WO1989004488A1 - Enzyme immunoassay for detecting hiv antigens in human sera - Google Patents

Enzyme immunoassay for detecting hiv antigens in human sera Download PDF

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Publication number
WO1989004488A1
WO1989004488A1 PCT/US1988/003698 US8803698W WO8904488A1 WO 1989004488 A1 WO1989004488 A1 WO 1989004488A1 US 8803698 W US8803698 W US 8803698W WO 8904488 A1 WO8904488 A1 WO 8904488A1
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WIPO (PCT)
Prior art keywords
hiv
immunoassay
test sample
antibody
antigen
Prior art date
Application number
PCT/US1988/003698
Other languages
French (fr)
Inventor
Kenneth H. Kortright
David E. Hofheinz
Meryl Ann Forman
Song Yea Lee
Paulette Elizabeth Smariga
Candie Sue Stoner
Original Assignee
Coulter Corporation
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Application filed by Coulter Corporation filed Critical Coulter Corporation
Priority to DE3853430T priority Critical patent/DE3853430T2/en
Priority to AU28148/89A priority patent/AU612760B2/en
Priority to EP89900430A priority patent/EP0386136B1/en
Priority to KR1019890701260A priority patent/KR890702038A/en
Publication of WO1989004488A1 publication Critical patent/WO1989004488A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/974—Aids related test
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/80—Fluorescent dyes, e.g. rhodamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/809—Multifield plates or multicontainer arrays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/825—Pretreatment for removal of interfering factors from sample
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/826—Additives, e.g. buffers, diluents, preservatives

Definitions

  • This invention provides an improved immunoassay for detecting HIV antigens in a human physiological fluid test sample which may have circulating antigens.
  • This invention in a preferred embodiment provides a unique enzyme immunoassay which can be performed for detecting HIV virus, HIV virus fragments or HIV in ⁇ fected cells.
  • This immunoassay provides a test result of positive or negative HIV antigen, as the case may be, with a greater degree of accuracy or sensitivity, consistency and in a shorter period of time than has been realized heretofore in an HIV antigen assay.
  • the assay embodying the invention uses solid-phase assay methodology in which a monoclonal antibody specific to certain HIV core antigens is coated on the solid surface.
  • HIV infec ⁇ tion commonly results in AIDS (Acquired Immune Deficiency Syndrome).
  • Reported increasing numbers of AIDS patients in the United States has caused the dis ⁇ ease to be recognized as approaching epidemic propor ⁇ tions with no known cure or vaccine.
  • the occurrence of AIDS has been increasing primarily in Europe and Afri ⁇ can countries of the world as the disease is trans- mitted through sexual activity, drug abuse, con ⁇ taminated organs in transplantation procedures and by blood transfusions.
  • Other strains of the HIV such as HTLV IV are developing through mutation of this virus.
  • Human antibody develops in 70-80% of the people exposed to HIV through sexual intercourse, blood trans ⁇ fusions, contaminated organs through transplantation or contaminated needles by drug abusers. The remaining 20-30% of exposed individuals who do not develop antibodies against the invading virus for a period of up to six months can remain undetected carriers in society. Therefore, if one is to effectively screen blood and organ donors, individuals, or blood products for containing the spread of AIDS, it is essential that the screening immunoassay be able to detect not only HIV antibody but also HIV virus itself and/or infected cells carrying the virus.
  • the assay embodying the invention successfully tests for the virus and virus infected cells.
  • the as ⁇ say can be performed in a single vessel such as a mi- crotiter plate well.
  • the patient's test sample comprises human plasma, i.e., whole blood from which all cells have been removed initially, or serum, i.e., plasma from which all clotting factors have been removed.
  • the assay provides a result of "positive” or "negative” as the case may be, by enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • the suitability of the use of the assay of this invention to identify AIDS infected patients is sig ⁇ nificantly greater than has been realized heretofore where an immunoassay tracks only antibodies to HIV in a physiological sample such as peripheral blood.
  • the test result is achieved in approximately four hours.
  • Lysing of viral concentrates to release viral proteins prior to using electrophoresis to spread viral proteins, i.e., antigens, on polyacrylamide gel is known with respect to a Western blot technique for con ⁇ firming the presence of only anti-HIV antibodies, not antigens, and is thus of no practical utility during that period of time prior to when no antibodies are present even though the patient has been infected with HIV.
  • an HIV viral lysate as a solid phase protein adhered " to a solid phase support, such as a microtiter plate formed of polystyrene is known with respect to an ELISA technique for screening for anti-HIV antibodies, not antigens, during the peri ⁇ od after HIV infection when no antibodies are present.
  • an enzyme immunoassay for testing HIV antigen in a human physiological fluid test sample such as plasma or serum or cell culture supernatant.
  • the immunoassay is used to test a single sample for HIV antigens wherein the methodology used is that of solid-phase as ⁇ say, e.g., enzyme-linked immunosorbent assay (ELISA) that uses microtiter strips, polystyrene beads, or fer- rous beads, for example, as support media.
  • ELISA enzyme-linked immunosorbent assay
  • the test sample or specimen of predetermined volume such as 200 micro- liters (--/L)
  • a cell lysing reagent is introduced and the mixture incubated for a suitable period of time at 37°C.
  • a quantity of preferably human anti-HIV antibody biotin complex reagent is added and incubated for a suitable period of time.
  • the sample is then sub ⁇ jected to incubation procedures which utilize selected developing reagents. The entire assay is completed in approximately four hours.
  • the immunoassay of this invention differs from that described in the related application in three major respects.
  • the use of the so-called "Spike” is eliminated.
  • the preferably human anti-HIV antibody biotin complex has a particular formulation.
  • the solid phase is coated with a unique monoclonal antibody iden- tified as the KC-57 monoclonal antibody.
  • an assay of a single sample for simultaneous detection of HIV antigen and/or HIV antibody in a human physiological test sample such as plasma or serum.
  • Microwells such as of a microtiter plate having microwell strips, are coated with HIV antibody obtained from sera which exhibit high titers of anti-HIV antibody.
  • the solid-phase support media i.e., the microtiter plate is prepared for the assay by coating the wells with purified anti-HIV antibody derived from a high titer mammalian serum or plasma source such as from a human, goat or other species.
  • Test sample is added to one coated microwell, or preferably several coated microwells, since duplicate tests are preferred.
