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DESIGN OF NOVEL TECHNIQUES TO PRODUCE PIG IGA HYBRIDOMAS

Objective

OBTENTION OF CELL STRAINS PRODUCING IGA MONOCLONAL ANTIBODIES AT HIGH LEVEL, IN ORDER TO STUDY THE PROTECTIVE MECHANISMS OF MUCOSA AGAINST INFECTIONS, AND TO PRODUCE VERY EFFICIENT ANTIBODIES FOR THE THERAPY OF INTESTINAL INFECTIONS.
Immunological studies, using differentiated pig cells, resulted in complete purification of T4-T8 and B pig lymphocytes by specific adherence to plastic. Endothelial cell clones, expressing the LacZ-antigen, were also obtained using a retroviral vector and IgA producing cells have been purified from the gut.

The aim of the project was to develop techniques to stimulate the appearance in pig gut of immunoglobulin A (IgA) producing cells and then to identify, isolate and purify them for in vitro immortalisation. Mouse monoclonal antibodies were prepared to markers of the pig immune response and used to monitor mucosal IgA responses, stimulated with live and live attenuated transmissible gastroenteritis virus (TGEV), recombinant Escherichia coli and proteins adjuvanted with cholera toxin. Techniques to isolate immune cells from the gut and gut associated lymphoid tissue were developed and these cells were subfractionated by panning using activated plastics. An in vitro technique to study the kinetics of the mucosal IgA response was developed and used to idenfity the optimum time for the isolation of IgA producing cells. Attempts to immortalise these cells by retrovirus transformation using a variety of techniques were not successful. Recently, we were able to fuse mouse myeloma cells with mesenteric lymph node cells isolated from TGEV injected pigs, and showed that these cells produce IgA anti-TGEV antibodies.
VERY FEW HYBRIDOMAS OBTAINED BY STANDARD IMMUNIZATION PROCEDURE PRODUCE IGA. IT WOULD BE USEFUL TO OBTAIN IGA REACTING HYBRIDOMAS IN PIGS TO STUDY PROTECTIVE MECHANISMS AGAINST VIRAL AND BACTERIAL DISEASES OF MUCOSAL SURFACES, AND ALSO TO DESIGN A NEW SIMPLE METHOD TO CONSTRUCT HYBRIDOMAS AND TO OBTAIN ANTIBODIES OF HIGH THERAPEUTIC VALUE TO CURE INTESTINAL INFECTIONS IN THE YOUNG ANIMAL.

THE OBJECTIVES OF THE PROJECT ARE :

1) STIMULATE IGA PRODUCING CELLS BY APPROPRIATE VACCINATION PROCEDURE (ANTIGENS : TRANSMISSIBLE GASTRO ENTERITIS VIRUS AND E.COLI).
2) OBTAIN PIG CELL LINES ABLE TO FUSE WITH LYMPHOCYTES.
3) TRANSFORM ISOLATED B CELLS INTO PERMANENT LINES BY FUSION WITH TRANSFORMED PIG CELLS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Institut National de la Recherche Agronomique (INRA)
Address
11 Rue Jean Nicot
75007 Paris
France

Participants (2)

University of Bristol
United Kingdom
Address
Senate House Tyndall Avenue
BS8 1TH Bristol
Université de Lyon I (Université Claude Bernard)
France
Address

69622 Villeurbanne