Monoclonal antibodies and sandwich ELISA for quantitation of HM-1 killer toxin.
To establish a method for quantitative analysis of HM-1 killer toxin (HM-1), two purified mouse monoclonal antibodies, 1F1 and 4A2, and rabbit polyclonal antiserum against HM-1 were prepared. Both monoclonal antibodies were classified as IgG1(kappa) subtype, and did not neutralize the killing activity of HM-1. By SPOTs analysis, the epitope of 1F1 was found in the sequence of CDPNTG with a corresponding sequence of 11-16 from N-terminal amino acid residues of HM-1, but the epitope of 4A2 was not determined. Using 4A2 and polyclonal antiserum, the sandwich enzyme-linked immunosorbent assay (ELISA) was applied to establish the quantitative determination of HM-1. The concentration of HM-1 was determined successfully at the range of 2.5-100 ng/ml. But in the case of 1F1, the method was not established. Genes were constructed to apply the system to the measurement of the secreted concentrations of mutant HM-1, and it was evident that the production of mutant toxins varied among HM-1 mutant genes. The findings of this study are unique in determinimg the epitope of monoclonal antibody against HM-1, and in quantifying the HM-1 using the spot analysis and sandwich ELISA methods.
Komiyama
Tadazumi
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan. tkomiyam@nigata-pharm.ac.jp
Zhang
Qing-Zhu
QZ
Miyamoto
Masahiko
M
Selvakumar
Dakshnamurthy
D
Furuichi
Yasuhiro
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007106:Immune Sera / Q000032:analysis; D055646:Killer Factors, Yeast; D051379:Mice; D009183:Mycotoxins / Q000032:analysis* / Q000276:immunology*; D010843:Pichia / Q000276:immunology
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D055646:Killer Factors, Yeast; D009183:Mycotoxins