  • Control wells are provided for normal, i.e., antigen and antibody negative, physiological fluid, e.g., normal human plasma or serum.
  • An antigen positive control well preferably is also used.
  • a known amount of HIV antigen, i.e. the "Spike" is added to each sample microwell and each con ⁇ trol well or wells and the so-combined sample and added antigen subjected to a lyse buffer to assure disruption of virus present in the test sample into its component parts. After a simple incubation at body temperature the microtiter plate is washed to remove material which might interfere with the signal producing system and the captured antigen is next allowed to react with anti-HIV antibody which has been conjugated with biotin.
  • kits In the practice of the preferred embodiment of this invention such a kit comprises: A 96 well polystyrene microtiter plate with the wells coated with anti-HIV antibody; A supply of plate covers; Lyse Buffer;
  • TMB Tetramethylbenzidien
  • TMB Substrate Buffer Normal Human Plasma Control
  • Normal Human Serum Control Normal Human Serum Control
  • the reagents are generally required to be prepared in advance so that they will be available at the ap ⁇ basementte steps in carrying out the exemplary preferred immunoassay procedure. These steps are as follows:
  • Peroxide-free octylphenopoly (ethyleneglycolether) e.g. Triton X-100, Boehringer Mannheim Biochemicals 5% Polyoxy thylenesorbiton monolaurate e.g. Tween 20, Sigma Chemical Co. 2%
  • HIV antigen content sufficient when diluted with antigen diluent to provide Spike of 50 picograms of HIV antigen per 20 //L of diluted reagent.
  • concentration of antigen in the Spike may be determined on the basis of P24 equivalent by use of a DuPont P24 Radioimmunoassay (RIA).
  • Antigen Diluent :
  • BSA Protease-Free Bovine Serum Albumin
  • Citric acid monohydrate 11.38g
  • IPX Wash Buffer (Diulte 1 to 10):
  • the immunoassay embodying the invention of the re- latedr-application preferably is performed in a familiar microtiter plate well.
  • a 200 microliter ( L) patient sample is tested.
  • the patient sample may comprise human plasma derived from whole blood from which the cells have been removed by centrifugat on, for instance, serum, or cultured cells.
  • the test sample of 200 tL is introduced into the well of a microtiter plate which has been coated with a heat inactivated purified anti-HIV antibody.
  • the purified anti-HIV antibody is derived from a high titer serum or plasma source. Such a source can be a human one.
  • a source can be a human one.
  • the mammalian antibody e.g., human anti-HIV antibody is diluted to 2.5 micrograms per mililiter in so-called "plating buffer".
  • the plating buffer is comprised of 250 mM of potassium phosphate at pH 6.0. 250 M I> of diluted antibody is pipetted into the well and stored at 4°C for approximately sixteen hours.
  • the well is then drained and washed three times in a phosphate buffered saline (PBS) wash solution.
  • PBS phosphate buffered saline
  • the well is then blocked for one hour at room temperature with 330 /L of a blocking reagent comprising 1% bovine serum albumin
  • BSA Bactease free
  • Sucrose 5% Sucrose plus 1% sodium ethyl- mercurithiosalicylate in 1 times PBS filtered through a 0.2.um filter.
  • the well is stored for up to six hours with the blocking solution at 4°C until dried.
  • the well is then drained, speed vacuum dried and stored at 4°C in a bag with dryite desiccant bag.
  • the well After incubation, the well is aspirated and washed three times in a conventional manner with wash buffer.
  • the anti-HIV antibody-biotin which is preferably in a lyophilized state is reconstituted and diluted to a 5% solution with biotin reagent diluent and 200 L added to the well and the plate incubated at 37°C for one hour.
  • the well After incubation, the well is aspirated and washed three times using the Wash Buffer reagent. Then 200 -uL of the streptavidin-peroxidase conjugate is added to the well, covered and incubated for thirty minutes at 37°C. After incubation, the well is aspirated and washed three times with the Wash Buffer. Then, 200 /L of TMB Substrate Solution is introduced to the well, covered and incubated for thirty minutes at room temperature.
  • the immunoassay embodying the invention provides for a quantitated patient sample including a predetermined quantity of a Spike of inactivated HIV viral antigen, which is detectable at a designated picogram sensitivity, to be introduced into an antibody coated microtiter well.
  • Various conventional incuba ⁇ tions follow, using prescribed human developing agents, such as, biotin covalently coupled to a mammalian specie derived anti-HIV antibody such as derived from a human.
  • the coupling is followed by a step of forming a biotin-avidin conjugated peroxidase complex after which the TMB substrate is added to provide colormetric visualization of the reaction.
  • HIV antibody biotin conjugate employed a long chain biotin.
  • concentration of Spike antigen and the antigen in the patient sample placed in the well which binds to the capture antibody coated walls of the well is then recognized by the anti-HIV antibody which is biotinylated and the complex then reacted with the avidin peroxidase and TMB substrate for visualization of the reaction. After stopping the reaction, the EIA readings can be taken to compare against control directed readings.
  • the reading obtained from the patient test sample is compared to the control readings such as those ob- tained from normal human plasma free of HIV antigen, anti-HIV antibody, or non-infectious virus subjected to the immunoassay procedure.
  • control readings such as those ob- tained from normal human plasma free of HIV antigen, anti-HIV antibody, or non-infectious virus subjected to the immunoassay procedure.
  • a series of tests were conducted in accordance with the above described improved ELISA protocol for simultaneous determination in a single test well of both HIV antigen and/or anti-HIV antibody.
  • the tests were conducted on plasma and serum derived from blood samples obtained at five medical institutes from patients suspected of being exposed to HIV, diagnosed as having an ARC or suspected of having, or diagnosed as having AIDS.
  • three 96 well mi- crotiter plates were used to perform 99 assays.
  • the samples were all subjected to a screening test by an ELISA technique other than that of the present invention and were also subjected to a confirmation test using a Western blot technique for analysis and identification of HIV proteins P24 and P120 before as ⁇ signing a "score" of positive or negative to the sample.
  • 70 or 70.7% were determined to be positive to either HIV antigen or anti-HIV antibody and 29 or 29.3% determined to be negative to either HIV antigen or anti-HIV antibody.
  • 6 samples or about 6.1% were found to be both HIV antigen and anti-HIV antibody positive.
