Characterisation of a monoclonal antibody to carp IL-1beta and the development of a sensitive capture ELISA

A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Mo...

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Bibliographic Details
Published in:Fish & shellfish immunology 2002-08, Vol.13 (2), p.85-95
Main Authors: Mathew, J A, Guo, Y X, Goh, K P, Chan, J, Verburg-van Kemenade, B M L, Kwang, J
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal - biosynthesis
Antibodies, Monoclonal - immunology
Blotting, Western - veterinary
Carps - immunology
Electrophoresis, Polyacrylamide Gel - veterinary
Enzyme-Linked Immunosorbent Assay - methods
Enzyme-Linked Immunosorbent Assay - veterinary
Female
Gene Library
Guinea Pigs
Hybridomas - chemistry
Hybridomas - immunology
Immunity, Cellular
Interleukin-1 - chemistry
Interleukin-1 - genetics
Interleukin-1 - immunology
Leukocytes - immunology
Lymphocyte Activation
Mice
Mice, Inbred BALB C
Recombinant Proteins - chemistry
Recombinant Proteins - immunology
Sensitivity and Specificity
Spleen - cytology
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Summary:A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1beta was developed with a detection limit of 10 ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1beta in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1beta is known to stimulate proliferation of T & B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.
ISSN:1050-4648