Title:  Monoclonal antibodies against Campylobacter jejuni and Campylobacter coli outer membrane antigens

United States Patent:  6,395,879

Assignee:  The Unites States of America as represented by the Secretary of Agriculture (Washington DC)

Filed:  March 26, 1999

The present invention is directed to a method of producing monoclonal antibodies that are highly specific for (1) unique epitopes of Campylobacter jejuni (Cj) only and (2) epitopes conserved between Campylobacter jejuni and Campylobacter coli (Cc) outer membranes; to specific monoclonal antibodies made by the methods of the instant invention; and uses thereof. The invention is drawn further to immunogens comprising the outer membrane complexes of Cj and Cc.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for an immunogen comprising outer membrane complexes from multiple strains of C. jejuni and C. coli, the immunogen inducing an immune response in animals, such as mice, enhancing the production of hybridomas specific for C. jejuni and C. coli epitopes, wherein the immunogen induces a broad range of antibodies against many outer membrane molecules of C. jejuni and C. coli, but not against other gram-negative enteric bacteria or non-jejuni/coli Campylobacter.

Monoclonal antibodies of the present invention made by using such an immunogen are specific for epitopes expressed on C. jejuni and C. coli, wherein the monoclonal antibody binds the C. jejuni and C. coli porin protein or carbohydrate bound to porin protein, the porin comprising the 45 kD and 35 kD monomeric forms and the trimeric form of the porin.

The invention also provides for monoclonal antibodies which bind specifically to a 43 kD protein expressed only on C. jejuni strains, or to carbohydrate bound to a 43 kD protein expressed only on C. jejuni strains.

The invention provides for a method of testing a sample for the presence of C. jejuni and/or C. coli comprising the steps of:

(a) exposing a sample suspected of containing C. jejuni and/or C. coli to a MAb which specifically binds C. jejuni and C. coli;

(b) detecting MAb-antigen binding, said binding being indicative of the presence of C. jejuni and/or C. coli in said sample; and

(c) capturing C. jejuni or C. coli with MAb-conjugated-magnetic beads or MAb-conjugated-polystyrene beads or MAb conjugated or bound to a solid matrix, and detection of bound C. jejuni or C. coli with a method selected from: a second specific MAb, a PCR-specific assay, mass spectrometry (MALDI-TOF), reporter phage specific for C. jejuni or C. coli, optical sensing, or electronic sensing.

The invention also provides for a method of immunizing animals by:

(a) preparing an immunogenic complex by isolating outer membrane complexes from at least two Campylobacter strains selected from at least one C. jejuni and at least one C. coli;

(b) pooling in approximately equivalent concentrations of the Campylobacter outer membrane complexes;

(c) immunizing animals intravenously; and

(d) screening antisera for Campylobacter antibody production.

The invention provides further for a method of making a monoclonal antibody specific for epitopes expressed on the outer membranes of C. jejuni and C. coli, wherein the monoclonal antibody binds a C. jejuni and C. coli porin protein or a porin protein-carbohydrate complex, the method comprising:

(a) preparing an immunogenic complex by isolating outer membrane complexes from at least two Campylobacter strains selected from the group consisting of C. jejuni and C. coli;

(b) pooling in approximately equivalent concentrations of the Campylobacter outer membrane complexes;

(c) immunizing animals intravenously;

(d) screening antisera for Campylobacter antibody production;

(e) fusing isolated spleen cells from animals positive for Campylobacter antibody production and myeloma cells;

(f) isolating hybridomas making antibodies that bind Campylobacter; and

(g) screening and identifying monoclonal antibodies that bind Campylobacter.

The present invention further provides for monoclonal antibodies made by such a method, wherein the MAb binds common epitopes of C. jejuni and C. coli.

The present invention is summarized further in that monoclonal antibodies specific for (1) unique epitopes of C. jejuni and (2) epitopes conserved between C. jejuni and C. coli outer membranes are produced by hybridomas formed by the fusion of cells from a mouse myeloma line and spleen cells from a mouse previously immunized with a mixture of outer membrane complexes (OMC) isolated from eight LPS serotype strains and two poultry strains of C. jejuni and C. coli. The monoclonal antibody designated as C731 is characterized in that it reacts with a protein having a molecular weight of approximately 44 kilodaltons and a carbohydrate moiety. The monoclonal antibody designated as C740 is characterized in that it reacts with a protein having a molecular weight of approximately 43 to 44 kilodaltons and a carbohydrate moiety.

The present invention is further directed to a process for producing monoclonal antibodies against Campylobacters comprising propagating a hybridoma formed by fusing a cell capable of producing antibodies against (1) C. jejuni or (2) C. jejuni and C. coli with a myeloma cell and harvesting the antibodies produced by the hybridoma.

It is an object of the present invention to produce monoclonal antibodies that are highly specific for (1) specific or distinct epitopes of C. jejuni and (2) epitopes shared between C. jejuni and C. coli outer membranes.

Another object of the present invention is to develop tests using monoclonal antibodies to assay for C. jejuni and C. coli isolated in pure culture and in clinical, food, and water samples.

It is further an object of the present invention to develop immunoassays for the detection of Campylobacter in food and in clinical specimens.

Another object of the present invention is to develop a procedure to isolate C. jejuni and C. coli from foods or other samples and to determine the prevalence of the organisms in the samples.

It is another object of the present invention to detect a neo-epitope of the outer membrane of C. jejuni, the neo-epitope defined here as an antigenic epitope comprising a porin protein-carbohydrate complex, wherein the neo-epitope is specific for C. jejuni and does not cross-react with other Campylobacters.

Because of their high specificity, the monoclonal antibodies may be a useful reagent for the detection of C. jejuni and/or C. coli in foods, clinical specimens, or in situ localization of the bacteria. The antibodies of the present invention may be used further to monitor environmental and/or waste water or various facilities for C. jejuni and/or C. coli contamination. The testing procedure would include, for example, enzyme-linked immunoassays (ELISAs), immunomagnetic capture, radioimmune assays, biosensor assays and other immunoassays including, but not limited to microscopic methods.

The monoclonal antibodies of the present invention may be used singly or in combination, such as in a cocktail mixture, to detect specific Campylobacter species, as neutralizing antibodies, or in a composition comprising one or more antibodies to be used in administering passive immunity to humans, livestock, poultry, or other animals.

The antibodies may be used further in side-by-side assays to determine whether the samples react only with the antibodies specific for both C. jejuni and C. coli or whether they react only with the antibodies that are specific for C. jejuni alone.

Another object of the invention is to provide an immunogen comprising outer membrane complexes from multiple strains of C. jejuni used as a vaccine which induces high levels of specific antibodies directed against C. jejuni and which protects against C. jejuni infection in humans, livestock, poultry, or other animals.

An additional object of the invention is to provide an immunogen comprising outer membrane complexes from multiple strains of C. jejuni and C. coli used as a vaccine which induces high levels of specific antibodies directed against both C. jejuni and C. coli and which protects against C. jejuni and C. coli infection in humans, livestock, poultry, or other animals.

Claim 1 of 8 Claims

We claim:

1. A monoclonal antibody designated as C731, renamed as C818, and deposited as ATCC HB-12651.

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