WO1987007957A1

WO1987007957A1 - Low-level detection of human immunodeficiency virus

Info

Publication number: WO1987007957A1
Application number: PCT/US1987/001510
Authority: WO
Inventors: Jacques Homsy; Jay A. Levy; Cecilia Cheng-Meyer
Original assignee: The Regents Of The University Of California
Priority date: 1986-06-20
Filing date: 1987-06-19
Publication date: 1987-12-30

Abstract

An immunoassay method for detecting low levels of Human Immunodeficiency Virus (HIV) protein in a fluid test sample comprising: (a) providing an indicator sample comprised of a purified and labeled indicator protein, said indicator protein being a synthetic protein that is immunologically cross-reactive with a selected HIV protein; (b) providing immobilized antibody on a solid support that biospecifically binds said selected HIV protein and said indicator protein; (c) incubating said immobilized antibody with said test sample; (d) incubating said immobilized antibody with said indicator sample after or simultaneously with said incubation with the test sample to provide indicator-contacted antibody; (e) separating any unbound indicator protein from said indicator-contacted antibody to provide separated antibody; and (f) measuring a signal related to the amount of said label bound to said separated antibody. Devices and test kits for use in the above immunoassay are also disclosed.

Description

LOW-LEVEL DETECTION OF HUMAN IMMUNODEFICIENCY VIRUS

Technical Field The present invention relates to an immunoassay for the detection of low levels of Human Immunodeficiency Virus protein.

Background of the Invention Acquired Immune Deficiency Syndrome (AIDS) is a disorder which has recently begun spreading in epidemic fashion within the United States, Europe and Central Africa. A human retrovirus given several different names, including Lymphadenopathy-Associated Virus (LAV), Human T-Lymphophotrophic Virus type ill (HTLV-III), and AIDS-Related Virus (ARV) , has been identified as the causative agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983) Science 220: 868-871; Fauci et al. (1984) Ann. Int. Med. 100: 92-106; Montagnier et al. iji Human T-Cell Leukemia Viruses, pp. 363-379 (Cold Spring Harbor Laboratory 1984); Popovic et al. (1984) Science 224: 497-500; Levy et al. (1984) Science 225: 840-842. ARV, LAV and HTLV-III are all strains of the same virus, which has been renamed Human Immunodeficiency Virus (HIV) by the International Committee on the Taxonomy of Viruses.

The discovery and characterization of HIV has prompted efforts to develop sensitive and specific assays for assessing infection by this virus., Although an indirect enzyme-linked immunoassay (ELISA) for detection of antibodies to HIV has been developed and is widely used for epidemiological screening, many questions have remained regarding the clinical significance of the presence of such antibodies in both symptomatic and asymptomatic individuals. J. Carran (1986) Science. Not only can the virus not be consistently isolated from antibody-positive individuals, but some cases of virus-positive, antibody-negative subjects have been reported. Levy et al. (1985) Ann. Int. Med. 103:694-699.

This lack of a clear correlation between antibody-positive individuals and active infection underscores the need for an assay that directly tests for the presence of HIV in individuals. The current method for determining whether an individual is actively infected is a reverse transcriptase (RT) assay. In this assay, a cell culture is infected with a sample from an_- individual, and then assayed every 3 days (up to.45 days) -for reverse transcriptase activity indicative of retrovirus growth. This method is prohibitively expensive for large-scale application. The major cost factor in the assay results from the inability to measure RT activity for a virus, such as HIV, directly in- clinical samples because the virus occurs in extremely low titer in infected individuals. This low titer requires the virus to be amplified by growing it in culture for up to 45 days with continued monitoring for RT activity. Amplification and handling adds greatly to the cost of the assay. Thus, it is not suitable for use diagnostically for a large number of patients suspected of being actively infected with HIV.

Recently, capture ELISA assays that detect the presence of viral antigen in culture supernatants have been developed. See. e.g., McDougal et al. (1985) J. Immun. Meth. 7_6.: 171-183; Higgins et al. (submitted for publication) . The nature of this sandwich capture system does not allow a sufficient sensitivity to detect the presence of virions any better than the widely used RT assay. Thus, it is unlikely that this assay could be employed diagnostically without the costly amplification ste .

A need exists, therefore, for an assay which directly detects the presence of HIV particles or protein in body fluid, as opposed to merely antibodies to HIV, and which does not require the time consuming and costly step of amplification. It is also desirable to employ an assay which does not require large volumes of body fluid from a patient. Thus, the desired assay should only require body fluid samples of 1 illiliter or less, and be able to detect the presence of viral protein at concentrations in the picomolar range. ^' It would also be extremely desirable if a sensitive assay could be developed that does not require radioactive labeling, but could use labels such as enzymes which produce a color change to indicate positive or negative results. Assays employing optical density from color development as the indicator signal, as opposed to radiation, will have greater applicability in a clinical setting since the user does not have to be equipped to handle radioactive materials.

