EP0227748A1 - Test for determining false positive reactions in a testing procedure to detect the presence of antibody to microorganisms. - Google Patents

Test for determining false positive reactions in a testing procedure to detect the presence of antibody to microorganisms.

Info

Publication number
EP0227748A1
EP0227748A1 EP19860903981 EP86903981A EP0227748A1 EP 0227748 A1 EP0227748 A1 EP 0227748A1 EP 19860903981 EP19860903981 EP 19860903981 EP 86903981 A EP86903981 A EP 86903981A EP 0227748 A1 EP0227748 A1 EP 0227748A1
Authority
EP
European Patent Office
Prior art keywords
test
cell line
microorganisms
infected
bound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19860903981
Other languages
German (de)
French (fr)
Inventor
Daniel H. Zimmerman
Sean P. O'neill
Gerald A. Bush
Judith A. Britz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alfa Wassermann Inc
Electro Nucleonics Inc
Original Assignee
Alfa Wassermann Inc
Electro Nucleonics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alfa Wassermann Inc, Electro Nucleonics Inc filed Critical Alfa Wassermann Inc
Publication of EP0227748A1 publication Critical patent/EP0227748A1/en
Withdrawn legal-status Critical Current

Links

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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • 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
    • 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

Definitions

  • TITLE TEST FOR DETERMINING FALSE POSITIVE REACTIONS IN A TESTING PROCEDURE TO DETECT THE PRESENCE OF ANTIBODY TO MICROORGANISMS.
  • This invention relates to the determination of the presence of antibody to microorganisms. It has particular application for use with a highly sensitive test that determines the presence of antibody, particularly to eliminate false positive reactions.
  • Electro- Nucleonics, Inc. of Fairfield, New Jersey, U.S.A., has developed an Enzyme Linked Immunosorbent Assay (ELISA) test kit for identification of antibodies to the Human T- Lymphotropic Virus Type-Ill (HTLV-III).
  • ELISA Enzyme Linked Immunosorbent Assay
  • the present invention has particular application to the identification of false positives which may be due to poor sample quality, differences in laboratory environment/technique or reaction with cellular material and other biological/chemical substances used in the manufacture of the VIRGO test kit.
  • a specificity test to determine false positive reactions is developed from the same cell line used in the primary antibody-determining VIRGO test, but which is non-infected with the antigen of interest.
  • a specificity test component is prepared from a concentrate of cellular material shed by uninfected H9 cells, the cell line used in the growth of the HTLV-III virus used in the VIRGO test kit. (The H9 cell line is described in the Popovic et al. article cited below. ) The concentrate is treated in a manner idencial to that used for the propagation of the HTLV-III coated microassay plates in the VIRGO test kit.
  • a ratio of the absorbance values on each plate distingiushes a specific from a non-specific antibody reaction on HTLV-III, and false positive indications are detected.
  • the VIRGO test kit marketed by Electro-Nucleonics, Inc. utilizes HTLV-III isolated in the laboratory of Dr. Robert Gallo (National Cancer Institute) and propagated from a seed stock according to procedures established by M. Popovic (Popovic, M. , M. G. Sarngadharan, E. Read, and R.C. Gallo, 1984. "Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS.” Science 224, 497-500.).
  • the cell line infected with HTLV-III is cultured and the culture supernatant is purified by centrifugation procedures.
  • the viral concentrate is then inactivated in a two-step procedure using chemical and physical treatments. Purified inactivated HTLV-III is absorbed onto wells of a microassay plate to complete the VIRGO test kit.
  • HTLV-III microassay plate serum or plasma samples diluted in a buffer are added to the HTLV-III microassay plate. If antibodies specific for HTLV-III are present in a sample under test, they will form stable complexes with the HTLV-III antigens on the plate. A goat anti-human IgG (Heavy and Light chain specific) labeled with horseradish peroxidase is added. If the antigen/antibody complex is present, the peroxidase conjugate will bind and remain in the well. Enzyme substrate is then added. Color will develop in wells containing antibody. No color develops in negative wells. An acid stop solution is added to each well and the color read on a microassay plate reader at 492 nm.
  • IgG Heavy and Light chain specific
  • CJDSTSTUTE SHEET Specificity test plates in accordance with the present invention are prepared by adsorbing a concentrate of cellular material shed by uninfected H9 cells, the same cell line used in the growth of the HTLV-III virus in producing the VIRGO test kit.
  • the H9 concentrate is treated in a manner identical to that used for the preparation of the HTLV-III coated microassay plates in the VIRGO test kit.
