EP0229811A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation

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Publication number
EP0229811A1
EP0229811A1 EP19860904296 EP86904296A EP0229811A1 EP 0229811 A1 EP0229811 A1 EP 0229811A1 EP 19860904296 EP19860904296 EP 19860904296 EP 86904296 A EP86904296 A EP 86904296A EP 0229811 A1 EP0229811 A1 EP 0229811A1
Authority
EP
European Patent Office
Prior art keywords
monoclonal antibody
vibrio
antigen
enzyme
fluorescent
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Withdrawn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
EP19860904296
Other languages
German (de)
English (en)
Inventor
Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
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.)
TECHNOLOGY LICENCE Co Ltd
Original Assignee
TECHNOLOGY LICENCE Co Ltd
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 TECHNOLOGY LICENCE Co Ltd filed Critical TECHNOLOGY LICENCE Co Ltd
Publication of EP0229811A1 publication Critical patent/EP0229811A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1239—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Vibrionaceae (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Vibrio will be described with particular reference to Vibrio cholerae. Vibrio cholerae is described in Zinsser Microbiology (17th ed.) at pp. 750-4. V. cholerae is a facultatively anaerobic organism with an optimum temperature ranging from 18 to 37° C. Its metabolism is both respiratory and fermentative. Vibrio chol ⁇ erae will grow on simple media providing a utiliz- able carbohydrate, inorganic nitrogen, sulfur, phosphorus, minerals, and adequate buffering. They grow best at pH 7.0 but tolerate alkaline ' conditions to pH 9.5, a property used in the design of ' isolation media. They are extremely sensitive * to an acid pH, and a pH of 6.0 or less will sterilize cultures.
  • the cholera enterotoxin is a complex molecule with a molecular weight of approximately 80,000 to 98,000. Choleragen is predominately protein (98 percent), with approximately one percent lipid and one percent carbohydrate.
  • A which is responsible for biologic activity
  • B which is responsible for the binding of toxin to cell membranes.
  • Sub- unit A consists of a single molecule of subunit Al which contains the toxic activity, and a single molecule of subunit A2 which may serve as a stabilizer of the A complex before its action on the cell.
  • One subunit A is noncovalent- ly linked to four to six of the B subunits.
  • Gml ganglioside an acidic glycolipid con ⁇ taining sialic acid and galactose, or other gangliosides of glycoproteins may be the natural receptors for the choleragen.
  • the action of choleragen is to activate membrane-bound adenyl cyclase, which converts to ATP to cyclic AMP.
  • the resulting increase in cyclic AMP produces a net flow of fluid and electrolytes from the cells of the small intestine into the lumen.
  • Whole choleragen does not enter the cell but rather remains associated with the cell membrane.
  • enterotoxins in this application will be referred to as enterotoxins Al, A2, Bl, and B2.
  • enterotoxins Al, A2, Bl, and B2 enterotoxins
  • virulent V. cholerae must be . able to adhere to the intestinal surfaces.
  • Studies on adherence show that virulent cells penetrate the intestinal mucus and attach to the microvilli at the brush border of the epithelial cells.
  • Motility may be involved in the adherence of V. cholerae, . since nonmotile varieties are unable to attach to the intestine.
  • Attachment antigens in this application will be referred to as attach ⁇ ment antigens 1 and 2.
  • the nonagglutinable vibrios can cause iso ⁇ lated as well as focal outbreaks of diarrhea but the volume of fluid loss does not approach that of classic cholera, and the disease is usually self-limiting.
  • cholera The primary defense in the control of cholera is the maintenance of adequate sewage treatment and water purification systems, together with the prompt detection and. treatment of patients and carriers. In countries with adequate sanita ⁇ tion, cholera is limited to imported cases.
  • Bacterial diarrhea caused by Vibrio cholerae is a common and often serious condition manifest- ing as fluid loss from the bowel, leading in many cases to dehydration and occasionally to death.
  • Epidemic infantile diarrhea is a serious problem that occurs in newborn nurseries and can result in high mortality rates.
  • diarrhea is the most common cause of death in early childhood in many tropical and subtropical
  • Dysentery is a severe form of diarrhea and can result in high mortality rates. Travel ⁇ ers 1 diarrhea (Turista) is the most common, and among the most feared, illness to threaten the traveler. Diarrhea is also commonly associat ⁇ ed with contaminated food and water. Severe diarrhea often brings the patient to the hospital or the physician's office. Acute diarrhea in adults can be so fulminant as to cause hypovolemic shock and death from the outpouring of fluid into the upper small bowel before the first diarrheal stool occurs.
  • Vibrio cholerae has serogroup 01, which subdivides on the basis of additional factors A, B, and C. Biotypes of serotype 01 are pgawa (A, B), inaba (A, C), and hikojima (A, B, C), plus a proposed serotype with only A. The extreme specificity of antigen-antibody reactions has made it possible to recognize differences between strains of a bacterial species that are indistinguishable on the basis of other phenotypic criteria.