Summary of the Invention It has been surprisingly discovered that a competition assay can provide, without amplification, a direct and quantitative assessment for the presence of HIV in fluid samples, such as those prepared from human body fluids, with enhanced sensitivity and good reliability. It has been discovered that it is possible to determine the presence or absence of viral protein in test samples having a concentration in the picomolar range. Furthermore, it has been determined that this sensitivity is retained even when the signal employed in the assay is color intensity and that there is no cross-reactivity with other human retroviruses. Thus, the assay of the present invention provides for the first time a diagnostic tool which can be employed to directly test for the presence of HIV in the body fluids of human patients without the expensive step of amplification.

The present invention is described in one or more of the following embodiments, including embodiments apparent therefrom to those of ordinary skill in the art.

In one embodiment, the present invention provides an immunoassay method for detecting low levels, of HIV protein in a fluid test sample comprising: (a) providing an indicator sample comprised of a labeled indicator protein, said indicator protein being a synthetic protein that is immunologically cross-reactive with a selected HIV protein; (b) providing immobilized antibody on a solid support that biospecifically binds said selected HIV protein and said indicator protein; (c) incubating said immobilized antibody with said test sample; (d) incubating said immobilized antibody with said indicator sample after or simultaneously with said incubation with the test sample to provide indicator-contacted antibody; (e) separating any unbound indicator protein from said indicator-contacted antibody to provide separated antibody; and (f) measuring a signal related to the amount of said label bound to said separated antibody. In another embodiment, the present invention is directed to an immunoassay method for directly detecting the presence of HIV protein in a fluid test sample prepared from a source selected from the group consisting of human cells and human body fluids comprising: (a) providing an indicator sample comprised of a labeled indicator protein, said indicator protein being a synthetic protein that is immunologically cross-reactive with a selected HIV protein; (b) providing immobilized antibody on a solid support that biospecifically binds said selected HIV protein and said indicator protein; (c) incubating said immobilized antibody with said test sample to provide test sample-contacted antibody; (d) incubating said test sample-contacted antibody with said indicator sample to provide indicator-contacted antibody; (e) separating any unbound indicator protein from said indicator-contacted antibodyt to provide separated antibody; and (f) measuring a signal related to the amount of said label bound to said separated antihody.

In another embodiment the present invention provides a device for use as a solid test support in an immunoassay method for the presence of Human Immunodeficiency Virus (HIV) gag protein in a fluid sample comprising a solid support member with a receptacle for receiving said fluid sample, the surface portion of said receptacle that contacts said fluid sample having immobilized thereon anti-HIV gag protein antibody in an amount that is partially, detectably and biospecifically saturated in antigen binding sites when contacted with a reference amount of a recombinant HIV gag protein, said reference amount in the range of about 4 picomoles to about 10 nanomoles. In yet another embodiment, the present invention is directed to a test kit for an immunoassay for detecting the presence of HIV protein in a fluid sample comprising the above device, an indicator mixture comprising recombinant HIV gag protein having a detectable label, and a substrate that produces a color change in the presence of an enzyme bound to said recombinant HIV gag protein.

These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art based upon the following disclosure.

Brief Description of Figures

Figure 1 shows the percent inhibition of binding for labeled, recombinant HIV p25gag by serial dilutions of unlabeled p25gag with various amounts of immobilized anti-HIV antibody.

Figure 2 is a schematic diagram of one embodiment of the present assay. In the depicted embodiment, the label is biotin and the signal (optical density from color development) arises from a reaction of an avidin-enzyme conjugate with a substrate.

Figure 3 shows the percent inhibition of binding in the present assay for labeled recombinant HIV p25gag by serial dilutions of test samples prepared from lysates of HIV-infected HUT-78 cells, unlabeled recombinant p25gag, supernatant of HIV-infected PMC or

HUT-78 (HIV fluid), normal serum spiked with HIV fluid. and HTLV-1 protein as a control.

Detailed Description of the Invention

The present invention provides a heterogeneous immunoassay for directly detecting the presence of Human Immunodeficiency Virus (HIV) protein in fluid samples. such as those prepared directly from human body fluids, human cells, and supernatant from human cell cultures. The assay of the present invention is sensitive enough to detect specifically HIV protein in fluid samples at levels in the picomolar (picogram per sample) range. The need to amplify HIV population in test samples, therefore, is eliminated. The assay of the present invention is quite rapid, requiring only a few hours to perform, as opposed to the 30 days required when amplification is necessary. The assay of the present invention is also sensitive enough to avoid the use of radiolabels, if desired. Since the assay of the present invention is inexpensive, fast, sensitive and can be employed with a color signal, it is suitable for the wide scale application of diagnosing active HIV infections.