  • H9 uninfected and HTLV-III infected cells are separately grown each as a standard suspension culture of between 2
  • the cells are re-fed every 2 - 3 days with an equal volume of fresh media of the same composition. In many cases this is accomplished by removing an equal volume of spent medium-containing cells.
  • the cells are normally removed by relative low speed (1000 xg) centrifugation. This so-called clarified extra ⁇ cellular fluid is then processed, or stored, at low temperature (+4 C or -20 C) until further processing.
  • the two samples are processed identically (insofar as possible).
  • the particular processing involves the use of a continuous flow centrifuge (RK).
  • the clarified fluid is pumped in at a constant flow rate and effluent is also removed at a similar flow rate.
  • the material (infected or uninfected) is banded in a sucrose density gradient. At the end of the run, the gradient is displaced by pumping 55% sucrose into the rotor. The gradient is collected in 25 - 30 equal volume fractions. The gradient is monitored
  • TE SHEET by use of a U.V. spectrophotometer recorder and also the density of each fraction is recorded.
  • the relevant fractions containing the viral materials are pooled, diluted and an equal volume of buffer to reduce the density and viscosity of the sucrose solution.
  • the virus is pelleted by high speed centrifugation and resuspended in Buffer devoid of sucrose. The material is then inactivated by the use of detergent and heat, samples removed for various quality control tests, aliquoted and stored at -70°C until use.
  • the uninfected fluid is identically processed and has all the same treatments and testing as described above for the infected fluid.
  • infected and uninfected materials are then coated onto solid supports by use of various buffers and then are packaged awaiting use in an assay.
  • the two materials infected primary test and non-infected specificity test are used identically in an immunoassay.
  • a light absorbance value of less than 0.100 indicates that the sample is nonreactive to HTLV-III antigen(s). If the detected absorbance indicates a possible positive reaction under the VIRGO procedure, the positive VIRGO test is preferably repeated and the sample is also identically processed using the H9 test plate. The VIRGO and H9 plate absorbances are then compared (ratio r) as follows:
  • a 1 and a_ are the detected light absorbances of the HTLV-III VIGRGO test plate and H9 specificity test plate, respectively, and K is a constant representing the intercept of the straight-line curve that results from a plotting of a 1 versus a terrain.
  • K is a constant representing the intercept of the straight-line curve that results from a plotting of a 1 versus a terrain.
  • k has been found to be 0.1.
  • the invention involves the use of a specificity test component prepared by using non-infected material of the same type as that used in producing infected materials for a primary test.
  • the invention obviously has application to other than the specific test procedure described above as the preferred embodiment of this invention.
  • soluble supports to which infected and uninfected materials are bound could be employed, e.g., soluble polymers which are polymerizable to complete detection, as in U.S. Patent No. 4,511,478.
  • testing criteria other than light absorbance as
  • SUBSTITUT SHEET in the above preferred embodiment could be employed, such as light reflectance, fluorescence, chemiluminescence, a precipitation pattern, to name some examples.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Les réactions positives fausses détectées dans un test très sensible visant à déterminer la présence d'anticorps de micro-organismes se distingue des réactions positives vraies par l'utilisation d'un test de contrôle de la spécificité. Le test conduisant à la réaction positive devant être examinée utilise une fraction exempte de cellules d'une lignée cellulaire infectée de micro-organismes. Une fraction exempte de cellules de la dite lignée de cellules non infectée de micro-organismes est utilisée dans le test de contrôle de la spécificité afin de détecter les réactions positives fausses. Les résultats des tests utilisant des fractions exemptes de cellules infectées et non infectées sont comparées afin de distinguer les réactions positives fausses des réactions positives vraies.False positive reactions detected in a very sensitive test to determine the presence of antibodies to microorganisms are distinguished from true positive reactions by the use of a specificity test. The test leading to the positive reaction to be examined uses a cell-free fraction of a cell line infected with microorganisms. A cell-free fraction of the so-called non-infected cell line of microorganisms is used in the specificity control test to detect false positive reactions. The results of tests using fractions free from infected and uninfected cells are compared in order to distinguish false positive reactions from true positive reactions.