  • Vibrio infections vary depending on the locus of the infection. It is estimated that in the United States and Europe many millions of cases of bacterial diarrhea occur annually of which several million are seen by a physician or admitt ⁇ ed to a hospital. Because of the self-limiting nature of the adult disease, most people do not seek treatment. Of the people seeking treat- ment, bacterial diagnosis of diarrhea is presently made by stool culture techniques. These tech ⁇ niques are generally performed onl ' y in hospitals and are slow, requiring one to three days. During this time the patient- is exposed, if treated, to the expense and potential hazard of inappropriate therapy.
  • a wide variety of isotopic and nonisotopic immunoassays have been utilized in conjunction with monoclonal antibodies to test for the pres- ence of an antigenic substance.
  • agglutination, immuno-fluorescent, chemilum- inescent or fluorescent immunoassay, immuno-e ⁇ ect- ron microscopy, radiometric assay systems, radio immunoassays, and enzyme-linked immunoassays are the most common techniques used with the - monoclonal antibodies. Other techniques include bioluminescent, fluorescence polarization, and photon-counting • immunoassays.
  • EIA enzyme-linked immunoassay procedure
  • the enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence .
  • the serotype of the infecting organism can be determined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
  • the present invention com ⁇ prises monoclonal antibodies specific for the antigens or species of Vibrio; in particular, the antigens or species of Vibrio cholerae; the antigens or species of Vibrio parahemolyticus; the antigens or species of Vibrio anguillerum, and the toxins of Vibrio cholerae, as well as a monoclonal antibody broadly cross-reactive with an antigen * for each species of the genus Vibrio.
  • the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Vibrio antigens, each comprising a monoclonal antibody against one of the above- mentioned antigens to Vibrio or to a particular species thereof and linked thereto an appro ⁇
  • the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom, or particle, or any other label.
  • the invention further comprises the process for diagnosing' the presence of Vibrio antigens or organisms in a specimen comprising contacting said specimen with the labeled monoclonal antibody in an appropriate immunoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a mono ⁇ clonal antibody for an antigen of Vibrio and a carrier or diluent, as well as kits containing at least one labeled monoclonal antibody to an antigen of a Vibrio.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells, from a mammal which has been immunized against the particular Vibrio antigen, with an appropri ⁇ ate myeloma cell line, preferably NSO (uncloned), P3NS1-Ag4/1, or Sp2/0 Agl4.
  • the resultant product is then cultured in a standard HAT (hypoxanthine, a inopterin, and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilizing immunoassay techniques which will be described below.
  • the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents
  • mice i.e., mice, rats, and rabbits
  • bovine, ovine, canine or the like
  • the mouse is first immunized by injection of the particular Vibrio antigen chosen generally for a period of approximately eleven weeks.
  • the mouse shows sufficient antibody produc ⁇ tion against the antigen, as determined by conven ⁇ tional assay, it is given a booster injection of the appropriate Vibrio antigen, and then killed so that the immunized spleen may be remov- ed.
  • the fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
  • the fused cells yielding an antibody which give a positive response to the presence of the particular Vibrio antigen are removed and cloned utilizing any of the standard methods.
  • the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Vibrio antigen.
  • the monoclonal antibody selected, which is specific for the particular Vibrio antigen or species, is then bound to an appropri ⁇ ate label. .
  • Amounts of antibody sufficient for labeling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue . culture or culture in vivo in mammals, such as mice.
  • the monoclonal antibodies may be labeled with a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
  • labels such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
  • the present invention will be described with reference to the use of an enzyme labeled monoclonal antibody.
  • Some of the enzymes ut ized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, or urease, and the like.
  • Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
  • EIA enzyme- linked immunosorbent assay
  • Fluorescent-immunoassay is based on the labeling of antigen or antibody with fluorescent probes. A nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled aatigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen. Examples of this particular type of fluorescent- immunoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous- systems such as the Substrate Labeled Fluorescent ' Immunoassay. The most suit ⁇ able fluorescent probe, and ' the one most widely used is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized for the probe- utilized in the particular assay and in- which the effect of scattering can be minimized.
  • Fluorescence polarization In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari ⁇ zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter range with respect to antigens in biological samples.
