Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2.
Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had kappa chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.
Adachi
Satoshi
S
Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
Hashimoto
Tomomi
T
Takeyoshi
Masahiro
M
Kato
Hirotomo
H
Iwata
Hiroyuki
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006168:Guinea Pigs; D006825:Hybridomas; D007376:Interleukin-2 / Q000032:analysis* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D007376:Interleukin-2