In its broad aspects, the present invention is directed to a heterogeneous competition assay that can detect the presence of extremely low levels of HIV protein in a fluid sample. The general methodology of competition assays is well-known in the art. It is usually based upon the competition between a labeled and unlabeled antigen for a limited amount of an immobilized antibody. In general, the assay of the present invention comprises contacting immobilized antibodies to a selected HIV protein with the fluid sample, and also contacting immobilized antibodies (simultaneously or afterwards) with a sample of a synthetic indicator protein that is immunologically cross-reactive with the selected HIV protein, but not with other retroviruses (e.g.. a unique recombinant HIV protein). After contacting the indicator protein with the immobilized antibody, unbound indicator protein is separated therefrom and the amount of indicator remaining bound to the immobilized antibody is measured. Using a negative control (i.e., a test sample having no HIV protein) as a reference, a reduction in the amount of indicator protein bound to the immobilized antibody indicates the presence of HIV protein in the test sample. By adjusting the amount of anti-HIV antibody employed in the assay, one can make the assay of the present invention quantitative down to an extremely low level (e.g., about 1 pM) . HIV, alternatively known as ARV, LAV and

HTLV-III, is a fairly well characterized virus. See, e.g., Pescador et al. (1985) Science 227: 484-492; ain-Hobson et al. (1985) Cell 40.: 9-17; Muesing et al. (1985) Nature 313: 450-458; Ratner et al. (1985) Nature 313: 277-284. Numerous HIV proteins have been identified in the internal core region of the virus (the gag region), including p25gag (sometimes referred to as p24), p55gag, pl6-18gag and p7-8gag. Two proteins in the.^' envelope protein region (the env region). gpl20env and gp41env, have been identified as well as their precursor, gpl60env. Several proteins, including reverse transcriptase, are encoded in the pol region, including p65pol, p48pol and p31pol.

The assay of the pres-ent invention is based on determining the presence or absence of one or more of the above viral proteins in a fluid sample. The selection of the appropriate HIV protein for the assay of the present invention is. based on the present disclosure, within the skill of the art. Various candidates can be screened in the assay described herein and their suitability readily determined. The selection of an appropriate protein will, of course, depend in part upon the intended application of the assay. For example, in certain circumstances it may be desirable to base the present assay on env proteins because of the strong antibody response in patients to envelope proteins. In an assay for diagnostically detecting the presence of virus in body fluids, it is desirable to use a protein that is both abundant and immunologically conserved among the various strains of HIV. Thus, it is preferred to test for the presence or absence of an internal core or gag protein of HIV, particularly p25gag. It may also be desirable to base the assay on more than one viral protein (e.g., using 2 or more indicator proteins). Another factor which can affect the selection of viral protein is the availability of immunologically cross-reactive indicator proteins or antibody, as described below. The immunoassay of the present invention is suitable for testing any appropriate fluid sample suspected of containing HIV particles or protein. The fluid samples may be prepared, for example, from any cell capable of being infected by HIV (e.g., HUT-78, H9, Jurkat, T-helper, glial cells, etc.) or any body fluid (e.g.. blood, saliva, semen, etc.) capable of containing the virus. Preferred sources from which fluid test samples are prepared include, but are not limited to, supernatant from human cell cultures susceptible of infection by HIV, human serum, and human peripheral mononuclear cells(PMC) containing the T-helper cells infected by HIV.

For diagnostic purposes, the source of fluid samples is a clinical sample, generally either serum or PMC (including T-cells) from a single human patient. An inherent advantage of the present invention is that these clinical samples need not be used to infect cells grown in culture to amplify the virus concentration; i.e.. the samples or the source of the samples can be "unamplified". HIV producing individuals will have very low concentrations of virus in these sources. Test samples prepared therefrom will sometimes have as little as about 0.01 pg/τιl viral protein (i.e., about 1 pg of viral protein in a 75 μl sample). The above concentration, as well as all concentrations of viral protein herein, unless otherwise indicated, refer to the concentration of the protein in the total and maximum volume at incubation with the immobilized antibody prior to the separation or washing step (referred to herein as the test or reference volume).

Fluid test samples are prepared according to their source. It is generally preferred to concentrate the virus in the sample if possible, for example, by centrifugation. Other treatments will depend upon the nature of the viral protein chosen. In assays using serum or cell culture supernatant as the source of the test sample, the serum or supernatant can be centrifuged:" in order to concentrate viral particles. The fraction containing the denser viral particles can be collected and used as the test sample. If the source of the test sample is cells, such as PMC, intact cells can be collected by centrifugation and then lysed, clarified by centrifugation, and the supernatant recovered for testing. When the assay is based o a core protein, it is preferred to expose the protein by disrupting the virus if the sample does not already contain exposed core protein. This can be done by any appropriate method, such as sonication or detergent treatment. The method of preparing test samples is, based on the present disclosure, within the skill of the art.