  • the particular significance of the immunoassay comprising the present invention will be appreciated from the fact that 17 of the samples determined to be "positive" were found to only be posi ⁇ tive with respect to HIV antigen.
  • test data compiled as a result of the Western blot confirmation tests performed appears to indicate that about 10% of the tests performed by the method of the present invention are subject to a false positive result and about 15% are subject to a false negative determination. It will be appreciated that the per- centage of false results may be attributable to factors which were not determined, such as human error in con ⁇ ducting the test or reading the result. Thus, the per ⁇ centage of apparent false results is not a true reflec ⁇ tion of the efficacy of the exemplary test run de- scribed herein.
  • the solid-phase support media is a microtiter plate prepared for the assay by coating the wells.with a novel monoclonal antibody which recognizes a common epitope of certain core antigens and no envelope HIV antigens in a human physiological test sample.
  • This monoclonal antibody was produced from a hybridoma or cell line developed at the laboratories of the common assignee by co-inventors common with those of this as ⁇ say invention.
  • the monoclonal antibody is identified as the KC-57 monoclonal antibody which recognizes an epitope common to the HIV antigens P55, P24 and partial breakdown products P39 and P33.
  • the KC-57 antibody does not bind the P18 core antigen or any other HIV as- sociated antigens.
  • a cell line which produces the KC-57 monoclonal antibody corresponding to that used in this invention has been deposited in the American Type Culture Collec- tion, Rockville, Maryland 20852 on November 6, 1987, concurrently with the filing of this application.
  • the cell line was assigned A.T.C.C. No. HB 9585.
  • Coating of one or more microtiter wells with the KC-57 monoclonal antibody is performed in substantially the same manner as described in the related applica ⁇ tion.
  • the KC-57 antibody is diluted to 2.5 micrograms per milliliter in the so-called "plating buffer”. 250 microliters of diluted monoclonal antibody is pipetted into the well, stored, washed, blocked, dried, speed drained and stored again as herein described.
  • a plurality of wells can be coated using commercially available automated pipetting apparatus.
  • a 200 microliter patient sample is tested, the sample being human serum or plasma derived from whole blood serum or culture cells.
  • the 200 microliter sample is pipetted into a well and 20zL of Lyse Buffer for lysing infected cells is added.
  • the well is cov ⁇ ered and then incubated for one hour at 37°C.
  • the well is aspirated and washed three times in a conventional manner with wash buffer.
  • 200 - ⁇ L of Anti-HIV Antibody-Biotin Complex reagent preferably lyophilized, is added in a reconstituted state.
  • the Anti-HIV antibody preferably is derived from a human source, i.e., a high titer serum or plasma source.
  • This reagent is reconstituted to a predeter ⁇ mined volume so that 200 /il * of the reconstituted reagent can be introduced to a well.
  • the plate is in ⁇ cubated for one hour at 37°C. Thereafter, the well is aspirated and washed three times using a Wash Buffer reagent.
  • the EIA readings taken can be compared against control directed readings, such as those obtained from normal human plasma or serum free of HIV antigen in a KC-57 monoclonal antibody coated well acting as a nega ⁇ tive control or those obtained from such a coated well into which 50 /L of a virus antigen reagent was intro- prised.
  • the latter procedure would provide a positive control.
  • the assay embodying the invention would employ a pair of positive control wells. 200 L of patient sample would be used in each of the above control well procedures.
  • an ELISA immunoassay is not the only one applicable to this invention. Thus, other methodologies as enumerated herein are considered to be applicable.
  • Table 2 reflects data developed to show the ac- curacy of the assay embodying the invention.
  • Normal human serum and normal human serum to which the p24 core antigen was added were assayed.
  • the levels of p24 spiked are itemized for each category of test conducted. At the 25 picogram (pg) and 6.25 pg per 200 microliters spiked level, 100% tested positive. At the 1.56 pg level, 87% tested positive. Normal hu ⁇ man serum did not test positive in 156 assays per ⁇ formed.
  • Table 3 demonstrates that the HIV antigen assay utilizing the KC-57 monoclonal antibody as a capture phase and human anti-HIV antibody as the detector phase identified all strains listed as being positive for HIV antigen content.
  • the above graph demonstrates the sensitivity achieved with t assay analysing p24 antigen into normal human plasma. This data was collected by six individual assays run on separate days. A positive value, twice the background absorbance was obtained at 1.2 pg/well or 6 pg/ml. Interassay variation ranged from 11-26 percent.
  • HIV antigen content sufficient when diluted with an appropriate diluent which will provide an Antigen Reagent concentration of 25 pg p24 per
  • BIOTIN REAGENT (4 X CONCENTRATE) 125 ML
  • Citric Acid Monohydrate 11.38 g
  • the immunoassay embodying the invention achieves important advantages.
  • the stability of the enzyme color reaction for a test is up to four (4) hours. This means that there will be no concern about checking a color determination after a test is completed since replication is possible for up to four hours there- after.
  • 90-91 tests can be conducted within an approximate four hour period since the wells will already be coated with KC- 57 monoclonal antibody.
  • Applicants are not aware of any HIV antigen assay capable of this productivity in so short a period of time.
  • the sensitivity and replication of results achieved with this assay is entirely without precedent in the art as known to ap- plicants.

Abstract

A solid-phase immunoassay is provided for determination of HIV antigens in human physiological fluid. The immunoassay is characterized by the coating of a solid substrate with a unique monoclonal antibody which recognizes a common antigenic determinant of a group of HIV core antigens and no HIV envelope antigens of HIV. The test sample preferably also is subjected to a lysing reagent prior to the incubation for uniformly dispersing antigens which may be present in the test sample.

Description

ENZYME IMMUNOASSAY FOR DETECTING HIV ANTIGENS IN HUMAN SERA
RELATED CASE This application is related to pending PCT ap- plication, International Serial No. PCT/US88/00877 filed March 14, 1988, titled SIMULTANEOUS ENZYME IM¬ MUNOASSAY FOR DETECTING ANTIGEN AND/OR ANTIBODY IN HU¬ MANS. The identified patent applications enjoy common ownership.