Description

TITLE: TEST FOR DETERMINING FALSE POSITIVE REACTIONS IN A TESTING PROCEDURE TO DETECT THE PRESENCE OF ANTIBODY TO MICROORGANISMS.
BACKGROUND AND BRIEF DESCRIPTION OF THE INVENTION
This invention relates to the determination of the presence of antibody to microorganisms. It has particular application for use with a highly sensitive test that determines the presence of antibody, particularly to eliminate false positive reactions.
The presence of circulating antibodies provides clinical data to indicate exposure of an individual to an antigenic agent and subsequent immune response. Recent eipdemiological evidence indicates that an infectious agent transmitted through intimate contact or use of infected blood or blood products may be the cause of the Acquired Immune Deficiency Syndrome (AIDS). Electro- Nucleonics, Inc., of Fairfield, New Jersey, U.S.A., has developed an Enzyme Linked Immunosorbent Assay (ELISA) test kit for identification of antibodies to the Human T- Lymphotropic Virus Type-Ill (HTLV-III). This test kit, marketed under the trademark VIRGO, detects antibody to HTLV-III and eliminates potentially infectious blood units from being used for transfusions or other therapeutic
4_. ■ __. SHEET procedures. Because of the high sensitivity of the test, false positive reactions may occur. • The present invention has particular application to the identification of false positives which may be due to poor sample quality, differences in laboratory environment/technique or reaction with cellular material and other biological/chemical substances used in the manufacture of the VIRGO test kit.
In the present invention, a specificity test to determine false positive reactions is developed from the same cell line used in the primary antibody-determining VIRGO test, but which is non-infected with the antigen of interest. In the case of HTLV-III virus, a specificity test component is prepared from a concentrate of cellular material shed by uninfected H9 cells, the cell line used in the growth of the HTLV-III virus used in the VIRGO test kit. (The H9 cell line is described in the Popovic et al. article cited below. ) The concentrate is treated in a manner idencial to that used for the propagation of the HTLV-III coated microassay plates in the VIRGO test kit. A repeatedly positive plasma or serum sample by the HTLV- III microassay plate and an H9 microassay plate, in accordance with the present invention, utilizing identical reagents. A ratio of the absorbance values on each plate distingiushes a specific from a non-specific antibody reaction on HTLV-III, and false positive indications are detected.
The invention will be more completely understood by reference to the following detailed description of a preferred embodiment thereof which is set forth in the
HEE context of determining false positive reactions in an ELISA test kit for identification of non-specific reactions to HTLV-III.
The VIRGO test kit marketed by Electro-Nucleonics, Inc., utilizes HTLV-III isolated in the laboratory of Dr. Robert Gallo (National Cancer Institute) and propagated from a seed stock according to procedures established by M. Popovic (Popovic, M. , M. G. Sarngadharan, E. Read, and R.C. Gallo, 1984. "Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS." Science 224, 497-500.). The cell line infected with HTLV-III is cultured and the culture supernatant is purified by centrifugation procedures. The viral concentrate is then inactivated in a two-step procedure using chemical and physical treatments. Purified inactivated HTLV-III is absorbed onto wells of a microassay plate to complete the VIRGO test kit.
To conduct a test, serum or plasma samples diluted in a buffer are added to the HTLV-III microassay plate. If antibodies specific for HTLV-III are present in a sample under test, they will form stable complexes with the HTLV-III antigens on the plate. A goat anti-human IgG (Heavy and Light chain specific) labeled with horseradish peroxidase is added. If the antigen/antibody complex is present, the peroxidase conjugate will bind and remain in the well. Enzyme substrate is then added. Color will develop in wells containing antibody. No color develops in negative wells. An acid stop solution is added to each well and the color read on a microassay plate reader at 492 nm.