In general, test samples are prepared so that the test volume is one milliliter or less. The preferred embodiment of the present invention is a micro-ELISA. Thus, test samples are prepared so that the final volume at which incubation with the antibody occurs is about 250 μl or less, and preferably in the range of about 50 μl to about 100 μl. The present invention employs an immobilized antibody to bind any of the chosen HIV protein in the test sample. The selection of the appropriate antibodies is within the skill of the art, and can include monoclonal and polyclonal antibodies. Since the titer of virus in the source of samples is generally quite low, it is preferred to use polyclonal antibody to the HIV protein in order to maximize antigen/antibody complex formation. As used herein, "polyclonal" antibody refers to conventional polyclonal antibodies (e.g., prepared from antiserum) , or a mixture of monoclonal antibodies. Polyclonals can be readily isolated from the pooled sera of antibody-positive individuals by standard techniques, or from antisera produced in an animal of choice. For example, rabbits may be immunized against HIV protein by serial exposure to the HIV protein through injection. Antibody is then prepared from sera of the immunized animals and partially purified by, for example, salt fractionation. The partially purified fraction will contain polyclonal anti-HIV protein antibody. Additional purification, by conventional techniques, may be desired.

The antibodies employed in the present invention can be immobilized on an appropriate solid test support by any appropriate technique. The solid test support can be any suitable insoluble carrier material for the binding of antibodies in immunoassays. Many such materials are known in the art. including but not limited to, nitrocellulose sheets or filters; agarose, resin, plastic (e.g., PVC, polystyr ne) or metal beads; plastic vessels; and the like. Many methods of immobilizing antibodies are known in the art. See, e.g., Silman et al. (1966) Ann. Rev. Biochem. 3_5_:873; Melrose (1971) Rev. Pure & App. Chem. 21:83; Cuatrecasas et al. (1971) Meth. Enzym. Vol. 22. Such methods include covalent coupling, direct adsorption, physical entrapment, and attachment to a protein-coated surface. In the latter method, the surface is first coated with a water-insoluble protein such as zein, collagen, fibrinogen, keratin, glutelin, etc. The antibody is attached by simply contacting the protein-coated surface with an aqueous solution of the antibody and allowing it to dry.

In general, the test support has a receptacle adapted to received the fluid test sample. Usually the receptacle has a volume in the neighborhood of about 1 ml to about 0.25 ml, or less. The surface of the receptacle which comes into contact with the fluid test—.^" samples has the anti-HIV protein immobilized thereon. In a preferred embodiment, the surface of the the receptacle which contacts the fluid test sample is also coated with a protein which is non-reactive with the HIV protein. This minimizes non-specific binding of the HIV protein of interest to the test support, thus maximizing the amount of the HIV protein from the test sample available for occupying antigen binding sites of the immobilized antibody.

Any combination of support and binding technique which leaves the antibody immunoreactive, yet sufficiently immobilized so that it is retained with any bound antigen during a washing, can be employed in the present invention. A preferred solid test support is a conventional plastic immunoassay plate or microtiter plate with wells of about 250μl volume coated with protein A, followed by absorption of the anti-HIV antibody. The wells are then coated with blocking protein (e.g., gelatin or bovine serum albumin).

The amount of immobilized anti-HIV antibody employed is important in determining the lower level of sensitivity of the assay. The level of sensitivity, as well as the quantitative range, are a matter of choice. The level of the HIV protein is usually so low in test samples prepared from human body fluids and cells, that it is important to minimize the number of available antigen binding sites on the test support in order to observe a detectable decrease in indicator protein binding. The amount of antibody employed is also selected based on the desire to have the assay quantitative over a specified range. For example, when the assay is designed to be quantitative in the neighborhood of about 1 pM, it will not be quantitative in a range around 100 pM; the 1 pM assay for a sample. ^■ containing lOOpM will still be qualitative because the assay will clearly show 100% inhibition of binding.

In the practice of the present invention for diagnostic purposes, an amount of antibody is immobilized on the test support that will be biospecifically, partially and detectably saturated at a selected concentration of HIV or indicator protein, generally about 100 pM or less, referred to herein as the reference or test concentration. This corresponds to about 2.5 pg/μl p25gag, which has a molecular weight of about 25kd. Since the indicator protein is selected to be immunologically cross-reactive with the HIV protein, the same amount of antibody should bind equal titers of HIV and indicator protein. In preferred embodiments, the amount of antibody is selected so as to be biospecifically, partially and detectably, saturated at reference concentrations of 10 pM or less, 1 pM or less, and 0.5 pM or less.

As used herein, the antigen binding sites of the antibody are "biospecifically" bound or saturated when an antigen/antibody complex is formed. The antigen binding sites of the antibodies are "partially" or "fully" saturated as used herein, when either a portion or all, respectively, of the possible antigen/antibody complexes for a given amount of antibody are formed. Antibody is "detectably" saturated or bound by a first antigen, as used herein, when a sufficient portion of the available antigen binding sites of the antibody are occupied by the first antigen, so that a second and immunologically cross-reactive antigen bearing a label gives a signal which indicates that less of the second antigen is bound when compared to the amount of second antigen bound when none of the antigen binding sites are occupied by the first antigen; i.e.. the presence of theT first antigen is measurable above background in a competition assay. For example, when the label is color intensity, generally about 10-20% or more of the antigen binding sites will have to be occupied before a detectable difference is noticed.

