WO1997039034A1 - Moyens pour la detection de bacteries de l'espece taylorella equigenitalis et applications biologiques - Google Patents
Moyens pour la detection de bacteries de l'espece taylorella equigenitalis et applications biologiques Download PDFInfo
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- WO1997039034A1 WO1997039034A1 PCT/FR1997/000649 FR9700649W WO9739034A1 WO 1997039034 A1 WO1997039034 A1 WO 1997039034A1 FR 9700649 W FR9700649 W FR 9700649W WO 9739034 A1 WO9739034 A1 WO 9739034A1
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- Prior art keywords
- monoclonal antibodies
- equigenitalis
- fragments
- species
- antibodies
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Classifications
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- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4233—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
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- 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
- C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the subject of the invention is means for the detection of bacteria of the genus Taylorella and their biological applications.
- ECM Since the outbreak of this disease in 1977 in Newmarket (Great Britain), CEM has spread among the equine population in the world (Europe, USA, Japan). ECM was initially characterized by the appearance of purulent vaginal discharge caused by acute endometritis. The epidemiology and clinical manifestations of the disease have now changed. There are only a few rare foci with an acute form of CEM; these are then contaminations of several mares belonging to the same harem. The clinical forms of metritis have, indeed, become rare and T. equigenitalis is mainly found in asymptomatic carriers or in the pre-clinical stage. The disease is transmitted by stallions which show no clinical symptoms. The ECM constitutes an obstacle to the international exchange of equines and its detection is recommended by the OIE (Office International des Epizooties, list B). Indirect means of screening such as serology have been abandoned by many countries such as the USA, Great Britain and France.
- OIE Office International des Epizooties, list B
- Direct means of screening are practiced: screening by bacteriological culture in many countries, screening by indirect immunofluorescence. In France, prophylactic measures include both bacteriological culture and
- IIF indirect immunofluorescence
- T. equigenitalis is a very fragile and very slow growing bacterium (the observation time of the seed boxes is at least 6 days). It is, moreover, liable to be inhibited by other bacteria of the flora examined.
- equigenitalis are themselves either too succinct and subject to variation (evidence of absence activity for the three classic enzymatic activities that T. equigenitalis presents), is too cumbersome to manage within the required time. Screening using only the bacteriological technique has therefore become a hazardous method of diagnosis. An undetermined percentage of healthy carriers is therefore considered to be uninfected each season.
- a second screening test for infection with T. equigenitalis was selected in France. This test is based on the identification of the bacterium by indirect immunofluorescence using antiserum produced on rabbits and anti-rabbit fluorescent antibodies. This screening test has the advantage of delivering its results much faster (24 to 48 hours) than a test by bacteriological culture.
- the inventors have sought to remedy these difficulties in detecting infection with T. equigenitalis, by developing new means making it possible to identify a bacterium of the species T. equigenitalis without the risk of either false positives or false negatives.
- the invention therefore aims to provide means for specific, highly reliable detection of T. equigenitalis, based on recognition of the defined antigen-antibody type. It also relates to the use of these means for the diagnosis, treatment and prophylaxis of diseases caused by T. equigenitalis.
- the means of the invention are monoclonal antibodies characterized in that they recognize an epi tope of a bacterium of the species T. equigenitalis.
- these antibodies do not exhibit cross-reactions with one or more epi topes of a Taylorella bacteria of a different species or of a bacteria of a different genus. They therefore make it possible to detect T. equigenitalis with certainty and, according to an aspect of great interest, using a single test.
- the monoclonal antibodies of the invention are also as obtained from hybrids, by fusion of non-secreting murine myeloma cells with spleen cells from immunized mice using of a strain of the inactive species T. equigenitalis or of extract (s) of such a strain, cloning and selection according to the property of their culture supernatant to recognize one or more epi topes of a bacterium of the species T. equigenitalis, and recovery of the antibodies sought, followed where appropriate by their purification.
- the invention also relates to the fragments of the mAbs defined above, more particularly their fragments Fv, Fab, F (ab ') 2.