  • Luminescence is the emission of light by an atom or molecule as an electron is transferred to the ground state from a higher energy state.
  • the free energy of a chemical reaction provides the energy required to produce an inter ⁇ mediate reaction or product in an electronically excited state. Subsequent decay back to the ground state is accompanied by emission of light.
  • Bioluminescence is the name given to a special form of che iluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction.
  • the best known chemiluminescent substance is luminol.
  • a further, aspect of the present invention is a therapeutic composition " comprising one or more of the monoclonal antibodies to the particular Vibrio antigen or species, as well as a pharmacologically acceptable carrier or diluent.
  • Such compositions can be used to treat humans and/or animals afflicted with some form of Vibrio infection and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individual being treated and the severity of the infection.
  • One or more of the monoclonal antibodies can be .assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens, or species of Vibrio in various speci ⁇ mens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Vibrio alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Vibrio and/or other bacteria.
  • conjugated or labeled mono ⁇ clonal antibodies for antigens and/or species of Vibrio and other gram-negative bacteria can be utilized in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible Vibrio or other gram-negative sepsis.
  • the monoclonal test is an advance over existing procedures in that it is more accurate than existing tests; it gives "same day” results, provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labor costs . and laboratory time required for
  • kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Vibrio.
  • kits comprising at least one labeled monoclonal antibody against a particular Vibrio antigen or species, as well as any appropriate stains, counterstains, or reagents.
  • Specific antigens or species to be detected in this kit include the characteristic antigens and toxins of Vibrio cholerae and the antigen or species of Vibrio parahemolyticus, Vibrio anguillerum.
  • Monoclonal diagnostics which detect the presence of Vibrio antigens can also be used in periodic testing of water sources, food sup ⁇ plies and food processing operations.
  • the present invention describes the use of the labeled monoclonal antibodies to determine the presence of a standard antigen
  • the invention can have many applications in diagnosing the presence of antigens by determining whether specimens such as urine, blood, stool, water, milk, and the like contain the particular Vibrio antigen. More particularly, the invention could be utilized as a public health and safety diagnos ⁇ tic aid, whereby specimens such as water or food could be tested for possible contamination.
  • the invention will be further illustrated in connection with the following examples which are set forth for purposes of illustration only and not by way of limitation.
  • the monoclonal antibodies of the present invention were prepared generally according to the method of Kohler and Milstein, supra.
  • DMEM Dulbeccos Modified Eagles Medium
  • FCS Foetal Calf Serum
  • PBS Phosphate Buffered Saline
  • TSB Tryptone Soya Broth
  • mice were injected with prepared Vibrio cholerae enterotoxin. They were given one intraperitoneal injection per week for three weeks and, after another week, one intravenous injection (5 ⁇ g prepared antigen/dose/mouse) . The mice were bled approximately six days after the last injection and the serum tested for antibodies by assay. The conventional assay used for this serum titer testing was the enzyme-linked immunosorbent assay system. When the mice showed -antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection intraperitoneally, three days prior to splenectomy.
  • Spleen cells from the immune mice were harvested three days after boosting, by conventional techniques.
  • the donor mouse selected was killed and surface-sterilised by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml DMEM to which had been added 3% FCS.
  • the spleen was then gently homogenised in a LUX homogenising tube until all cells had been released from the membrane, and the cells were washed in 5 ml 3% FCS-DMEM.
  • the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension were then made in 3% FCS-DMEM.
  • the myeloma cell line used was NS0 (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using, as tissue culture medium, DMEM containing 3% FCS.
  • the spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS-DMEM.
  • 0.1 ml of the ' suspension was diluted to 1 ml and a haemacytometer with phase microscope was used.
  • 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
  • the resultant cell pellet was placed in a 37°C water-bath. 1 ml of a 50 w/v solution of polyethylene glycol 1500 (PEG) in saline Hepes, pH approximately 7.5, was added, and the mixture gently stirred for approximately 1.5 minutes. 10 ml serum-free tissue culture medium DMEM were then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS. 10 ⁇ l of the mixture were placed in each of 480 wells of standard multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium
  • the wells were kept undisturbed and cultured at 37°C in 9% CO- air at approximately 100% humidity.
  • the wells were analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days.
  • screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described below.
  • agar method a freshly-prepared stock solution of sterile 1.2% agar in double distilled water with an equal volume of double-strength DMEM and additives was maintained at 45 C. This solution (10 ml) was then aliquoted into 10 cm Petri dishes, to form a base layer. An overlay of equal volumes of agar and cells in 18% FCS-DMEM was spread evenly over the base. The cells were allowed to multiply for approximately 10 days at 37 C, 7-9% CO_, 95% RH.
  • Viable separate colonies were picked off the agar surface and placed into 60 wells of a 96-well microtitre tray in 18% FCS-DMEM. After a further period of growth, the supernatants were assayed for specific antibody by the standard enzyme immunosorbent assay.
  • limiting dilution method was used.
  • dilutions of cell suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve one cell/well and one-half cell/well in a 96-well microtitre plate.
  • the plates were incubated for 7-14 days at 37 C, 97% RH, 7-9% C0 2 until semi-confluent.
  • the supernatants were assayed for specific antibody by the standard enzyme immunoabsorbent assay.
  • the clones were assayed by the enzyme immunoassay method to determine antibody production.
  • the monoclonal antibodies from the clones were screened by the standard techniques for binding to the enterotoxin, prepared as in the immunisation, and for specificity. Specificity to the beta sub-unit of cholera toxin was observed, as were negative reactions to 13. coli labile toxin and boiled strains of IS. coli.
  • the EIA immunoassay noted above may be used.
  • mice were primed with pristane, for at least
  • the precipitate was harvested by centrifugation at 10,000 g for 10 minutes.
  • the precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) .
  • the solution was dialysed versus 2x1000 ml of the same buffer, at +4°C.
  • the dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer.
  • the monoclonal antibody was eluted with phosphate/EDTA buffer.
  • Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml of PBS pH 7.4, at +4°C. After dialysis, the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% solution of glutaraldehyde in PBS was added. The conjugation mixture was left at room temperature for 1.5 hours.
  • glutaraldehyde was removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25 * M) column, previously equilibrated in PBS.
  • the conjugate was eluted with 3.5 ml PBS and then dialysed against 2 x 2000 ml of Tris buffer (50 mM Tris, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide) at +4°C.
  • Tris buffer 50 mM Tris, 1 mM magnesium chloride, pH 8.0 plus 0.02% sodium azide
  • To the dialysed conjugate was added 1/lOth its own volume of 10% BSA in Tris buffer.
  • the conjugate was then sterile-filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial, and stored at +4°C.
  • the enzyme immunoassay method was used for testing. This method comprises coating the wells of a standard polyvinyl chloride (PVC) microtitre tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenyl phosphate.
  • PVC polyvinyl chloride
  • the monoclonal antibodies were found to be specific for the enterotoxin.
  • the monoclonal antibody was tested and shown to be of the Class IgG..
  • the particular epitopic site to which the antibody attaches to the antigen can also be determined.
  • the same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate.
  • Example 1 The procedure of Example 1 was repeated, to give a monoclonal antibody broadly cross-reactive with each species of the genus Vibrio.
  • Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Anticorps monoclonaux du gène Vibrio, les anticorps marqués, compositions et kits les contenant, et leur utilisation dans le diagnostic de l'antigène et à des fins thérapeutiques.
EP19860904296 1985-07-16 1986-07-16 Anticorps monoclonaux et leur utilisation Withdrawn EP0229811A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8517896 1985-07-16
GB858517896A GB8517896D0 (en) 1985-07-16 1985-07-16 Monoclonal antibodies

Publications (1)

Publication Number Publication Date
EP0229811A1 true EP0229811A1 (fr) 1987-07-29

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EP19860904296 Withdrawn EP0229811A1 (fr) 1985-07-16 1986-07-16 Anticorps monoclonaux et leur utilisation

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EP (1) EP0229811A1 (fr)
JP (1) JPS63500594A (fr)
GB (1) GB8517896D0 (fr)
WO (1) WO1987000534A1 (fr)

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EP0819435A1 (fr) * 1996-07-16 1998-01-21 K.U. Leuven Research & Development Immunisation passive de poissons et de crustacés et émulsions d'immunoglobulines utilisées à cet effet
JP4886122B2 (ja) * 2001-05-31 2012-02-29 日水製薬株式会社 腸炎ビブリオ検出用培地

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Publication number Priority date Publication date Assignee Title
US4461829A (en) * 1981-09-14 1984-07-24 Miles Laboratories, Inc. Homogeneous specific binding assay element and lyophilization production method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8700534A1 *

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Publication number Publication date
GB8517896D0 (en) 1985-08-21
WO1987000534A1 (fr) 1987-01-29
JPS63500594A (ja) 1988-03-03

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