CJDSTSTUTE SHEET Specificity test plates in accordance with the present invention are prepared by adsorbing a concentrate of cellular material shed by uninfected H9 cells, the same cell line used in the growth of the HTLV-III virus in producing the VIRGO test kit. To produce specificity test plates, the H9 concentrate is treated in a manner identical to that used for the preparation of the HTLV-III coated microassay plates in the VIRGO test kit.
To explain the above in more detail, the H9 uninfected and HTLV-III infected cells are separately grown each as a standard suspension culture of between 2
5 and 10 x 10 cells and media (RPMI 1640) supplemented with
10% Fetal Bovine Sera. The cells are re-fed every 2 - 3 days with an equal volume of fresh media of the same composition. In many cases this is accomplished by removing an equal volume of spent medium-containing cells. The cells are normally removed by relative low speed (1000 xg) centrifugation. This so-called clarified extra¬ cellular fluid is then processed, or stored, at low temperature (+4 C or -20 C) until further processing.
Since the material from the uninfected cells is to be used in a specificity test, the two samples (infected and uninfected) are processed identically (insofar as possible). The particular processing involves the use of a continuous flow centrifuge (RK). The clarified fluid is pumped in at a constant flow rate and effluent is also removed at a similar flow rate. The material (infected or uninfected) is banded in a sucrose density gradient. At the end of the run, the gradient is displaced by pumping 55% sucrose into the rotor. The gradient is collected in 25 - 30 equal volume fractions. The gradient is monitored
TE SHEET by use of a U.V. spectrophotometer recorder and also the density of each fraction is recorded. The relevant fractions containing the viral materials are pooled, diluted and an equal volume of buffer to reduce the density and viscosity of the sucrose solution. The virus is pelleted by high speed centrifugation and resuspended in Buffer devoid of sucrose. The material is then inactivated by the use of detergent and heat, samples removed for various quality control tests, aliquoted and stored at -70°C until use.
The uninfected fluid is identically processed and has all the same treatments and testing as described above for the infected fluid.
The infected and uninfected materials are then coated onto solid supports by use of various buffers and then are packaged awaiting use in an assay. The two materials (infected primary test and non-infected specificity test) are used identically in an immunoassay.
In accordance with the VIRGO test procedure, a light absorbance value of less than 0.100 indicates that the sample is nonreactive to HTLV-III antigen(s). If the detected absorbance indicates a possible positive reaction under the VIRGO procedure, the positive VIRGO test is preferably repeated and the sample is also identically processed using the H9 test plate. The VIRGO and H9 plate absorbances are then compared (ratio r) as follows:
STITUTE SHEET a1 - k
=r a2
where a1 and a_ are the detected light absorbances of the HTLV-III VIGRGO test plate and H9 specificity test plate, respectively, and K is a constant representing the intercept of the straight-line curve that results from a plotting of a1 versus a„. In this example of HTLV-III, k has been found to be 0.1.
The results are interpreted as follows:
HTLV-III VIRGO test plate absorbance Ratio r Interpretation
-^>- 0.100 ^ 3.0 Sample is exhibiting a nonspecific reaction
__Z- 0.100 _^3.0 Reactive to HTLV-III antigen(s)
In summary, the invention involves the use of a specificity test component prepared by using non-infected material of the same type as that used in producing infected materials for a primary test. The invention obviously has application to other than the specific test procedure described above as the preferred embodiment of this invention. For example, soluble supports to which infected and uninfected materials are bound could be employed, e.g., soluble polymers which are polymerizable to complete detection, as in U.S. Patent No. 4,511,478. ^ Further, testing criteria other than light absorbance as
SUBSTITUT SHEET in the above preferred embodiment could be employed, such as light reflectance, fluorescence, chemiluminescence, a precipitation pattern, to name some examples. The invention, accordingly, should be taken to be defined by the following claims.
SUBSTITUT