- the mAbs of the invention and, where appropriate, their fragments, are further characterized in that they are capable of recognizing proteins of T. equigenitalis from the group comprising proteins such as the proteins of 150, 120, 52.7 or 22 (LPS) kDa.
- the means of the invention are immunogenic proteins characterized in that they are capable of interacting with said mAbs or their fragments. These proteins are obtained, thanks to the so-called mAbs or their fragments, from T. equigenitalis, or by synthesis.
- the means of the invention are anti -antibodies (hereinafter referred to as anti -AcM for short) and the fragments of these anti -anti co rps, these anti -AcM and their fragments being characterized in what they are capable of interacting with mAbs or their fragments defined above.
- the invention therefore provides a method for obtaining and selecting MAb defined above, characterized in that it comprises 1: cell fusion of non-secreting murine myeloma with spleen cells from mice immunized using '' a strain of the species T. equigenitalis or extract (s) of such a strain, screening using a revelation technique, such as, in particular, indirect immunofluorescence, hybridomas whose culture supernatants react positively with a bacterium of the species T. equigenitalis or a fragment thereof this,
- the invention also relates to the application of the above technique for the production of anti-mAb antibodies.
- the invention relates to strains of hybridomas as obtained according to the methods defined above.
- the mAb fragments and the anti-mAb can be easily obtained using conventional enzymatic techniques.
- the contacting step is carried out under conditions in particular of duration, temperature, buffer, allowing the establishment of an antigen-antibody type reaction.
- markers are used, for example fluorescent, enzymatic, radioactive or luminescent markers. It will be noted that the judicious choice of a particular mAb, or of a fragment of this mAb, makes it possible to directly identify a given epi tope of T. equigenitalis in a sample or a culture to be analyzed. By using an immunogenic protein or an anti-mAb antibody or a fragment of the latter, a prior contact of the sample or of the culture with the bacteria will be demonstrated.
- the absence of cross-reactions of the mAbs of the invention and their fragments with epithets of bacteria of the genus Taylorella other than T. equigenitalis, and bacteria of a different genus, is advantageously taken advantage of for the diagnosis of pathologies linked to T. equigenitalis.
- the invention therefore also relates to the use of said mAbs and their fragments for the diagnosis of an infection with T. equigenitalis, more particularly contagious equine metritis, characterized in that it comprises: - bringing into contact one or more mAbs of the invention, or their fragments, with a biological sample, and
- the reagents in particular the markers or buffers, allowing the revelation of the targeted immunological reaction, and, optionally, reagents for blocking non-antigen-antibody reactions such as mouse serum,
- the mAbs and their fragments defined above can be used in therapy to combat infection by T. equigenitalis, and more particularly against contagious equine metritis.
- the invention thus also relates to pharmaceutical compositions containing one or more mAbs, or their fragments, defined above, as drug vectors or as passive immunotherapy agents, alone or in combination with pharmaceutically inert vehicles. It also relates to their use for the development of biosensors.
- the invention relates to the use of immunogenic proteins and anti-mAbs or their fragments for the preparation of vaccine compositions preventive of infection by T. equigenitalis.
- the vaccine compositions of the invention are characterized in that they contain at least one immunogenic protein or an anti-mAb or their fragments, as defined above, in an amount sufficient to elicit an immune response, in association with excipients physiologically acceptable.
- FIGS. 1 to 3 respectively represent:
- Example 1 Obtaining and selecting hybridomas capable of producing anti-T monoclonal antibodies. equigenitalis strains of T. equigenitalis used for immunization
- the two reference strains RI-16 and R2-19 therefore exhibit the properties generally observed for all the strains of T. equigenitalis studied in the prior art and are therefore used for the immunization of mice.
- the reference strains Rl-16 and R2-19 are washed twice in 0.1 M PBS buffer, pH 7.4, and inactivated by heating at 56 ° C for 75 min. The cells are then diluted in PBS, until bacterial suspensions of optical density 0.77 to 380 nm. They are then divided into aliquots and stored at -80 ° C until use.