TECHNICAL FIELD
This invention provides an improved immunoassay for detecting HIV antigens in a human physiological fluid test sample which may have circulating antigens. This invention in a preferred embodiment provides a unique enzyme immunoassay which can be performed for detecting HIV virus, HIV virus fragments or HIV in¬ fected cells. This immunoassay provides a test result of positive or negative HIV antigen, as the case may be, with a greater degree of accuracy or sensitivity, consistency and in a shorter period of time than has been realized heretofore in an HIV antigen assay. The assay embodying the invention uses solid-phase assay methodology in which a monoclonal antibody specific to certain HIV core antigens is coated on the solid surface.
BACKGROUND ART
HTLV III infection, now referred to as HIV infec¬ tion, commonly results in AIDS (Acquired Immune Deficiency Syndrome). Reported increasing numbers of AIDS patients in the United States has caused the dis¬ ease to be recognized as approaching epidemic propor¬ tions with no known cure or vaccine. The occurrence of AIDS has been increasing primarily in Europe and Afri¬ can countries of the world as the disease is trans- mitted through sexual activity, drug abuse, con¬ taminated organs in transplantation procedures and by blood transfusions. Other strains of the HIV such as HTLV IV are developing through mutation of this virus.
Separate tests for the presence of HIV antibody or HIV antigen have been developed by investigators, in¬ cluding scientists at the National Institute of Health (NIH). Assays for detecting HIV antibody in plasma and serum have received clinical approval for commer¬ cialization. Assays for detecting HIV antigen are known for research use but to our knowledge none has received clinical approval for commercialization. These tests separately and independently analyze the plasma, serum or blood cell culture supernatant of screened individuals for HIV antigen or human anti-HIV antibody content which binds the HIV antigen, see Anti- HIV Testing: Screening and Confirmation Tests, Medical Laboratory Products, April 1987, p. 16-18, for a recent review article comparing several such separate tests. Human antibody develops in 70-80% of the people exposed to HIV through sexual intercourse, blood trans¬ fusions, contaminated organs through transplantation or contaminated needles by drug abusers. The remaining 20-30% of exposed individuals who do not develop antibodies against the invading virus for a period of up to six months can remain undetected carriers in society. Therefore, if one is to effectively screen blood and organ donors, individuals, or blood products for containing the spread of AIDS, it is essential that the screening immunoassay be able to detect not only HIV antibody but also HIV virus itself and/or infected cells carrying the virus. Heretofore difficulty has been encountered with false-positive results which may occur due to non¬ specific antibodies or to antibodies directed against antigens in supporting cell cultures used to obtain su- pernatants containing viral particles in sufficient quantity as is generally necessary to overcome the less than desirable sensitivity of prior tests.
The assay embodying the invention successfully tests for the virus and virus infected cells. The as¬ say can be performed in a single vessel such as a mi- crotiter plate well. Preferably, the patient's test sample comprises human plasma, i.e., whole blood from which all cells have been removed initially, or serum, i.e., plasma from which all clotting factors have been removed. The assay provides a result of "positive" or "negative" as the case may be, by enzyme immunoassay (EIA). The suitability of the use of the assay of this invention to identify AIDS infected patients is sig¬ nificantly greater than has been realized heretofore where an immunoassay tracks only antibodies to HIV in a physiological sample such as peripheral blood. The test result is achieved in approximately four hours.
Lysing of viral concentrates to release viral proteins prior to using electrophoresis to spread viral proteins, i.e., antigens, on polyacrylamide gel is known with respect to a Western blot technique for con¬ firming the presence of only anti-HIV antibodies, not antigens, and is thus of no practical utility during that period of time prior to when no antibodies are present even though the patient has been infected with HIV. Similarly, the use of an HIV viral lysate as a solid phase protein adhered"to a solid phase support, such as a microtiter plate formed of polystyrene, is known with respect to an ELISA technique for screening for anti-HIV antibodies, not antigens, during the peri¬ od after HIV infection when no antibodies are present. However, it has not been recognized that the lysing of HIV in a patient's blood sample in contact with a unique monoclonal antibody coated solid phase in the presence of a mammalian biotinylated anti-HIV antibody can provide for the relatively rapid, sensitive quantitative measurement of HIV antigens in a single test sample as realized by this invention.
DISCLOSURE OF THE INVENTION An enzyme immunoassay is provided for testing HIV antigen in a human physiological fluid test sample such as plasma or serum or cell culture supernatant. In a preferred embodiment of the invention, the immunoassay is used to test a single sample for HIV antigens wherein the methodology used is that of solid-phase as¬ say, e.g., enzyme-linked immunosorbent assay (ELISA) that uses microtiter strips, polystyrene beads, or fer- rous beads, for example, as support media. In a par¬ ticularly preferred embodiment, the test sample or specimen of predetermined volume, such as 200 micro- liters (--/L), is introduced into a well of a microtiter plate which has been coated with a unique monoclonal antibody which recognizes the same epitope of a group of core antigens. A cell lysing reagent is introduced and the mixture incubated for a suitable period of time at 37°C. A quantity of preferably human anti-HIV antibody biotin complex reagent is added and incubated for a suitable period of time. The sample is then sub¬ jected to incubation procedures which utilize selected developing reagents. The entire assay is completed in approximately four hours.
The immunoassay of this invention differs from that described in the related application in three major respects. The use of the so-called "Spike" is eliminated. The preferably human anti-HIV antibody biotin complex has a particular formulation. The solid phase is coated with a unique monoclonal antibody iden- tified as the KC-57 monoclonal antibody.
