WO1986002736A1 - Procede d'analyse immunologique pour petites molecules - Google Patents

Procede d'analyse immunologique pour petites molecules Download PDF

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Publication number
WO1986002736A1
WO1986002736A1 PCT/US1984/001737 US8401737W WO8602736A1 WO 1986002736 A1 WO1986002736 A1 WO 1986002736A1 US 8401737 W US8401737 W US 8401737W WO 8602736 A1 WO8602736 A1 WO 8602736A1
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WO
WIPO (PCT)
Prior art keywords
antibody
small molecule
small
antibodies
nonidentical
Prior art date
Application number
PCT/US1984/001737
Other languages
English (en)
Inventor
Paul H. Ehrlich
Alexander Krichevsky
Original Assignee
Eks, 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 Eks, Inc. filed Critical Eks, Inc.
Priority to EP19840904238 priority Critical patent/EP0198826A4/fr
Priority to PCT/US1984/001737 priority patent/WO1986002736A1/fr
Priority to AU36157/84A priority patent/AU3615784A/en
Priority to JP50414284A priority patent/JPS63501096A/ja
Publication of WO1986002736A1 publication Critical patent/WO1986002736A1/fr

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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

Definitions

  • This invention relates to a method for performing two-site immunoassays for small molecules.
  • small molecules are those im unogens or haptens which were previously considered too small for binding two antibody molecules and hence were widely believed not to be detectable using this type of assay.
  • the invention is believed to be applicable to virtually all immunoassay procedures, including traditional immunoassay procedures as well as those most recently developed.
  • a preferred assay method is one in which one antibody is present in solid phase and the other antibody is labelled and in liquid phase.
  • the present invention provides a method for detecting accurately very minute amounts of such agriculturally and medically important low molecular weight molecules as steroids, mycotoxins, antibiotics, small hormones and small peptides.
  • Two-site immunoassays have many advantages over single-site methods, including increased sensitivity, specificity, speed and stability of the assay [A. White, "Monoclonal Antibodies for Steroid Immunoassay,” IN: .H. Hunter and J.E.T. Corrie (Eds.), Immunoassays for Clinical Chemistry, Edinburgh, London, Melbourne and New York, Churchill Livingstone (1983) p. 500] .
  • immunoradiometric assay IRMA
  • a two-site immunoassay requires that two antibodies be able to bind simultaneously to the molecule to be assayed. It has generally been believed that in order to bind two antibodies simultaneously, the molecule of interest roust be of a certain minimum size, since, when one antibody binds to one epitope on a small imraunogen or hapten, this antibody can sterically block any other antibody binding a
  • H.A. Katus and others used a "double sandwich" radioimmunoassay to measure two different epitopes against human myosin light chain in order to differentiate between cardiac-and skeletal-myosin light chains. ["Increased Specificity in Human
  • a two-site immunoassay requires the presence of at least two epitopes on the same molecule with enough distance between these epitopes to allow two antibodies to bind simultaneously without steric hindrance.
  • the length required to avoid steric interference and permit the combination of a hapten or immunogen with two antibody molecules is thought to be at least 20 to 26 angstroms.
  • This invention is based on the discovery that two-site immunoassays can be performed to detect or measure the concentration of small antigens and small haptens of a size previously considered too small to be assayed using two-site immunological assay methods.
  • the small molecules that are within the scope of this invention range in size from molecular weights of about 250 to 2500-3000, preferably between about 275 and 1000. The most preferred range is between about 275 and 500.
  • molecular weight is at least illustrative of the small molecules that are the subject of the present invention. and furthermore, we believe that the molecules in the above-mentioned molecular weight ranges are within the bounds that are unexpected by those skilled in the art.
  • two-site immunoassays are particularly useful to determine the presence and concentration of small molecules which are important in agriculture and medicine.
  • clinically relevant small molecules which can be tested using two-site immunoassays include steroids, such as aldosterone, cortisone, estradiol, progesterone, and testosterone, small hormones such as triiodothyronine and lutenizing hormone-releasing factor (LH-RH) , as well as antibiotics such as gentiroicins and penicillins.
  • mycotoxins like aflatoxins and trichothecenes, can be detected at very minute concentrations using two-site methods, and these assays are useful in testing grain and other agricultural products.
  • Small peptides such as aspartame and the previously mentioned hormones are also suitable to be assayed by two-site methods.