- mice are injected 0.5 ml of Rl-16 and R2-19 bacterial suspension emulsified with the complete Freund's adjuvant (2 mice per strain) into BALB C adult mice. A booster injection is given on the 14th day with the same preparation. On the 21st day, the mice are immunized with 0.2 ml of suspension without adjuvant by the intravenous route and the spleen cells are collected 2 days later.
- Hybridomas are produced according to the standard procedure described by Kohler and Milstein (see reference below).
- Hybridoma growth is observed in 820 of the 1020 wells used (81.37%). IIF tests are carried out on 60 of these 820 wells to detect hybridomas producing the desired monoclonal antibodies. screening of hybridomas and monoclonal antibodies produced
- Unimmunized mouse serum is used as a negative control.
- the FITC mouse antiserum conjugate is incubated with each bacterial strain to serve as a conjugate control.
- the hybridomas of these wells are cloned by the limit dilution method in order to obtain a single cell per well in a 96-well tissue culture plate using the HT-DMEM medium and feeder cells.
- Single clone wells are screened with IIF and positive cells are frozen in liquid nitrogen.
- tissue culture supernatants of hybridomas are buffered by the addition of Tris IM, pH 8.0 (vol. 1/20) and sodium azide (0.02%). Aliquots are made and stored at -20 ° C.
- Example 2 Characterization of the anti-T monoclonal antibodies. equigenitalis
- the supernatants of the 14 hybridoma clones obtained according to Example 1 are tested by IIF according to the capacity of their supernatants to recognize other bacterial strains than the two reference strains R-16 and R-19 used for immunization, namely: the 7 wild strains of T.
- Staphyloccoccus aureus Pseudomonas fluorescens and Klebsiella pneumoniae. These bacteria are grown on a Columbia-based blood-agar medium.
- the 14 monoclonal antibodies tested recognize the seven wild strains of T. equigenitalis. 3 of them give a weaker positive response, namely 7 B7.1; 7 B7.10 and 10C9.6. None of the 14 monoclonal antibodies tested recognizes one of the 8 bacterial strains which do not belong to the species T. equigenitalis.
- the EN extracts of the T. equigenitalis strains were dissolved in a sample solvent (Tris.HCl 0.1 M pH 6.8; glycerol 10%; SDS 2%; ⁇ -mercaptoethanol 2 mM and bromophenol blue 0.01% ) in order to obtain a protein concentration of 1 mg / ml, then were brought to a boil at 100 ° C for 5 min (extract under denaturing conditions of T. equigenitalis, ED).
- a sample solvent Tris.HCl 0.1 M pH 6.8; glycerol 10%; SDS 2%; ⁇ -mercaptoethanol 2 mM and bromophenol blue 0.01%
- LPS Lipopolysaccharide extract
- the coloration of the total proteins by colloidal gold was used.
- the membranes were immersed for 30 min in a blocking solution (3% gelatin in 20 mM Tris and 0.5 M NaCl) and rinsed with gentle stirring in a washing solution (20 mM Tris; NaCl, 0.5 M; Tween R 20 0.05%).
- the membranes were then brought into contact with monoclonal antibody solutions diluted from 1/100 to 1/1000 in the antibody buffer (Tris 20 mM; 0.5 M NaCl, Tween R 20 0.05% gelatin 1% ) for 180 min at 25 ° C.
- the binding of monoclonal antibodies to the peptide bands was visualized using alkaline phosphatases (AP) conjugated with IgG immunoglobulins. goat (heavy and light chains) anti-mice (BLoRad, dilution to 1/2000) and using a substrate solution for PA (EoRad).
- AP alkaline phosphatases
- FIG. 2 illustrates an immunoblot between the bacterial proteins and the mAbs according to the invention on the one hand and the positive serum of mice on the other hand.
- the positive serum collected from immunized mice reacts with 5 proteins of the strain R-19: 120 kDA; 52.7 kDA; 33.4kDA; 17.5 kDA and 22 (LPS) kDA.