BEST MODE FOR CARRYING OUT THE RELATED INVENTION
In the preferred embodiment of the related ap¬ plication there is provided an assay of a single sample for simultaneous detection of HIV antigen and/or HIV antibody in a human physiological test sample, such as plasma or serum. Microwells, such as of a microtiter plate having microwell strips, are coated with HIV antibody obtained from sera which exhibit high titers of anti-HIV antibody. The solid-phase support media, i.e., the microtiter plate is prepared for the assay by coating the wells with purified anti-HIV antibody derived from a high titer mammalian serum or plasma source such as from a human, goat or other species. Test sample is added to one coated microwell, or preferably several coated microwells, since duplicate tests are preferred. Control wells are provided for normal, i.e., antigen and antibody negative, physiological fluid, e.g., normal human plasma or serum. An antigen positive control well preferably is also used. A known amount of HIV antigen, i.e. the "Spike" is added to each sample microwell and each con¬ trol well or wells and the so-combined sample and added antigen subjected to a lyse buffer to assure disruption of virus present in the test sample into its component parts. After a simple incubation at body temperature the microtiter plate is washed to remove material which might interfere with the signal producing system and the captured antigen is next allowed to react with anti-HIV antibody which has been conjugated with biotin. Following a subsequent incubation with streptavidin-peroxidase reagent, color development of bound enzyme is revealed using an appropriate sub- strate. Resultant optical densities are proportional to the relative amount of HIV antigen, and/or anti-HIV antibody, occurring in the test sample and antigen pos¬ itive control or the absence thereof in the test sample and controls. As is customary, the antibody coated solid-phase support and generally all of the reagents necessary for carrying out an assay are provided in kit form. In the practice of the preferred embodiment of this invention such a kit comprises: A 96 well polystyrene microtiter plate with the wells coated with anti-HIV antibody; A supply of plate covers; Lyse Buffer;
Antigen Reagent "Spike"; Antigen Diluent;
Anti-HIV Antibody-Biotin Reagent; Biotin Reagent Diluent; Streptaviden-Horseradish Peroxidase (HRPO)
Conjugate; 3, 3' , 5, 5' Tetramethylbenzidien (TMB) reagent;
TMB Substrate Buffer; Normal Human Plasma Control; Normal Human Serum Control;
Antigen Positive Control (50 pg antigen per 20 /*L)
10X Wash Buffer; Hydrogen Peroxide Solution; and Stopping Solution.
Glossary of Reagents
The reagents are generally required to be prepared in advance so that they will be available at the ap¬ propriate steps in carrying out the exemplary preferred immunoassay procedure. These steps are as follows:
Lyse Buffer:
Peroxide-free octylphenopoly (ethyleneglycolether) e.g. Triton X-100, Boehringer Mannheim Biochemicals 5% Polyoxy thylenesorbiton monolaurate e.g. Tween 20, Sigma Chemical Co. 2%
Sodium Ethylmercurithiosalicylate e.g. Thimersol, Sigma Chemical Co. 0.05%
Dye, FDC Blue No. 1 0.9 mg/ml Distilled water Q.S. 1 L
Antigen Reagent:
HIV antigen content sufficient when diluted with antigen diluent to provide Spike of 50 picograms of HIV antigen per 20 //L of diluted reagent. The concentration of antigen in the Spike may be determined on the basis of P24 equivalent by use of a DuPont P24 Radioimmunoassay (RIA). Antigen Diluent:
Protease-Free Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) 1.0%
Sodium azide 0.1% Dye, FDC Red No. 3 0.5 mg/ml
Anti-HIV Antibody-Biotin Reagent:
Human anti-HIV Antibody-Biotin Conjugate (prefer bly lyophi1ized)
Biotin Reagent Diluent: For 1L
Triton X-100 0.5%
Normal Human Serum (heat inactivated) 0.5% Tween 0.2%
Thimersol 0.5%
10X PBS (lOmM phosphate, 145 mM NaCl pH 7.2)100.ml Distilled Water Q.S.1L
Streptavidin-HRPO Conjugate:
Calbiochem-Behring Corp.
TMB Reagent:
TMB 1.Og DMSO (dimethyl sulfoxide) Q.S. 100.0ml
TMB Dilution Buffer:
Na2HP047H20 24.13g
Citric acid monohydrate 11.38g
Chloracetamide 1.OOg Distilled water Q.S. 1L pH adjusted to 4.4 Stopping Solution:
18M concentated H2S04 100.0 ml
Distilled water 800.00 ml
Hydrogen Peroxide Solution:
30% H202
IPX Wash Buffer (Diulte 1 to 10):
Tween 20 10.0ml
10X PBS 0.99L
1% Chloracetamide 10.Og
Depending upon the nature of the reagents, as well as the protocol, it will be appreciated by those skilled in the immunoassay art that the concentration of individual reagents can be varied widely once it is appreciated that a significant aspect of this invention resides in the step of adding a Spike consisting of a known amount of antigen of the single binding pair being assayed.
IMMUNOASSAY PROCEDURE:
The immunoassay embodying the invention of the re- latedr-application preferably is performed in a familiar microtiter plate well. In the preferred embodiment, a 200 microliter ( L) patient sample is tested. The patient sample may comprise human plasma derived from whole blood from which the cells have been removed by centrifugat on, for instance, serum, or cultured cells. The test sample of 200 tL is introduced into the well of a microtiter plate which has been coated with a heat inactivated purified anti-HIV antibody.
The purified anti-HIV antibody is derived from a high titer serum or plasma source. Such a source can be a human one. To coat a well, the mammalian antibody, e.g., human anti-HIV antibody is diluted to 2.5 micrograms per mililiter in so-called "plating buffer". The plating buffer is comprised of 250 mM of potassium phosphate at pH 6.0. 250 MI> of diluted antibody is pipetted into the well and stored at 4°C for approximately sixteen hours. The well is then drained and washed three times in a phosphate buffered saline (PBS) wash solution. The well is then blocked for one hour at room temperature with 330 /L of a blocking reagent comprising 1% bovine serum albumin
(BSA) (Protease free), 5% Sucrose plus 1% sodium ethyl- mercurithiosalicylate in 1 times PBS filtered through a 0.2.um filter. The well is stored for up to six hours with the blocking solution at 4°C until dried. The well is then drained, speed vacuum dried and stored at 4°C in a bag with dryite desiccant bag.
Although the coating procedure has been described for a single well, conventional microtiter plates are available with a plurality of wells. Also available is equipment for coating a plurality of wells. Thus, the immunoassay embodying the invention can be performed utilizing such available equipment with a plurality of wells.
To the 200 μ test sample in the well is added 20 /L of Lyse Buffer for lysing the infected cells and 20 L of Human Antigen Working Solution which is the antigen reagent diluted with antigen diluent to the preselected 50 pg antigen per 20 ML . The well is then covered and mixing of ingredients in a suitable shaker is performed for one minute. The mixed materials are then incubated for one hour at 37°C.
After incubation, the well is aspirated and washed three times in a conventional manner with wash buffer. The anti-HIV antibody-biotin which is preferably in a lyophilized state is reconstituted and diluted to a 5% solution with biotin reagent diluent and 200 L added to the well and the plate incubated at 37°C for one hour.
