (rabbit polyclonal F [ab '] _Two Anti-HIV-1 [active ingredient: anti-p24 antibody at a concentration of 2-6 μg / ml]) Antibody dilution containing 175 μl (2.25% BSA, 7.5% calf serum, 7.5% goat serum, 25% human heavy mineralized plasma) , 0.1% sodium azide) was added to each well and incubated for 60 minutes at room temperature without rotation. After incubation, the wells were washed eight times with wash buffer as described above. Then, 150 μl of a conjugate dilution (described in Example 2) containing a mixture of HRPO-labeled recombinant HIV-1 p41 protein (pTB 319) and HRPO-labeled goat anti-rabbit IgG (described in Example 2) was added to each well. In addition, the plate was incubated at room temperature for 60 minutes without rotation. Wash wells 8 times with wash buffer (described above), then dH _Two Rinse with O. Then 125 μl of OPD substrate was added to each well and the wells were incubated for 10 minutes at room temperature in the dark. The reaction was stopped by adding 125 μl of stop reagent (described in Example 2). The absorbance of each well was read at 490 nm with reference to 630 nm. The cut-off value of 0.025 OD + mean OD of the negative control was determined. A sample was considered reactive (positive) if the sample / cut-off value was greater than 1. The data from these assays is shown in Table 2. In Table 2, "OD" means optical density reading, "S / CO" means sample / cutoff ratio, "Result" means test decision, and "NC" means negative control. And "PC" means a positive control. * pg / ml-picogram / ml As the data in Table 2 shows, the assay of the present invention can detect the presence of HIV antibodies and / or HIV antigens in a seroconversion panel. When compared individually with the HIV-1 / 2 antibody test and the HIV-1 antigen test, the method of the present invention had higher detection sensitivity than the single test based on the antigen or antibody test. Comparing the results of the method of the present invention shown in Table 2 with the results obtained by combining the HIV-1 / 2 antibody test and the HIV antigen test, the method of the present invention shows that all the samples reactive in each test were analyzed. Could be detected. Example 5 Coating Microparticles Simultaneously with HIV Antibody and HIV Antigen Capture Reagent Both monoclonal anti-HIV p24 antibodies (31-42-19 and 31-90-25) as described above and the recombinant HIV-1 p41 antigen (HIV- 1 The p41 recombinant protein pTB319 and the HIV-2 p41 recombinant protein pJC104) were combined, and each was prepared as a 0.01 M carbonate buffer (pH 9.5) at a concentration of 150 μg / ml (Seradyne Inc., Indianapolis, Indiana). Co., Ltd.) was simultaneously coated on a 0.5% suspension (weight / volume) of polystyrene fine particles at room temperature (15-30 ° C.) for 2 hours. The microparticle suspension was gently centrifuged and the microparticle pellet was resuspended in 0.05 M Tris buffer (pH 8.0) to remove excess unbound protein. This wash was repeated until there was no unbound protein. After blocking the microparticles with 10 mg / ml casein 0.01 M Tris (pH 8.0), 0.15 M NaCl at 56 ° C. for 18-24 hours, the microparticles were washed again as described above, 0.05 M Tris (pH 8.0), 0.15 M Diluted to a 0.015% suspension (weight / volume) in M NaCl, 1% BSA, 15% sucrose and 0.1% sodium azide. Example 6 Simultaneous detection of HIV antibody and HIV antigen using microparticles Any system using microparticles may be used. _x The <RTIgt; (R) </ RTI> microparticle enzyme immunoassay (MEIA) system is used. Abbo tt IM _x The MEIA system is available from Abbott IM (available from Abbott Diagnostic Division, a division of the applicant). _x It is described in detail in the (registered trademark) operation familiarization manual. In this assay, 100 μl of the 0.15% suspension prepared in Example 5 was mixed with 100 μl of a test sample suspected of containing HIV-1 and / or HIV-2 antibodies and / or HIV-1 antigen, and Abbott IM _x Incubate at 40 ° C. for 10 minutes in a reaction