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. 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.
Mathew
J A
JA
Animal Health Biotechnology Laboratory, Institute of Molecular Agrobiology, National University of Singapore.
Guo
Y X
YX
Goh
K P
KP
Chan
J
J
Verburg-van Kemenade
B M L
BM
Kwang
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D015153:Blotting, Western / Q000662:veterinary; D002347:Carps / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D015723:Gene Library; D006168:Guinea Pigs; D006825:Hybridomas / Q000737:chemistry / Q000276:immunology; D007111:Immunity, Cellular; D007375:Interleukin-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007962:Leukocytes / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology
D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D011994:Recombinant Proteins