- Immobilon R PVDF (Sigma) membranes were pre-wetted with 100% methanol solution for
- the binding of the monoclonal antibodies to the dot blot membranes was revealed using PA conjugated to goat IgG immunoglobulins (heavy and light chains) anti-mice and using a substrate solution for PA (BLoRad) .
- the 14 monoclonal antibodies are compared in dot-blot with the extracts EN and ED of the strain R-19.
- These 8 monoclonal antibodies can therefore constitute reagents suitable for the detection of antigens of T. equigenitalis and, more particularly, for the diagnosis of CEM.
- Such antibodies can be used to characterize bacteria of the Taylorella genus in any biological preparation using denaturing conditions.
- the immunotype kit from Sigma which consists of strips of ni trocellulose pre ⁇ coated with anti-isotype antibodies of mouse immunoglobulins. After additional incubations, the identity The results obtained appear in column 9 of Table III.
- the 14 monoclonal antibodies produced are part of IgM for 5 of them, IgG2b for 4 of them, IgG3 for 3 of them and IgGl for 2 of them.
- Both antibody and indirect immunofluorescence screening techniques namely the "polyclonal antibody” technique and the technique which is the subject of the present invention, have both screened for T. equigenitalis isolated by bacteriological culture.
- the specificity of the monoclonal antibodies according to the invention, used in the context of indirect immunofluorescence, is better than that of polyclonal antibodies (94% vs 60%).
- Example 5 Elimination of non "antigen-antibody” reactions.
- Nonspecific reactions can sometimes be obtained between antibodies and Staphylococcus protein intermediary (protein A for S. aureus and protein G for Streptococci of groups C and G).
- the reactions are not of the antigen-antibody type.
- FITC were separated from the unlabeled molecules by passage through a Sephadex G25 column (Pharmacia).
- the slides are incubated for 1 hour at 37 ° C. with the FITC-labeled monoclonal antibodies according to the invention described above.
- the slides After washing and final rinsing, the slides are mounted in glycerin, buffered and examined under a fluorescence microscope. These three blocking techniques give fluorescence for the T. equigenitalis slides and do not give fluorescence for the non-specific binding slides (S. aureus and Streptococcus).
- the mAbs are precipitated by adding saturated ammonium sulfate to the final concentration of 50%. After centrifugation, the precipitate is resuspended in PBS, then filtered through Sephadex® G75 gel (Pharmacia) and finally purified by affinity chromatography on a column of protein A- Sepharose® CL-4B
- mice BUJEV'C mice are immunized by 1 SC injection of a mixture of equal parts of 50 ⁇ g of polymerized mAb and complete Freund's adjuvant. 2 subsequent injections are given 2 weeks apart, one with incomplete Freund's adjuvant, and the other without any adjuvant and peritoneally.
- microplates (Maxisorb, Nunc) are incubated for 16 h at 4 ° C with 100 ⁇ l / well of a suspension of 0.2 ⁇ g / ml of Fab in carbonate buffer pH8.
- the microplates are washed 3 times with P BS-Tween 20® (0.05%), pH 7.2, then the non-specific sites are blocked by a solution of BSA 2% in P BS-Tween 20® for 30 min at 37 ° C.
- the culture supernatants of the hybridomas are incubated 1 H at 37 ° C.
- the reaction is revealed by an anti-mouse conjugate labeled with peroxidase and its substrate.
- the AcM2 antibodies of the selected hybridomas, then their corresponding Fab fragments are purified according to the methods described below.
- the Fab fragments are coupled to the limpet hemocyanin keyhole (KLH, Sigma) by incubation for 16 h at 4 ° C. in a 0.05% glutaraldehyde (Sigma) solution, in a ratio of 1/1.
- the reaction is stopped with a 0.02M glycine solution and the conjugates are dialyzed against PBS.
- the protein is dosed at 25-100 ⁇ g per dose of vaccine and the vaccine is supplemented with alumina hydroxide as an adjuvant.