After incubation, the well is aspirated and washed three times using the Wash Buffer reagent. Then 200 -uL of the streptavidin-peroxidase conjugate is added to the well, covered and incubated for thirty minutes at 37°C. After incubation, the well is aspirated and washed three times with the Wash Buffer. Then, 200 /L of TMB Substrate Solution is introduced to the well, covered and incubated for thirty minutes at room temperature.
Then, 50 AL of 2 Molar sulfuric acid is added to the well to stop the reaction. The optical density of the solution in the well is then read on a microtiter plate reader at a wavelength of 450 nanometers using 570 nanometers as a reference where dual wavelength ca¬ pability is available. If dual wavelength capability is not utilized, the plate can be read at both wavelengths and the 450 nanometer readings corrected by subtracting the 570 nanometer readings.
The immunoassay embodying the invention provides for a quantitated patient sample including a predetermined quantity of a Spike of inactivated HIV viral antigen, which is detectable at a designated picogram sensitivity, to be introduced into an antibody coated microtiter well. Various conventional incuba¬ tions follow, using prescribed human developing agents, such as, biotin covalently coupled to a mammalian specie derived anti-HIV antibody such as derived from a human. The coupling is followed by a step of forming a biotin-avidin conjugated peroxidase complex after which the TMB substrate is added to provide colormetric visualization of the reaction. A particularly favor¬ able degree of sensitivity for the test was noted when the HIV antibody biotin conjugate employed a long chain biotin. The concentration of Spike antigen and the antigen in the patient sample placed in the well which binds to the capture antibody coated walls of the well is then recognized by the anti-HIV antibody which is biotinylated and the complex then reacted with the avidin peroxidase and TMB substrate for visualization of the reaction. After stopping the reaction, the EIA readings can be taken to compare against control directed readings.
The reading obtained from the patient test sample is compared to the control readings such as those ob- tained from normal human plasma free of HIV antigen, anti-HIV antibody, or non-infectious virus subjected to the immunoassay procedure. We have established the reference point for a test result using a patient sample as an increase or decrease of about 30%. Thus, if an optical density reading decreases more than 30% from the OD of the Spike, the determination is antibody positive. If the reading increases more than 30%, the determination is antigen positive.
While an ELISA using a combination of ligand and receptor, i.e., biotin-avidin protocol for providing a detectible signal for the immunological reactions has been described in the preferred methodology, it will be appreciated that numerous other methodologies may be employed for visualizing the resultant immunological reactions. In this regard, other labels or markers may be employed for visualizing the resultant immunological reactions, such as radionuclides, other enzymes, fluoresσers, chemiluminescers, enzyme substrates, par¬ ticles, e.g., magnetic particles, combination of ligands and receptors other than biotin and avidin. A series of tests were conducted in accordance with the above described improved ELISA protocol for simultaneous determination in a single test well of both HIV antigen and/or anti-HIV antibody. The tests were conducted on plasma and serum derived from blood samples obtained at five medical institutes from patients suspected of being exposed to HIV, diagnosed as having an ARC or suspected of having, or diagnosed as having AIDS. In the test series, three 96 well mi- crotiter plates were used to perform 99 assays.
With respect to microtiter plate 1, there were 32 tests run and 50 pg per 20 /l spike used to establish the base line optical density exhibited an optical density (OD) that equals 0.894 + 0.061. Using an ar- bitrary guideline of 30% deviation in optical density below and above 0.894, it was established for purposes of the assay that OD of < 0.626 equals anti-HIV antibody positive and OD of > 1.16 equals HIV antigen positive.
Similarly with respect to plate 2, 34 tests were run and the 50 pg Spike has an OD of 0.715 + 0.56. An OD reading of < 0.500 was considered to be anti-HIV antibody positive and QD of > 0.93 was considered to be HIV antigen positive.
By the same rationale, with respect to plate 3, 33 tests were run and the 50 pg Spike had an OD of 0.655 +_ 0.59 whereby an OD of < 0.459 was considered to be anti-HIV antibody positive an OD of > 0.852 was consid- ered to be HIV antigen positive.
The samples were all subjected to a screening test by an ELISA technique other than that of the present invention and were also subjected to a confirmation test using a Western blot technique for analysis and identification of HIV proteins P24 and P120 before as¬ signing a "score" of positive or negative to the sample. Of the 99 samples, 70 or 70.7% were determined to be positive to either HIV antigen or anti-HIV antibody and 29 or 29.3% determined to be negative to either HIV antigen or anti-HIV antibody. Of the 70 samples found to be positive, 6 samples or about 6.1% were found to be both HIV antigen and anti-HIV antibody positive. However, the particular significance of the immunoassay comprising the present invention will be appreciated from the fact that 17 of the samples determined to be "positive" were found to only be posi¬ tive with respect to HIV antigen.
It is a significant advance in immunoassay to have the ability to simultaneously screen a physiological fluid sample in a single test vessel for both antigen and antibody of a single binding pair. In the preferred embodiment of the invention, this enables si¬ multaneous screening, by use of a single test well, a test sample of plasma or serum from donated blood for both anti-HIV antibody and HIV antigen or more impor¬ tantly the presence of only HIV antigen. The critical need to identify donated blood containing only HIV antigen during that period of time between a person being infected with HIV and when antibodies to the HIV are raised or raised to a sufficient level to be detected by a screening test, need hardly be empha¬ sized.
The test data compiled as a result of the Western blot confirmation tests performed appears to indicate that about 10% of the tests performed by the method of the present invention are subject to a false positive result and about 15% are subject to a false negative determination. It will be appreciated that the per- centage of false results may be attributable to factors which were not determined, such as human error in con¬ ducting the test or reading the result. Thus, the per¬ centage of apparent false results is not a true reflec¬ tion of the efficacy of the exemplary test run de- scribed herein.
BEST MODE FOR CARRYING OUT THIS INVENTION
This invention utilizes the individual procedures, reagents and apparatus to a substantial degree de¬ scribed in the related application. However, there are certain reagents used and not used which represent im¬ portant departures from the assay protocol of the re¬ lated apprication and which contribute to realizing the novel immunoassay for detecting HIV antigens in a patient embodying this invention. The solid-phase support media is a microtiter plate prepared for the assay by coating the wells.with a novel monoclonal antibody which recognizes a common epitope of certain core antigens and no envelope HIV antigens in a human physiological test sample. This monoclonal antibody was produced from a hybridoma or cell line developed at the laboratories of the common assignee by co-inventors common with those of this as¬ say invention. The monoclonal antibody is identified as the KC-57 monoclonal antibody which recognizes an epitope common to the HIV antigens P55, P24 and partial breakdown products P39 and P33. The KC-57 antibody does not bind the P18 core antigen or any other HIV as- sociated antigens.