  • small molecules which can be assayed using the methods described herein may be haptens and therefore not immunogenic unless coupled to a protein or other suitable group. Because of the small size of the molecules which are the subject of the present discovery, in some cases it may be necessary to construct two conjugated small molecules, that is, the small molecule to be assayed is conjugated by attachment of a protein or other suitable group to two different sites on the small molecule. Then, these two sets of conjugated small molecules are used to immunize at least two mice, or other suitable animals, to produce either polyclonal antisera or monoclonal antibodies as described above. One mouse or other suitable animal can be immunized with these two sets of conjugated small molecules to raise two monoclonal
  • OMPI _ antibodies suitable to practice the present invention.
  • Methods of constructing conjugated haptens are well known in the art and are often varied to suit the particular hapten to be studied.
  • a procedure for making two conjugates, at different positions, to the steroid, aldosterone, will be given in the experimental examples which follow.
  • an immunoradiometric assay would proceed as follows: antibody A attached to a solid support is brought into contact with radiolabelled antioody B mixed with a known quantity of small molecule X; the mixture of antibodies A and B molecule as well as small hapten X is incubated; subsequently, the liquid phase is removed and washed from the solid phase; and the solid support is checked for radioactivity. Radioactivity will be found in association with the solid support only it antibodies A and B are both capable of binding simultaneously to small molecule X.
  • the same two-site immunometric assay used to test for simultaneous binding can then be used to assay a
  • a "forward" assay in which the antibody ' bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody:antigen complex, is employed. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen if any, and then contacted with a solution containing a known quantity of labelled antibody.
  • the solid support is washed a second time to remove the unreacted labelled antibody.
  • the washed solid support is tested to detect the presence of labelled antibody, for example, by
  • emitted radiation if the label is a radioactive element The amount of labelled antibody detected is compared to that for a negative control sample known to be free of the antigen. Detection of labelled antibody in amounts substantially above the background levels indicated by the negative control is interpreted to indicate the presence of the suspect antigen. It is best that several dilutions of the antigen-containing fluid be tested, since a great excess of antigen will give a background reading. Dilution of the sample would effectively eliminate this possibility. Quantitative determinations can be made by comparing the amount of labelled antibody with that obtained for standard samples containing known quantities of the antigen.
  • This kind of assay is frequently referred to as a
  • the particular system of one solid phase/one liquid phase, two-site immunometric assay used is not limited and workers skilled in the art will consider many variations.
  • many substances are suitable as the immobilizing support for the solid phase in this type of assay such as polyvinyl chloride microtiter plates, filter paper, plastic beads, or test tubes made from polyethylene, polystyrene, polypropylene or other suitable material.
  • Antibodies can also be linked to particular materials such as agarose, cross-linked dextran and other
  • a liquid phase antibody can be radiolabelled with 125I using the procedures of D. M. Klinman and J.
  • a small molecule which is the subject matter of the present application', may have enough symmetry in its conformation or chemical structure to
  • 25 invention is merphyrin. In most other cases, two nonidentical antibodies, that is, two antibodies which do not bind to the same epitope, would be required.
  • Example 3 A detailed description of an immunoradiometric 30 assay as contemplated for use in connection with the present discovery is given below as Example 3.
  • Two-site immunoassays other than the immunoradiometric or sandwich assays described above, are 35 also suitaole to carry out the teaching of the present
  • a mixture of two monoclonal antibodies can be tested in a cooperative immunoassay, as described in the Ehrlich and Moyle Science article and the Ehrlich et al. Journal of Immunology article previously discussed. If a synergistic effect using this type of assay is observed, this result can be taken as a demonstration that the two nonidentical monoclonal antibodies are capable of binding to the same small molecule simultaneously; and therefore, these particular antibodies and the same assay procedures can be used to test a collected sample.
  • Antibodies that display a synergistic effect in regard to a specific antigen are often capable of forming an extremely stable complex with that antigen.
  • Two monoclonal antibodies to a small steroid for use in a sandwich immunoassay can be raised by immunizing one mouse or other suitable animal using the method of Zimmering and co-workers [P.E. Zimmering et al. , Biochemistry, Vol 6 (1967) pp. 154-164] . Once antisera are thus raised, monoclonal antibodies can be produced according to the methods described by H. Zola and D.