cell to generate a reaction mixture. HIV antibodies and / or HIV antigens bind to the microparticles in the antibody / antigen / microparticle complex. Transfer 150 μl of the reaction mixture onto a glass fiber matrix where the microparticles are retained by irreversible binding. The antibody / antigen / microparticle complex was then biotinylated recombinant HIV-1 and HIV-2 recombinant p41 antigen (pTB319 and pJC104 described above) and biotinylated F (Ab ') _Two The anti-HIV-1 p24 is reacted with 50 μl of a probe consisting of 0.05 M Tris (pH 8.0), 2% BSA, 0.25% saponin and 0.1% sodium azide for 10 minutes at 40 ° C. 50 μl of an antibody conjugate consisting of goat anti-biotin alkaline phosphatase in 0.1 M Tris (pH 8.0), 0.5 M NaCl, 0.9% Brij-35®, 1.0% BSA and 0.1% sodium azide, React with the biotin probe / antibody / antigen / particle complex at 40 ° C for 10 minutes. Next, these fine particle complexes were washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCl and 0.1% sodium azide, and the biotin probe / antibody / antigen fine particle complex was washed with methyl umbelliferyl phosphate as a substrate. After reacting with 50 μl of ze (MUP, the present applicant), the fluorescence of the product methylumbelliferone (MU) is measured. The MU generation rate is proportional to the concentration of the analyte in the test sample. Example 7 Separate Coating of Microparticles with HIV Antibody and HIV Antigen Capture Reagent In this example, various analyte capture reagents (Seradyne Inc., Indianapolis, Indiana) are separately coated on polystyrene microparticles. Each reagent may be coated separately or in various combinations. After coating each analyte capture reagent on its respective microparticle, the various coated microparticles are stored together and used in the assay. In this example, two types of monoclonal anti-HIV p24 antibodies (31-42-19 and 31) were placed on microparticles separate from microparticles simultaneously coated with recombinant HIV-1 and HIV-2 p41 antigens (pTB319 and pJC104). -90-25) is coated together. The exact amount can vary, but generally the coating procedure is approximately as described in Example 5. After blocking physically separated microparticles with 0.01 M Tris (pH 8.0), 10 mg / ml casein in 0.15 M NaCl at 56 ° C. for 18-24 hours, the microparticles were as described in Example 5. And stored together and diluted to a 0.015% suspension (wt / vol) in 0.05M Tris (pH 8.0), 0.15M NaCl, 1% BSA and 15% sucrose. During the storage stage, the microparticles may be stored in various ratios to affect the sensitivity and specificity of the assay to optimize the use of the microparticles. Example 8 Simultaneous Detection of HIV Antibody and HIV Antigen on Microparticles Separately Coated with HIV Antibody and HIV Antigen Capture Reagent Any system using microparticles may be used. _x The <RTIgt; (R) </ RTI> microparticle enzyme immunoassay (MEIA) system is used. Abbo tt IM _x The MEIA system is available from Abbott IM (available from Abbott Diagnostic Division, a division of the applicant). _x It is described in detail in the (registered trademark) operation familiarization manual. In this assay, 100 μl of the 0.15% suspension prepared in Example 5 was mixed with 100 μl of a test sample suspected of containing HIV-1 and / or HIV-2 antibodies and / or HIV-1 antigen, and Abbott IM _x Incubate at 40 ° C. for 10 minutes in a reaction cell to generate a reaction mixture. HIV antibodies and / or HIV antigens bind to the microparticles in the antibody / antigen / microparticle complex. Transfer 150 μl of the reaction mixture onto a glass fiber matrix where the microparticles are retained by irreversible binding. The antibody / antigen / microparticle complex was then biotinylated recombinant HIV-1 and HIV-2 recombinant p41 antigen (pTB319 and pJC104 described above) and biotinylated F (Ab ') _Two Anti-HIV-1 p24 is reacted with 50 μl of a probe consisting of 0.05 M Tris (pH 8.0), 2% BSA, 0.25% saponin and 0.1% sodium azide at 40 ° C. for 10 minutes. 