STATEMENT OF DEPOSIT
A cell line which produces the KC-57 monoclonal antibody corresponding to that used in this invention has been deposited in the American Type Culture Collec- tion, Rockville, Maryland 20852 on November 6, 1987, concurrently with the filing of this application. The cell line was assigned A.T.C.C. No. HB 9585.
Coating of one or more microtiter wells with the KC-57 monoclonal antibody is performed in substantially the same manner as described in the related applica¬ tion. The KC-57 antibody is diluted to 2.5 micrograms per milliliter in the so-called "plating buffer". 250 microliters of diluted monoclonal antibody is pipetted into the well, stored, washed, blocked, dried, speed drained and stored again as herein described. A plurality of wells can be coated using commercially available automated pipetting apparatus.
A 200 microliter patient sample is tested, the sample being human serum or plasma derived from whole blood serum or culture cells. The 200 microliter sample is pipetted into a well and 20zL of Lyse Buffer for lysing infected cells is added. The well is cov¬ ered and then incubated for one hour at 37°C.
After incubation, the well is aspirated and washed three times in a conventional manner with wash buffer. 200 -αL of Anti-HIV Antibody-Biotin Complex reagent, preferably lyophilized, is added in a reconstituted state. The Anti-HIV antibody preferably is derived from a human source, i.e., a high titer serum or plasma source. This reagent is reconstituted to a predeter¬ mined volume so that 200 /il* of the reconstituted reagent can be introduced to a well. The plate is in¬ cubated for one hour at 37°C. Thereafter, the well is aspirated and washed three times using a Wash Buffer reagent. The procedure of adding streptavidin-peroxidase conjugate, incubation and adding 200 -_L of the TMB Substrate Solution is as described in the related application. Then, 50 -uL of Molar Sulfuric Acid is added to a well to stop the reaction and the optical density of the solution is then read on a microtiter plate reader at a wavelength of 450 nanometers using 570 nanometers as a reference where dual wavelength capability is available. If dual wavelength capability is not utilized, the plate can be read at both wavelengths and the 450 nanometer readings corrected by subtracting the 570 nanometer readings. The EIA readings taken can be compared against control directed readings, such as those obtained from normal human plasma or serum free of HIV antigen in a KC-57 monoclonal antibody coated well acting as a nega¬ tive control or those obtained from such a coated well into which 50 /L of a virus antigen reagent was intro- duced. The latter procedure would provide a positive control. Preferably, the assay embodying the invention would employ a pair of positive control wells. 200 L of patient sample would be used in each of the above control well procedures. As pointed out in the related application, an ELISA immunoassay is not the only one applicable to this invention. Thus, other methodologies as enumerated herein are considered to be applicable.
TABLE 1
SUMMARY OF HIV ANTIGEN ASSAY RESULT
Initial Repeatable
HIV NEGATIVE # Tested False Positive False Positive
Figure imgf000018_0001
Subtotal 360 8
HIV POSITIVE # Tested Antigen Positive % Positive
Diagnosed AIDS* 61 27 44 Blood Bank 37 6 16
Subtotal 98 33 34
* Some samples from patients on AZT therapy.
Referring to Table 1, normal and abnormal HIV neg¬ ative serum samples tested are listed and the number of samples tested is noted. The total number of samples tested was 360 of which Initial False Positive values were seen to be eight in number. Repeated tests of the eight samples indicated they were truly negative. Al¬ though 25% of the 12 rheumatoid factor containing sera were positive in this assay initially, they were not repeatable. It is believed that contaminating material in these sera may have created a false impression of high incidence of initial positives.
With respect to the HIV POSITIVE data displayed, sera from 98 humans known to be serologically positive by Western Blot analysis, both asymptomatic and diag¬ nosed AIDS patients, were tested. The percent positive determinations in each category of sera tested is listed. 44% of the diagnosed AIDS samples were posi- tive for circulating p24 antigen. In Deborah A. Paul, et al., J. Med Virol., 22:357:363 (1987), 69% of diag¬ nosed AIDS patients were found to have circulating HIV antigen. The difference between these two determina¬ tions may be attributed to the AZT therapy ( "zidovudine", formerly known as azidothymidine), which is believed to account for rapid decline in the cir¬ culating HIV antigen levels. The 16% of asymptomatic individuals found to be positive correlates well with the 19% determination in Deborah A. Paul, supra.
TABLE 2
HIV ANTIGEN ASSAY - p24 SPIKED SAMPLE RESULTS
p24 Antigen Positive Number Number Calculated Mass/200 -l Performed Positive Recover %
25 pg
6.25 pg 1.56 pg 0 pg
Figure imgf000020_0001
Table 2 reflects data developed to show the ac- curacy of the assay embodying the invention. Normal human serum and normal human serum to which the p24 core antigen was added (called "spiked") were assayed. The levels of p24 spiked are itemized for each category of test conducted. At the 25 picogram (pg) and 6.25 pg per 200 microliters spiked level, 100% tested positive. At the 1.56 pg level, 87% tested positive. Normal hu¬ man serum did not test positive in 156 assays per¬ formed.
TABLE 3
KC-57 HIV ANTIGEN CAPTURE ELISA
RESULTS
+ + + + + +
Figure imgf000021_0001
Table 3 demonstrates that the HIV antigen assay utilizing the KC-57 monoclonal antibody as a capture phase and human anti-HIV antibody as the detector phase identified all strains listed as being positive for HIV antigen content.