When the source of the anti-HIV protein antibody is polyclonal antisera, it will usually be necessary to calibrate the amount of immunoglobulin immobilized on the test support for each batch of antiserum because the titer of anti-HIV antibody in each batch may vary. Most sources of polyclonal antisera are heterogeneous. It is straightforward, however, to determine an appropriate amount by titration against a reference sample containing either native HIV protein or the indicator protein of choice. Thus, the determination of the appropriate amount of protein immobilized on the test support can, in view of the present disclosure, be readily determined by one of ordinary skill.

As discussed above, the assay of the present invention employs competition for the binding sites of the immobilized antibody between a synthetic indicator protein and any cross-reactive HIV protein in the sample. Thus, the indicator protein is "immunologically cross-reactive" with the chosen HIV protein; i.e. the indicator protein and the selected HIV protein have a common epitope which is recognized by the chosen anti-HIV protein antibodies and unique to HIV. The affinities of the antibody for each of the proteins is preferably comparable. While purified native HIV protein could in theory be employed, its production is in a sufficiently purified state is not practical. It is preferred to use synthetic HIV protein produced by recombinant or chemical methods to guarantee the specificity of the assay. The use of recombinant protein is particularly preferred since it can be prepared inexpensively compared to chemical methods.

Recombinant protein will often times be slightly different from native protein in, for example, terminal amino acid sequences or glycosilation. It can be produced, however, in such a way as to mimic immunologically the HIV protein. The recombinant indicator protein could be completely or partially homologous to native HIV protein amino acid sequence since it need only preserve the common epitope(s). It is preferred to employ an indicator protein that is substantially homologous to a native HIV protein, particularly when polyclonal antibody is employed. The production of recombinant HIV protein in known in the art. See, e.g., Cabradilla et al. (1986) Bix>/Technology 4_:128-133; Chang et al. (1985) Bio/Technology 3_:905-909; Crowl et al. (1985) Cell 4^:979-986; Chang et al. (1985) Science 228:93-96; Chang et al. (1985) Nature 315:151-154; Pescador et al. (1985) Science 227:484-492. Expression of a p25gag protein is taught in Stei er et al. (1986) Virology 150:283-290. and European Patent Application Publication No. 181,150 (Application No. 85307860.8, published 14 May 1986), as well as both yeast and bacterial expression systems. The selection of the appropriate expression system, e.g. bacterial, yeast, mammalian cells, etc., is within the skill of the art.

In the practice of the assay disclosed herein, a signal related to the amount of indicator protein bound by the immobilized antibody is measured. This generally requires some detectable label to be associated with the indicator protein incubated with the antibody. .The label can be any type that allows for the^" detection of the protein. For example, it can be the label itself which directly produces a measurable signal, or the label can be later associated specifically with a second label which produces a measurable signal. Examples of the former are radiolabeled indicator proteins (e.g. 35S, 14C, etc.), or fusion proteins composed of the antigenic indicator protein and another amino acid sequence derived from an enzyme which retains its active properties. The latter form of labeling (where the label on the indicator protein does not directly produce the measured signal) is exemplified by a fusion protein wherein the heterologous amino acid sequence is recognized by an antibody that does not bind native HIV protein from the test sample. This allows detection of the indicator protein by contact with antibo.dies (labeled with a detectable signal) to the heterologous portion of the fusion protein. Another example of indirect labeling is the avidin-biotin system in which the indicator protein would be biotinylated, and later contacted with avidin conjugated to a detectable signal..

The label on the indicator protein allows a signal to be measured which is related to the amount of indicator protein in a sample. Common signals are radiation levels (when radioisotopes are used), optical density (e.g., enzyme reactions) and fluorescence (fluorescing compounds). It is preferred, as mentioned above, to employ a signal such as optical density (or color intensity) which does not require the handling of radioactive materials. Color intensity related to the amount of indicator protein in the sample can be produced, for example, by using the avidin-biotin labeling system described above, where the indicator protein is biotinylated and the avidin is conjugated to an enzyme that produces a color reaction.

Alternatively, one can use an enzyme labeled antibody that is biospecific for the indicator protein, but not biospecific for native HIV protein (e.g., biospecific for the heterologous portion of a fusion indicator- protein).