  • Example 1 It is not always possible to raise two different antibodies by the procedures of Example 1; in many cases, two different hapten conjugates must be used as immunogens, since the method of coupling a small hapten may not allow the production of two distinct antioodies that can bind simultaneously.
  • the immunization of one animal with two conjugates would result in the production of two types of antibodies.
  • the two populations of antibodies may not be separable (or at least considerable effort may be necessary to separate them) . Therefore, the use of two different animals would provide an easy method of keeping the two antibody populations separate.
  • the two conjugates could be injected into one animal because separation of different types of antibodies would be'effected by cloning of cell lines.
  • aldosterone An example of two conjugates of one hapten is aldosterone.
  • Aldosterone -18,21-dihemisuccinyl- bovine serum albumin can be prepared according to the method of Erlanger et al. [Journal of Biological Chemistry, Vol. 228 (1957) pp. 713-727] . Immunization protocols are given in the same article. The production of 3-oxime aldosterone conjugates is described by J.T. McKenzie and J.A. Clements [Journal of Clinical Endocrinology and Metabolism, Vol. 38 (1974) p. 622] . Use of this conjugate as an immunogen is described by T. Ogihara et al.
  • An immunoradiometric assay with one solid phase ' antibody and one radiolabelled liquid phase antibody can be carried out as follows: Monoclonal antibodies A and B raised to a small hapten X (as in Examples 1 and 2) are purified from ascites fluid by well-known procedures such as the methods of H. Zola and D. Brooks, cited previously. To test whether antibodies A and B can bind simultaneously to small hapten X, fifty microliters of 0.1 mg/ml antibody A in 0.01 M potassium phosphate, 0.15 M NaCl, 0.02% sodium azide, pH 7.5 is incubated overnight at
  • Antibody B is radiolabelled with 125I, by the method
  • Antibody A solution is removed from the microtiter plate, then the wells in the plate are rinsed with distilled water three times and the wells are dried by banging the plates on a paper towel.
  • a sample containing a known quantity of small hapten X is mixed with radiolabelled antibody B, and fifty microliters of the mixture containing approximately 20,000 to 50,000 cpm of ra ⁇ iolabelled antibody B is added to each microtiter plate well. After an incubation of about 17-24 hours at 20°C, the radioactive solution is aspirated and the wells washed as before. Each well is cut from the plate with a scissor and counted in a gamma counter. Radioactivity attached to a well greater than background counts (background counts would be the amount of radioactivity bound even if small
  • hapten X was not mixed with antibody B before antibody B was added to the microtiter wells) , depends on the ability of antibodies A and B to bind simultaneously to small hapten X. It is best to use several concentrations of hapten X, because an excess amount of hapten will give background radiation bound to the plastic. If any of the concentrations give higher than background radioactivity, then antibodies A and B can bind hapten X simultaneously.
  • the presence or unknown concentration of small hapten X in a sample is determined in a similar manner.
  • This sample is mixed with radiolabelled antibody B while antibody A is adsorbed to a solid support, and the subsequent steps listed above are conducted.
  • the amount of radioactivity attached to a well is proportional to the amount of small hapten X in the sample, and its concentration can be judged by comparing this amount of radioactivity bound to the solid support with the amount bound when known concentrations of small hapten X are assayed in this system.
  • a cooperative immunoassay with two liquid phase antioodies starts with fifty microliters of radiolabelled small hapten X and fifty microliters of a sample containing the unlabelled small hapten to be measured (both in 1% horse serum, 99% 0.01M potassium phosphate, 0.15 M NaCl, 0.02% sodium azide, pH 7) are mixed with 100 microliters 0.3M potassium phosphate (pH 7.5) .
  • the antibody mixture is composed as follows: the amount of each antibody that had previously been determined to bind 50% of radiolabelled small hapten X in the absence of unlabelled small hapten X by the procedure described here (wherein each individual antibody replaces the mixture of antibodies) are mixed in a 1:1 ratio.
  • the antibodies, buffers and radiolabelled and unlabelled small hapten X are incubated one hour at 37°C followed by 18 hours at 5°C.
  • Ten microliters of 50% normal mouse serum in phosphate buffered saline is then added to each assay tube.
  • An appropriate amount of rabbit anti-mouse IgG antiserum is then added. (The appropriate amount is determined by performing the described assay with no unlabelled small hapten X and varying amounts of rabbit anti-mouse IgG antiserum. The amount of antiserum that precipitates the highest amount of radioactivity is the appropriate amount.)
  • the tubes are incubated 10 minutes at 37°C and then one hour at room temperature.
  • the precipitate is sediraented by centrifugation, the supernatant is aspirated, and the radioactivity in the precipitate is counted and is proportional to the amount of small hapten X present in the sample, that is, the lower the count, the higher the concentration of small hapten X present in the sample.