TABLE 4
SPECIFICITY OF KC-57 MONOCLONAL ANTIBODY
Figure imgf000021_0002
The above table demonstrates the reactivity of KC- 57 antibody against other infectious agents when run in the HIV Antigen Assay. No reactivity is seen with any of these agents. COMPARISON OF HIV ISOLATES SPIKED INTO NORMAL HUMAN PLASMA
Figure imgf000022_0001
CONCENTRATION P24 EQUIVALENT/O.2ml PLASMA The above graph demonstrates representative standard curves developed from assays using normal human sera spiked with culture media taken from human lymphocyte cultures infected with two isolates of HIV, BAGAL and LAV, and purified p24 antigen from a LAV infected human T cell line. p24 antigen concentrations were established using the Dupont p24 RIA. Sensitivity for each preparation is shown to be less than 10 pg/ml. P24 SPIKED IN NORMAL HUMAN PLASMA
Figure imgf000023_0001
CONCENTRATION OF P24 (pg/0.2ml)
The above graph demonstrates the sensitivity achieved with t assay analysing p24 antigen into normal human plasma. This data was collected by six individual assays run on separate days. A positive value, twice the background absorbance was obtained at 1.2 pg/well or 6 pg/ml. Interassay variation ranged from 11-26 percent. GLOSSARY OF REAGENT CHANGES
ANTIGEN REAGENT
HIV antigen content sufficient when diluted with an appropriate diluent which will provide an Antigen Reagent concentration of 25 pg p24 per
50 -ul. Addition of 50 M Antigen Reagent to 0.2 ml normal human plasma, human serum or culture media will provide a positive control with an ap¬ proximate absorbance value (450/570) of 1.00 _+ 0.250.
BIOTIN REAGENT (4 X CONCENTRATE) 125 ML
Triton X-100 2.0%
Tween 20 0.8%
Normal Human Serum (heat inactivated) 2.0% Non-fat Dry Milk 20.0%
Thimerosol 0.25 g
10 x PBS (100 mM, 1.45 m NaCl) 50.0 ml
Anti-HIV antibody Biotin complex 0.3125 mg
Distilled Water QS 125.0 ml
TMB DILUENT BUFFER
Na2HPO47H20 24.13 g
Citric Acid Monohydrate 11.38 g
Chloracetamide 1.0 g
Distilled H20 QS 1L
Adjust pH to 4.4
Add 0.15 ml 30% H202
To demonstrate the consistency or repeatability of the HIV antigen assay of the inven¬ tion, a series of six assays were conducted over a pe- riod of three days. Zero levels or negative controls and 25 picograms of P24 core antigen were the samples assayed. The average and standard deviation (x +_ S.D. ) of the negative control was 0.069 +_ 0.013 with an 18% intra-assay variability. Inter-assay variability was
20% with an average and standard deviation (x ;+ S.D.) of 0.069 0.017. The 25 picogram level demonstrated an intra-assay variability of 1% with an average and standard deviation (x +_ S.D.) of 1.149 + 0.012. The inter-assay variability was 11% with an average and standard deviation (x + S.D.) 1.149 +_ 0.128.
The immunoassay embodying the invention achieves important advantages. The stability of the enzyme color reaction for a test is up to four (4) hours. This means that there will be no concern about checking a color determination after a test is completed since replication is possible for up to four hours there- after. For a 96 well microtiter plate provided in a kit with the requisite reagents for performing the as¬ say and using five (5) wells as control wells, 90-91 tests can be conducted within an approximate four hour period since the wells will already be coated with KC- 57 monoclonal antibody. Applicants are not aware of any HIV antigen assay capable of this productivity in so short a period of time. The sensitivity and replication of results achieved with this assay is entirely without precedent in the art as known to ap- plicants.

Claims

1. An immunoassay for detecting HIV antigens in a human physiological fluid test sample which may have circulating antigen, said assay comprising:
(a) contacting the test sample with a solid sur- face to which is bound a monoclonal antibody capable of linking with a common antigenic determinant of plurality of HIV antigens and in¬ troducing a quantity of preferably a human anti- HIV antibody labelled to provide a useful detec- tible signal for a test sample; and
(b) incubating the resultant complexes and sub¬ jecting same to a conjugated signal producing com¬ bination which is capable of yielding a quantita¬ tively measurable signal correlated to the signal for a normal test sample to indicate either antigen positive or negative for the test sample.
2. The immunoassay of claim 1 including the step of contacting the test sample with a lysing reagent for uniformly dispensing antigens which may be present in the test sample prior to introducing the labelled antibody.
3. The immunoassay of claim 1 in which the step (b) is performed by ELISA methodology
4. The immunoassay of claim 1 in which said monoclonal antibody is the KC-57 monoclonal antibody.
5. The immunoassay of claim 1 in which the sig¬ nal producing combination of step (b) is a glycoprotein bound antibody conjugate and said signal is a color detection signal.
6. The immunoassay of claim 5 in which said con¬ jugate is a biotinylated antibody.
7. A kit for use in performing an immunoassay according to claim 1 comprising in combination:
(a) a solid surface to which is bound the KC-57 monoclonal antibody capable of linking with a cor- responding antigenic determinant common to certain HIV antigens; (b) an amount preferably of a human anti-HIV antibody complex for providing a useful detectible signal for a test sample; (c) an amount of a lysing agent for uniformly dis¬ pensing viral antigens which may be present in a test sample; and
(.d) materials necessary for visualizing im¬ munological reactions resulting from use of the kit in performing the immunoassay.
8. The kit described in claim 7 in which said monoclonal antibody is the KC-57 monoclonal antibody produced from a hybridoma, samples of which are on deposit with the American Type Culture Collection, Rockville, Maryland and assigned A.T.C.C. No. HB 9585.
PCT/US1988/003698 1987-11-06 1988-10-24 Enzyme immunoassay for detecting hiv antigens in human sera WO1989004488A1 (en)

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DE3853430T DE3853430T2 (en) 1987-11-06 1988-10-24 ENZYME IMMUNOTEST METHOD FOR DETECTING HIV ANTIGENS IN HUMAN SERES.
AU28148/89A AU612760B2 (en) 1987-11-06 1988-10-24 Enzyme immunoassay for detecting hiv antigens in human sera
EP89900430A EP0386136B1 (en) 1987-11-06 1988-10-24 Enzyme immunoassay for detecting hiv antigens in human sera
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EP0386136A1 (en) 1990-09-12
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DE3853430D1 (en) 1995-04-27
CN1021375C (en) 1993-06-23
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ZA888170B (en) 1990-07-25
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MX168290B (en) 1993-05-14
US4886742A (en) 1989-12-12
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ES2009368A6 (en) 1989-09-16
JPH03502241A (en) 1991-05-23

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