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Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/155,982 US6858209B2 (en) | 1996-04-12 | 1997-04-11 | Means for detecting bacteria of the taylorella equigenitalis species and their biological applications |
AU26416/97A AU708879B2 (en) | 1996-04-12 | 1997-04-11 | Means for detection of bacteria of the species Taylorella equigenitalis and their biological applications |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR96/04623 | 1996-04-12 | ||
FR9604623A FR2747387B1 (fr) | 1996-04-12 | 1996-04-12 | Moyens pour la detection de bacteries du genre taylorella et applications biologiques |
Publications (1)
Publication Number | Publication Date |
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WO1997039034A1 true WO1997039034A1 (fr) | 1997-10-23 |
Family
ID=9491167
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR1997/000649 WO1997039034A1 (fr) | 1996-04-12 | 1997-04-11 | Moyens pour la detection de bacteries de l'espece taylorella equigenitalis et applications biologiques |
Country Status (4)
Country | Link |
---|---|
US (1) | US6858209B2 (fr) |
AU (1) | AU708879B2 (fr) |
FR (1) | FR2747387B1 (fr) |
WO (1) | WO1997039034A1 (fr) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1986002360A1 (fr) * | 1984-10-19 | 1986-04-24 | Technology Licence Company Limited | Anticorps monoclonaux et leur utilisation |
Family Cites Families (5)
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US4444879A (en) * | 1981-01-29 | 1984-04-24 | Science Research Center, Inc. | Immunoassay with article having support film and immunological counterpart of analyte |
US4600711A (en) * | 1982-08-18 | 1986-07-15 | The University Of Kentucky Research Foundation | Composition for topical and infusion treatment of wounds and burns |
US4483851A (en) * | 1982-08-18 | 1984-11-20 | The University Of Kentucky Research Foundation | Treatment for contagious equine metritis |
US4540660A (en) * | 1983-04-07 | 1985-09-10 | Daryl Laboratories, Inc. | Substrate for fluoroimmunoassay of biological fluids |
US5891438A (en) * | 1992-10-30 | 1999-04-06 | The Regents Of The University Of California | Method for stimulating production of variable region gene family restricted antibodies through B-cell superantigen vaccination |
-
1996
- 1996-04-12 FR FR9604623A patent/FR2747387B1/fr not_active Expired - Fee Related
-
1997
- 1997-04-11 US US09/155,982 patent/US6858209B2/en not_active Expired - Fee Related
- 1997-04-11 AU AU26416/97A patent/AU708879B2/en not_active Ceased
- 1997-04-11 WO PCT/FR1997/000649 patent/WO1997039034A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1986002360A1 (fr) * | 1984-10-19 | 1986-04-24 | Technology Licence Company Limited | Anticorps monoclonaux et leur utilisation |
Non-Patent Citations (3)
Title |
---|
A. MACMILLAN ET AL.: "Antibodies reactive with Taylorella equigenitalis in equine sera.", THE VETERINARY RECORD, vol. 118, no. 20, 17 May 1986 (1986-05-17), LONDRES, GRANDE BRETAGNE, pages 562, XP000615390 * |
D. GRADINARU ET AL.: "Production and characterization of monoclonal antibodies against Taylorella equigenitalis.", VETERINARY RESEARCH, vol. 28, no. 1, 1997, PARIS, FRANCE, pages 65 - 76, XP002038656 * |
M. EGUCHI ET AL.: "Passive hemagglutination test for detection of antibodies against Taylorella (Haemophilus) equigenitalis in sera of mares.", VETERINARY MICROBIOLOGY, vol. 18, no. 2, October 1988 (1988-10-01), AMSTERDAM, PAYS-BAS, pages 155 - 161, XP000615355 * |
Also Published As
Publication number | Publication date |
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FR2747387A1 (fr) | 1997-10-17 |
AU2641697A (en) | 1997-11-07 |
FR2747387B1 (fr) | 1998-07-03 |
AU708879B2 (en) | 1999-08-12 |
US20020037879A1 (en) | 2002-03-28 |
US6858209B2 (en) | 2005-02-22 |
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