50 μl of an antibody conjugate consisting of goat anti-biotin alkaline phosphatase in 0.1 M Tris (pH 8.0), 0.5 M NaCl, 0.9% Brij-35®, 1.0% BSA and 0.1% sodium azide And a complex of biotin probe / antibody / antigen / fine particle at 40 ° C. for 10 minutes. Next, these fine particle complexes were washed six times with 0.05 M Tris (pH 8.0), 0.3 M NaCl and 0.1% sodium azide, and the biotin probe / antibody / antigen fine particle complex was washed with methyl umbelliferyl phosphate as a substrate. After reacting with 50 μl of ze (MUP, the present applicant), the fluorescence of the product methylumbelliferone (MU) is measured. The MU generation rate is proportional to the concentration of the analyte in the test sample. The assay of the present invention may be further optimized using different combinations of antigen or antibody capture or probe reagents, varying the assay conditions and / or incubation times, and other methods, reagents and conditions known to those skilled in the art. It is thought that it can be converted. Thus, various other antibody capture reagents (eg, HIV p24, gp120, gp160, p17, etc.) can be used. Changing the antibody capture reagent may require the use of a different antigen capture reagent. All of these variations are considered to be within the scope of the present invention. Furthermore, although some of the assays described in the examples used automated systems, it is well within the scope of the present invention that manual methods or other automated analyzers can be used or adapted in the assays of the present invention. Is within.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Stone, Barbara, L             United States, Illinois 60035, High             Land Park, Dayfield Ro             Code 771 (72) Inventors Liyu, Douglas J.             Deer, Illinois 60640, United States             Go, North Wayne 5447 (72) Inventors Harrington, Susan K             United States, Illinois 60087, Wolves             Kugan, Stanley Avenue 322 (72) Inventor Dawson, Jyoji Jei             United States, Illinois 60048, Riva             Tevil, Spree Use Wood, 15670 (72) Inventor Wu Ping             United States, Illinois 60035, High             Land Park, Oak Street             2680

Claims (1)

[Claims] 1. a) a test sample comprising: i) at least one HIV antibody capture reagent specific for an HIV antibody analyte and attached to a solid phase; ii) at least one HIV antibody analyte specific and attached to a solid phase. One HIV antigen capture reagent, iii) an HIV antibody indicator reagent that is specific for an HIV antibody analyte and consists of one member of a binding pair labeled with a signal generating compound, and iv) an HIV antigen analyte. An HIV antigen-indicating reagent / HIV antibody-indicating substance complex and / or an HIV-antigen-capturing reagent complex that is simultaneously or sequentially contacted with an HIV antigen-indicating reagent consisting of one member of a binding pair labeled with a signal-generating compound, and / or an HIV antigen-capturing reagent And / or b) detecting said signal-generating compound associated with said complex to detect an HIV antibody analyte and / or HIV antigen in a test sample. Determining the presence or absence of an analyte An assay for simultaneously detecting the presence or absence of an HIV antigen analyte and / or an HIV antibody analyte that may be present in a test sample. 