Numerous enzymes/substrate combinations are known in the immunoassay art that can produce a suitable signal, such as color intensity or development. See, e.g., U. S. Pat. NOS 4,323,647 & 4.190,496, the disclosures of which are incorporated herein by reference. A preferred and well-known enzyme/substrate combination is horseradish peroxidase and o-phenylenediamine. The above described components of the assay (i.e., the test sample, the immobilized antibody and indicator protein) are then contacted in the proper order. The immobilized antibody can be incubated with the test sample and indicator protein simultaneously when it is not necessity to maximize sensitivity. To achieve an optimum level of detection, however, the immobilized antibody should be first incubated with the test sample for a sufficient period to allow all of the antigen/antibody complexes to form between the bound antibody and any HIV protein present in the sample. Then the immobilized antibody is incubated with a sample of the indicator protein (purified of other immunologically cross-reactive proteins) in an amount that would saturate all the available antigen binding sites. When the test sample is prepared from a source that may contain any anti-HIV antibody (e.g., serum), the sample should be incubated with the immobilized antibody first ^• the immobilized antibody is then washed to remove any antibody from the sample, and then the immobilized antibody is incubated with the indicator protein. As used herein, "incubations" mean contacts between antibodies and antigens under conditions that allow for the formation of antigen/antibody complexes (e.g., proper pH, temperature, time, medium, etc.).

After incubation with the indicator protein, the immobilized antibody and any unbound indicator protein are separated, usually by washing the test support under conditions, as known to those in the art, that will remove indicator protein that is not biospecifically bound to the test support, but allow biospecifically bound protein to remain. After this separation step (as mentioned above), the amount of indicator protein remaining on the test support is determined by measuring the signal related to the amount of label on the test support. The signal obtained is compared to the signal that results when there is no HIV protein in the test sample. When the signal indicates that a relatively lower amount of indicator protein has bound to the test support (i.e., binding inhibition), then the assay indicates that there is HIV protein in the source of the test sample.

The present invention is also directed to a device that can be employed as a test support in the assay of the present invention. In general, the device comprises a support member with a receptacle for receiving the fluid test sample, with its surface portion that contacts the fluid sample having the immobilized thereon the anti-HIV protein antibody in the amount discussed above. It is generally adjusted so that it is partially, detectably and biospecifically saturated with an amount of recombinant HIV core protein^" in the range of about 4 picomoles to about 10 nanomoles per receptacle (about 1 picogram to about 250 picogram/per receptacle for p25gag) . Preferably, the antibody is partially, detectably and biospecifically saturated by core protein in the range of about 10 picomoles to about 1 nanomole. Examples of suitable amounts within this range include, but are not limited to, 40 picomoles, 100 picomoles, 200 picomoles, and 400 picomoles per receptacle. Examples of appropriate support members are well known in the art, and include any suitable immunoassay plate. A particularly preferred support member is a micro-immunoassay plate having about 250 μl wells to which protein can be bound.

The present invention also provides test kits which can be used in the practice of the abo,ve immunoassay. In general, these test kits will contain the device described above, and several mixtures, including an indicator mixture containing the indicator protein in the appropriate amount and with a detectable label, as well as a substrate that will produce a color change in the presence of an enzyme that is or becomes bound to the indicator protein. The test kits may also optionally contain instructions describing the method of the above immunoassay.

EXAMPLES

The following examples are intended for illustrative purposes only, and are not to limit the scope of the present invention. For example, variation in the source, type or method of production of antibody; different labels and/or signals; test supports of various materials and configurations; different immobilization methods; and different indicator proteins may be employed without departing from the scope of the present invention.

The following is an example of determining the amount of immobilized antibody from a particular source to use in an assay according to the present invention. Unless otherwise indicated, volume of a solution used, for incubation or coating is 75 μl for examples I-III. igG antibodies were purified from a high titer anti-HIV serum by protein A chromatography. Concentration of the purified IgG was measured by adsorption at 280 n . A 96-well Immunolon-2 microtiter plate (Dynatech, Alexandria, Virginia) (250 μl/well) was coated overnight at room temperature with serial dilutions of the purified IgG in NaHCO buffer, pH 9.6, at concentrations ranging from 5 to 0.1 μg/ml. After washing the plate 3 times with phosphate buffered saline (PBS), pH 7.4, the wells were incubated with serial dilutions of biotinylated recombinant p25gag (see Example II) at concentrations ranging from 4 to 0.01 μg/ml for 30 min at room temperature. The recombinant protein was dissolved in PBS containing 1% gelatin, 0.2% Tween 20 (Gelatin-Tween) , and 0.0001% sodium dodecyl sulfate (SDS).

The plates were washed again with PBS containing 0.05% Tween 20 (PBS-Tween) and incubated for 30 min at room temperature with horseradish peroxidase (HRP)-conjugated avidin (Cappel, Cooper Biomedical, Malvern, Pennsylvania) diluted 1:500 in Gelatin-Tween. After repeating the previous wash with PBS-Tween, a substrate solution containing 0.4 mg/ l o-phenylenediamine (Sigma) in citrate-phosphate buffer, pH 5.0, mixed with 0.006% (v/v) H-O- was added to the wells. The color reaction was allowed to develop for 10 to 20 min in the dark, and then stopped by the addition of 25 μl of 4N sulfuric acid in each well. The plates were read at 492 nm in a micro-ELISA spectrophotometer (Titertek Multiskan. Flow Laboratories) .