Claims

1. A test kit for confirming the presence of antibody to microorganisms in test material to be used in conjunction with a fraction of a microorganism-infected cell line which has given a positive reaction to said test material and comprising a fraction of said cell line non- infected by said microorganisms and substantially free of whole cells and bound to a support.
2. A test lot according to claim 1, in combination with, as a separate test constituent to be individually reacted, a fraction of a microorganism-infected cell line bound to a support and substantially free of whole cells.
3. A test kit according to claim 1 or 2, in which said fractions are bound to insoluble supports.
4. A test kit according to claim 1 or 2, in which said fractions are bound to soluble supports.
5. A test kit according to claim 1 or 2, in which the microorganisms are HTLV-III antigens and said cell line comprises the leukaemic cell line H9.
6. A method of confirming the presence of antibody to microorganisms in. test material comprising contacting test material with a fraction of a cell line infected by a microorganism and substantially free of whole cells and bound to a support, and sensing a characteristic of the contacted material, and, in the event of a reaction sensed as positive, contacting said test material with a fraction of said cell line non-infected by said microorganism and substantially free of whole cells and bound to a support, and sensing the characteristic thereof, and comparing said sensed characteristics.
7. A method according to claim 6, in which said sensed characteristic is light absorbance.
8. A method according to claim 7, in which the ratio of said light absorbances is determined.
9. A method according to claim 8, in which the following ratio is determined:
(a1 - k)/a-,. where a1 is the first-mentioned of said light aabbssoorrbbaanncceess,, aanndd aa„„ iiss tthhee sseeccoonndd--mmee:ntioned one of said light absorbances, and k is a constant.
10. A method according to claim 9, in which the constant k is substantially equal to 0.1.
11. A method according to claim 6, in which the microorganisms are HTLV-III antigens and said cell line comprises the leukaemic cell line H9.
EP19860903981 1985-06-17 1986-06-04 Test for determining false positive reactions in a testing procedure to detect the presence of antibody to microorganisms. Withdrawn EP0227748A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74576085A 1985-06-17 1985-06-17
US745760 1985-06-17

Publications (1)

Publication Number Publication Date
EP0227748A1 true EP0227748A1 (en) 1987-07-08

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Application Number Title Priority Date Filing Date
EP19860903981 Withdrawn EP0227748A1 (en) 1985-06-17 1986-06-04 Test for determining false positive reactions in a testing procedure to detect the presence of antibody to microorganisms.

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AU (1) AU5990286A (en)
WO (1) WO1986007633A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3609217A1 (en) * 1986-03-19 1987-09-24 Boehringer Mannheim Gmbh METHOD AND REAGENT FOR DETERMINING A REACTION PARTNER OF AN IMMUNOLOGICAL REACTION
US6461825B1 (en) 1987-09-30 2002-10-08 Sanofi (Societe Anonyme) Immunometric assay kit and method applicable to whole cells
FR2621128B1 (en) * 1987-09-30 1994-05-06 Sanofi IMMUNOMETRIC ASSAY KIT AND METHOD FOR WHOLE CELLS

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8324800D0 (en) * 1983-09-15 1983-10-19 Pasteur Institut Antigens

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8607633A1 *

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AU5990286A (en) 1987-01-13
WO1986007633A1 (en) 1986-12-31

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Inventor name: ZIMMERMAN, DANIEL, H.