2. a) a test sample comprising: i) at least one HIV antibody capture reagent specific for an HIV antibody analyte and attached to a solid phase; ii) at least one HIV antibody analyte specific and attached to a solid phase. One HIV antigen-capturing reagent, and iii) an HIV antigen-indicating reagent that is specific to the HIV antigen analyte and is one member of a binding pair labeled with a signal-generating compound, and is simultaneously contacted with the HIV antigen-indicating reagent to form a first mixture. B) removing said first mixture for a time and under conditions sufficient to produce an HIV antibody capture reagent / HIV antibody analyte complex and an HIV antigen capture reagent / HIV antigen analyte / HIV antigen indicator reagent complex; Incubating; c) contacting said complex with an HIV antibody indicator reagent which is specific for said HIV antibody analyte and is one member of a binding pair labeled with a signal producing compound to form a second mixture; d. ) HIV antibody capture reagent / HIV antibody analyte / HIV antibody Incubating said second mixture for a time and under conditions sufficient to produce a body indicator reagent complex, and e) detecting said signal producing compound associated with said complex by detecting said HIV antigen in a test sample. An assay for simultaneously detecting the presence or absence of an HIV antigen analyte and / or an HIV antibody analyte in a test sample, comprising the step of determining the presence or absence of an analyte and / or the HIV antibody analyte. 3. The assay according to claim 1 or 2, wherein the HIV antibody capture reagent and the HIV antigen capture reagent bind to the same solid phase. 4. The assay according to claim 1 or 2, wherein the HIV antibody capture reagent binds to a first solid phase and the HIV antigen capture reagent binds to a different second solid phase. 5. The HIV antigen analyte is an HIV p24 gag antigen, and the HIV antigen capture reagent is a monoclonal antibody, a polyclonal antibody, a recombination-induced antibody, a monoclonal antibody fragment, a polyclonal antibody fragment, and a group consisting of fragments of a recombination-induced antibody. The assay according to claim 1 or 2, which is an anti-HIV p24 antibody selected from the group consisting of: 6. The assay according to claim 5, wherein the monoclonal anti-HIV p24 antibody fragment and the polyclonal anti-HIV p24 fragment are F (Ab ') ₂ anti-HIV p24 antibody fragments. 7. 7. The assay of claim 6, further comprising a hapten attached to the F (Ab ') ₂ fragment. 8. The assay according to claim 5, wherein the anti-HIV p24 antibody is a mixture of monoclonal antibodies secreted by the hybridoma cell lines ATCC accession numbers HB9725 and HB9726. 9. The HIV antibody analyte is an anti-HIV-1 antibody and / or an anti-HIV-2 antibody, and the HIV antibody capture reagent is a mixture of HIV-1 p41 env protein and HIV-2 p41 env protein. The assay according to 2. 10. 10. The assay according to claim 9, further comprising a hapten attached to said HIV-1 p41 env protein and said HIV-2 p41 env protein. 11. The assay according to claim 10, wherein the HIV-1 p41 env protein and the HIV-2 p41 env antigen are produced recombinantly or synthetically. 12. The HIV antibody capture reagent comprises at least one recombinant HIV-1 p41 antigen and at least one HIV-2 p41 antigen, and the HIV antigen capture reagent comprises at least one anti-HIV p24 antibody. 3. The assay according to 1 or 2. 13. 13. The assay of claim 12, wherein said anti-HIV p24 antibody binds to a first solid phase and said recombinant HIV-1 p41 antigen and HIV-2 p41 antigen bind to different second solid phases. 14． a) A mixture obtained by contacting a test sample with a solid phase to which at least one anti-HIV-1 p24 antibody, at least one recombinant HIV-1 p41 antigen and at least one HIV-2 p41 antigen are attached. B) anti-HIV-1 p24 antibody / HIV-1 p24 antigen complex and / or HIV-1 p41 antigen / anti-HIV-1 p41 antibody and / or HIV-2 p41 antigen / anti-HIV-2 p41 antibody Incubating the mixture for a time and under conditions sufficient to form a complex; c) i) generating an anti-HIV-1 p24 antibody / HIV-1 p24 antigen / anti-HIV-1 p24 antibody complex; Contacting the anti-HIV-1 p24 antibody / HIV-1 p24 antigen complex with an anti-HIV-1 p24 antibody capable of specifically binding to the HIV-1 p24 antigen under sufficient time and conditions; ii) signal An indicator reagent consisting of an anti-species antibody labeled with a product compound is brought into contact with the anti-HIV-1 p24 antibody / HIV-1 p24 antigen / anti-HIV-1 p24 antibody complex, and iii) testing the generated signal HIV-1 p24 antigen in sample To determine the presence of the HIV-1 p24 antigen in the test sample by detecting that it is present; and d) i) the HIV-1 p41 antigen / anti-HIV-1 p41 antibody complex and / or Contacting the HIV-2 p41 antigen / anti-HIV-2 antibody complex with an indicator reagent comprising an HIV-1 p41 antigen labeled with a signal generating compound and an HIV-2 p41 antigen labeled with a signal generating compound; and ii) generating The detected signal is detected as an indication of the presence of the anti-HIV-1 p41 antibody and / or the HIV-2 p41 antibody in the test sample, whereby the anti-HIV-1 p41 antibody and / or anti-HIV in the test sample is detected. -2 An assay for simultaneously detecting the presence or absence of the HIV-1 p24 antigen, anti-HIV-1 p41 antibody and anti-HIV-2 p41 antibody in a test sample, which comprises determining the presence or absence of the p41 antibody. 15. a) A mixture obtained by contacting a test sample with a first solid phase to which at least one anti-HIV-1 p24 antibody is attached, and a second solid phase to which recombinant HIV-1 p41 antigen and HIV-2 p41 antigen are attached B) anti-HIV-1 p24 antibody / HIV-1 p24 antigen complex and / or HIV-1 p41 antigen / anti-HIV-1 p41 antibody complex and / or HIV-2 p41 antigen / anti-HIV-2 incubating the mixture for a time and under conditions sufficient to produce a p41 complex; c) i) producing an anti-HIV-1 p24 antibody / HIV-1 p24 antigen / anti-HIV-1 p24 antibody complex Contacting the anti-HIV-1 p24 antibody / HIV-1 p24 antigen complex with an anti-HIV-1 p24 antibody capable of specifically binding to the HIV-1 p24 antigen for a time and under conditions sufficient for: ii) Contacting an indicator reagent consisting of an anti-species antibody labeled with a signal-generating compound with the anti-HIV-1 p24 antibody / HIV-1 p24 antigen / anti-HIV-1 p24 antibody complex, and iii) generating the signal HIV-1 p24 antigen in test sample The presence or absence of the HIV p24 antigen in the test sample by detection as an indication of its presence; and d) i) the HIV-1 p41 antigen / anti-HIV-1 p41 antibody complex and / or HIV-2 contacting the p41 antigen / anti-HIV-2 p41 antibody complex with an indicator reagent comprising an HIV-1 p41 antigen labeled with a signal generating compound and an HIV-2 p41 antigen labeled with a signal generating compound; and ii) the generated signal By detecting as an indication that the anti-HIV-1 p41 antibody and / or HIV-2 p41 antibody is present in the test sample, the anti-HIV-1 p41 antibody and / or HIV-2 p41 antibody in the test sample Assay for simultaneously detecting the presence of an HIV-1 p24 antigen and / or an anti-HIV-1 p41 antibody and / or an anti-HIV-2 p41 antibody in a test sample, which comprises determining the presence or absence of the antibody. 16. The assay according to claim 14 or 15, wherein the anti-HIV-1 p24 antibody is a mixture of monoclonal antibodies secreted by the hybridoma cell lines ATCC accession numbers HB9725 and HB9726. 17． a) at least one solid phase to which an HIV antigen capture reagent comprising at least one anti-HIV p24 antibody and an HIV antibody capture reagent comprising at least one HIV p41 antigen are attached; b) HIV antigen analyte An indicator reagent for an HIV antigen analyte, which is a specific member of a binding pair labeled with a signal-generating compound, and c) a member of a binding pair labeled with a signal-generating compound, which is specific for an HIV antibody analyte. A test kit for simultaneously detecting an HIV antigen analyte and / or an HIV antibody analyte in a test sample comprising an HIV antibody analyte indicating reagent comprising: 18. The HIV antigen capture reagent comprises at least one anti-HIV p24 antibody attached to a first solid phase, and the HIV antibody capture reagent comprises at least one HIV p24 antigen attached to a second solid phase. The test kit of claim 17.