The results are shown in Figure 1. The concentrations of biotinylated recombinant p25 and purified IgG that gave a sufficient color development (optical density 0.3-0.5 above a background <0.07) with the lowest amount of IgG on the plate were chosen as the working dilutions. Under the above-described conditions, this corresponded to a concentration of approximately 0.5 μg/ml of antibodies and 0.7 μg/ml of biotinylated recombinant p25gag. A 0.5 μg/ml coating dilution of antibodies gave the best inhibition curve with serial dilutions (down to 10 —8) of HIV culture fluids averaging 10 cpm/ l of RT activity. I I

The following describes the preparation of immunoassay plates for use in an assay according to the present invention.

The microtiter plates (Example I) were coated with a solution containing 4 μg/ml of protein A (Sigma Chemicals, St. Louis. Missouri), for 1 hr at room temperature to minimize internal variations in the amount of antibody coating the wells. The plates were then washed 3 times with PBS before the solution containing IgG ( Example I ) at the desired concentration was incubated in each well overnight at room temperature as described in Example I. After washing 3 times with PBS-Tween, uncoated sites on the plates were then blocked with a 4% gelatin solution, pH 7.4, containing 0.05% Tween-20 for 1 hr at 37°-C. The plates were then washed again.

III

The following describes a protocol which can be employed in an assay according to the present invention wherein a recombinant HIV gag protein is the selected HIV protein, and test samples are prepared from human body fluids, human cells, and human cell culture supernatants.

When the source of test samples were HIV-infected peripheral mononuclear cells (PMC) obtained from AIDS patients, supernatants of PMC in culture were filtered through 0.45 μ centrex filters (Schleicher & Schuell) prior to the addition of Nonidet P-40 (NP-40) (Sigma Chemicals) in a 1:100 ratio. After incubating at 4°C for 30 min with intermittent shaking, these samples were ready for testing.

Samples were also prepared from cell lysates of, for example, HUT-78 cultures infected with HIV (e.g., American Type Culture Collection, Rockville,

Maryland, accession no. CRL 8597) or PMC isolated from

AIDS patient. Approximately 10 cells were washed 3 times with Hanks Buffer and then resuspended in 1 ml of lysing buffer (0.1 M Tris, pH 8.0, 0.1% Triton-X 100) for 30 min on ice. Lysates were clarified by centrifugation at 3,000 g for 10 min, and the supernatant used for testing.

Test samples were also prepared from human sera. Sera from patients were either tested directly at a 1:10 dilution, or concentrated by centrifugation and resuspension. When concentrated, 1.5 ml of serum was filtered (0.45 μ as above) and centrifuged at 12,000 g for 2 hr at 4°C (Microfuge 12, Beckman Instruments). .

The supernatants were removed and the pellets stored at -70°C until testing. At least 30 min before assaying, pellets were resuspended in 0.15 ml of PBS buffer containing 1% NP-40 and incubated at 4°C with intermittent shaking.

Microtiter plates were prepared as described in Example II by incubation with 0.5 μg/ml concentration of the IgG of Example I. Yeast-produced recombinant p25gag (Chiron Corporation, Emeryville.

California) (accd, Steiner et al. (1986) Virology

150:283-290; Eur. Pat. 181,150) was biotinylated according to the procedure described by Guesdon et al

(1979) J. Histochem. & Chytochem. 27(8) :1131-1139, using a molar ratio of amino groups to

D-biotin-N-hydroxy-succinimide ester (Boehringer Manheim

Biochemicals, Indianapolis, Indiana) of 1:5., The scheme of the assay is shown in Figure 2. The microtiter plates were incubated at 37°C with 67 μl of a test sample prepared as described above. After 40 min, 8 μl of a lOx solution (7 μg/ml) of biotinylated recombinant p25gag was added to each well containing test fluid (except for wells containing patient serum) , and the plates incubated for 20 min at room temperature. When the test sample was patient serum, 75 μl of the serum diluted 1:10 in Gelatin-Tween was incubated with the immobilized antibody, removed after 40 min, the plate washed 3 times with PBS-Tween, and then incubated with 75 μl of a lx (0.7 μg/ml) solution of biotinylated recombinant p25gag protein. The remainder of the assay, including washing, incubation with HRP-avidin, and the color reaction, was conducted as described iri Example I.

Figure 3 shows the results of the assay as conducted with test samples prepared from HI,V inf^ecte.d cell culture supernatant (HUT-78 and PMC), ("HIV fluid"), unlabeled recombinant p25gag, HUT-78 ce'll lysates, normal serum spiked with HIV fluid, and HTLV-I(control) . A 50% inhibition of binding of biotinylated recombinant p25gag to the antibodies (OD₅ ) was obtained with (i) between a 10 —4 ^■ and i ι I0^r dilution of HIV fluids, (ii) 10 picograms of recombinant p25gag/well, and (iii) 150 cells of HIV-infected HUT-78 cell lysates (ca.75% of cells shown to be infected by IFA) . Viral p25gag could be detected in serum samples spiked with HIV culture fluids tested at 1:10 dilution with a similar sensitivity as that achieved with the HIV culture supernatants. A log increase in detection was gained by concentration of virions from the sera by centrifugation and resuspension prior to testing. Twenty-six sera from patients pertaining to various clinical/risk groups were tested by competition ELISA for HIV p25gag. Antibodies were measured by standard indirect ELISA or immunoblot. Four sera were inoculated in human PMC cultures and the cultures monitored for viral reverse transcriptase activity. The results are shown in the table below:

Table

Patient p25gag Antibody Diag/risk RT(xlO cpm/ml)

GS13 + + ARC 385.4 (16d)

GS21 +_, + ARC 3.9 (12d)

4708 — + 0

4710 — + 71.8 (19d)

4711 + + 27.2 (24d)

DJ/M + +

DJ/R + + -

DJ/T + +

DJ/W - +

H23 + 01

H208 - + KS

H228 + + ARC

-360 + + ARC

H456 +.

H709 - + Het

H729 +. ARC

K7 — - C

K24 — - HH

K25 ^ - HH

K27 + - HH

K32 - - HH

K33 — + HH

K34 — + HH

K35 - + HH.

K36 + + HH

K37 — + HH

Diag/Risk: diagnostic/risk group

01: opportunistic infections

KS: Kaposi sarcoma

ARC: AIDS-related complex

HH: healthy homosexual

Het: healthy heterosexual

C: healthy control patient

Variations of the above embodiments will be readily apparent to those of ordinary skill in the art. Thus, it is intended that the scope of the present invention be limited solely by the following claims.

Claims

1. An immunoassay method for detecting low levels of Human Immunodeficiency Virus (HIV) protein in a fluid test sample comprising:

(a) providing an indicator sample comprised of a purified and labeled indicator protein, said indicator protein being a synthetic protein that is immunologically cross-reactive with a selected HIV protein;

(b) providing immobilized antibody on a solid support that biospecifically binds said selected HIV protein and said indicator protein; (c) incubating said immobilized antibody with said test sample;

(d) incubating said immobilized antibody with said indicator sample after or simultaneously with said incubation with the test sample to provide indicator-contacted antibody;

(e) separating any unbound indicator protein from said indicator-contacted antibody to provide separated antibody; and

(f) measuring a signal related to the amount of s/aid label bound to said separated antibody.

2. The method of claim 1 wherein said indicator protein is made by recombinant means.

3. The method of claim 2 wherein said selected

HIV protein is a gag protein.

4. The method of claim 3 wherein said gag protein is p25gag.

5. An immunoassay for directly detecting the presence of Human Immunodeficiency Virus (HIV) protein in a fluid test sample prepared from a source selected from the group consisting of human cells and human body fluids comprising:

(b) providing immobilized antibody on a solid support that biospecifically^' binds said selected HIV protein and said indicator protein; (c) incubating said immobilized antibody with said test sample to provide test sample-contacted antibody;

(d) incubating said, test sample-contacted antibody with said indicator sample -to provide indicator-contacted antibody;

(f) measuring a signal related to the amount of said label bound to said separated antibody.

6. The method of claim 5 wherein said indicator protein is produced by recombinant means.

7. The method of claim 6 wherein said selected

HIV protein is a gag protein.

8. The method of claim 7 wherein said gag protein is p25gag.

9. The method of claim 7 wherein said test sample has been treated to disrupt virus particles.

10. The method of claim 6 wherein said signal is optical density.

11. The method of claim 6 wherein said immobilized antibodies are polyclonal.

12. The method of claim 6 wherein said source is human T cells from a single patient.

13. The method of claim 6 wherein said source is human serum from a single patient and said test sample-contacted antibody is separated from any immunoglobulin in said test sample before said incubation with the indicator sample.

14. A device for use as a solid test support in an immunoassay method for the presence of Human

Immunodeficiency Virus (HIV) gag protein in a fluid sample comprising a solid support member with a receptacle for receiving said fluid sample, the surface portion of said receptacle that contacts said fluid sample having immobilized thereon anti-HIV gag protein antibody in an amount that is partially, detectably and biospecifically saturated in antigen binding sites when contacted with a reference amount of a recombinant HIV gag protein, said reference amount in the range of about 4 picomoles to about 10 nanomoles.

15. The device of claim 14 wherein said antibody is polyclonal.

16. The device of claim 14 wherein said receptacle can hold a maximum of about 1 ml or less of said fluid sample.

17. The device of claim 14 wherein said receptacle can hold a maximum of about 250 μl or less of said fluid sample.

18. A test kit for an immunoassay for detecting the presence of HIV protein in a fluid sample comprising the device of claim 14, an indicator mixture comprising recombinant HIV gag protein having a detectable label, and a substrate that produces a color change in the presence of an enzyme bound to said recombinant protein.

19. The kit of claim 18 wherein said immobilized antibody is anti-HIV p25gag antibody and said recombinant HIV gag protein is p25gag..