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Abstract

Procédé permettant d'exécuter des analyses immunologiques à deux sites sur de petites molécules, considérées jusqu'à présent trop petites pour lier deux molécules d'anticorps et que l'on croyait impossibles à détecter en utilisant ce type d'analyse. On estime qu'il est possible d'appliquer ce procédé virtuellement à toutes les procédures d'analyse immunologiques, aussi bien aux procédures conventionnelles qu'à celles développées tout dernièrement. Ce procédé permet de détecter avec précision des quantités très faibles de molécules de faible poids moléculaire aussi importantes sur le plan médical et agricole que les stéroïdes, les mycotoxines, les antibiotiques, les petites hormones et les petits peptides.
PCT/US1984/001737 1984-10-29 1984-10-29 Procede d'analyse immunologique pour petites molecules WO1986002736A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP19840904238 EP0198826A4 (fr) 1984-10-29 1984-10-29 Procede d'analyse immunologique pour petites molecules.
PCT/US1984/001737 WO1986002736A1 (fr) 1984-10-29 1984-10-29 Procede d'analyse immunologique pour petites molecules
AU36157/84A AU3615784A (en) 1984-10-29 1984-10-29 Immunoassay method for small molecules
JP50414284A JPS63501096A (ja) 1984-10-29 1984-10-29 小型分子の免疫測定法

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PCT/US1984/001737 WO1986002736A1 (fr) 1984-10-29 1984-10-29 Procede d'analyse immunologique pour petites molecules

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4772551A (en) * 1985-12-26 1988-09-20 Neogen Corporation Method and test kit for detecting a trichothecene using novel monoclonal antibodies
US4828985A (en) * 1984-06-12 1989-05-09 Cambridge Patent Developments Limited Antibodies against the complex of a small molecule and its binding protein, their preparation and their use in diagnostic methods
EP0589487A1 (fr) * 1989-03-14 1994-03-30 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux contre des petites molécules organiques
US5639624A (en) * 1989-03-14 1997-06-17 Board Of Regents Of The University Of Nebraska Monoclonal antibodies specific for metallic cations and method therefor
EP0892271A1 (fr) * 1997-07-14 1999-01-20 Universiteit Gent Laboratorium voor Bromatologie Faculteit Farmaceutische Wetenschappen Détection des mycotoxines par l'immunoessai enzymatique basé sur l'écoulement à travers une membrane
EP0893690A1 (fr) * 1997-07-14 1999-01-27 Universiteit Gent Laboratorium voor Bromatologie Faculteit Farmaceutische Wetenschappen Détection des mycotoxines par immunoessai enzymatique à flux traversant sur une membrane

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021182555A1 (fr) * 2020-03-12 2021-09-16 日立化成ダイアグノスティックス・システムズ株式会社 Anticorps de détection de l'aldostérone et méthode de dosage immunologique de l'aldostérone

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US4016043A (en) * 1975-09-04 1977-04-05 Akzona Incorporated Enzymatic immunological method for the determination of antigens and antibodies
US4036823A (en) * 1975-04-28 1977-07-19 Biological Developments, Inc. Barbituric acid antigenic conjugates, their preparation, antibodies and use
US4078049A (en) * 1977-02-28 1978-03-07 Hoffmann-La Roche Inc. Acetylcholine assay
US4172124A (en) * 1978-04-28 1979-10-23 The Wistar Institute Method of producing tumor antibodies
US4189463A (en) * 1978-04-07 1980-02-19 Hoffmann-La Roche Inc. Immunoassay for zoxazolamine
US4191738A (en) * 1978-04-07 1980-03-04 Hoffmann-La Roche Inc. Immunoassay for N-desmethyldiazepam
US4361647A (en) * 1980-05-22 1982-11-30 Palo Alto Medical Research Foundation Sandwich immunoassay and compositions for use therein
US4376110A (en) * 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4471058A (en) * 1982-07-26 1984-09-11 Board Of Trustees Operating Michigan State University Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4036823A (en) * 1975-04-28 1977-07-19 Biological Developments, Inc. Barbituric acid antigenic conjugates, their preparation, antibodies and use
US4016043A (en) * 1975-09-04 1977-04-05 Akzona Incorporated Enzymatic immunological method for the determination of antigens and antibodies
US4078049A (en) * 1977-02-28 1978-03-07 Hoffmann-La Roche Inc. Acetylcholine assay
US4189463A (en) * 1978-04-07 1980-02-19 Hoffmann-La Roche Inc. Immunoassay for zoxazolamine
US4191738A (en) * 1978-04-07 1980-03-04 Hoffmann-La Roche Inc. Immunoassay for N-desmethyldiazepam
US4172124A (en) * 1978-04-28 1979-10-23 The Wistar Institute Method of producing tumor antibodies
US4361647A (en) * 1980-05-22 1982-11-30 Palo Alto Medical Research Foundation Sandwich immunoassay and compositions for use therein
US4376110A (en) * 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US4471058A (en) * 1982-07-26 1984-09-11 Board Of Trustees Operating Michigan State University Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies

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Title
Archives of Biochemistry and Biophysics, Volume 220, issued January 1983, pages 318-320, HUANG et al, "Many Monoclonal Antibodies with an Apparent Specificity for Certain Lung Cancers are Directed against a Sugar Sequence Found in Lacto-N-Fucopentoasose III". *
Cancer Research, Volume 42, issued January 1982, pages 150-154, MAZAURIC et al, "Monoclonal Antibody-Defined Human Lung Cell Surface Protein Antigens". *
Cancer Research, Volume 44, issued February 1984, pages 681-687, VARKI et al, "Antigens Associated with a Human Lung Adenocarcinoma Defined by Monoclonal Antibodies". *
CHEMICAL ABSTRACTS, Vol. 99, No. 15, issued 1983, October 10, W.M. HUNTER, 'Recent Advances in Radioimmunoassay and Related Procedures', page 323, column 1, the Abstract No. 99:118630c, Radioimmunoassay Relat. Proced. Med., Proc. Int. Symp. 1982, 3-21, IAEA. *
CHEMICAL ABSTRACTS, Vol. 99, No. 23, issued 1983, December 5, W.M. HUNTER et al. 'Immunoassay of Human Luteinizing Hormone'. page 196, column 1, the Abstract No. 99:188641f, Brit UK Pat. Appl. GB 2,111,201, 29 June 1983. *
Federation Proceedings Volume 41, issued April 1982, page 409, Abstract 816, SOBOL et al, "Monoclonal Antibody which Distinguishes Human Lung Epidermoid and Adenocarcinomas from Small Cell Lung Cancers". *
Hybridoma, Volume 3, issued 1984, pages 119-130, SAJI et al, "Monoclonal Antibodies Specific for two Different Histological Types of Human Lung Carcinoma". *
In Vitro, Volume 17, issued December 1981, pages 1058-1070, MINNA et al, "Metho s for Production of Monoclonal Antibodies with Specificity for Human Lung Cancer Cells". *
Journal of Immunological Methods, Vol. 42, No. 1, issued 1981, M. UOTILA et al, 'Two-Site Sandwich Enzyme Immunoassay with Monoclonal Antibodies to Human alpha-Fetoprotein' pages 11-15. *
Journal of Immunology, Volume 131, issued July 1983, pages 497-502, MULSHINE et al, "Monoclonal Antibodies that Distinguish Non-Small Cell Lung Cancer". *
Nature, Vol. 256, issued 07 August 1975, G. KOHLER et al., 'Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity', pages 495-497. *
Proceeding of the National Academy of Sciences, USA, Volume 78, issued July 1981, pages 4591-4595, CUITTITTA et al, "Monoclonal Antibodies that Demonstrate Specificity for Several Types of Human Lung Cancer". *
See also references of EP0198826A4 *
Seminars in Oncology, Volume 10, issued March 1983, pages 3-19, GAZDAR et al, "the Biology of Non-Small Cell Lung Cancer". *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4828985A (en) * 1984-06-12 1989-05-09 Cambridge Patent Developments Limited Antibodies against the complex of a small molecule and its binding protein, their preparation and their use in diagnostic methods
US4772551A (en) * 1985-12-26 1988-09-20 Neogen Corporation Method and test kit for detecting a trichothecene using novel monoclonal antibodies
EP0589487A1 (fr) * 1989-03-14 1994-03-30 Board Of Regents Of The University Of Nebraska Anticorps monoclonaux contre des petites molécules organiques
US5503987A (en) * 1989-03-14 1996-04-02 Board Of Regents Of The University Of Nebraska Monoclonal antibodies specific for small moieties and method for their use
US5639624A (en) * 1989-03-14 1997-06-17 Board Of Regents Of The University Of Nebraska Monoclonal antibodies specific for metallic cations and method therefor
US5807695A (en) * 1989-03-14 1998-09-15 Board Of Regents Of University Of Nebraska Metallic cation binding polypeptides and methods therefor
EP0892271A1 (fr) * 1997-07-14 1999-01-20 Universiteit Gent Laboratorium voor Bromatologie Faculteit Farmaceutische Wetenschappen Détection des mycotoxines par l'immunoessai enzymatique basé sur l'écoulement à travers une membrane
EP0893690A1 (fr) * 1997-07-14 1999-01-27 Universiteit Gent Laboratorium voor Bromatologie Faculteit Farmaceutische Wetenschappen Détection des mycotoxines par immunoessai enzymatique à flux traversant sur une membrane

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EP0198826A1 (fr) 1986-10-29
EP0198826A4 (fr) 1989-03-23
JPS63501096A (ja) 1988-04-21

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