Preparation of polyclonal antibody against E.coli endotoxin and application of icELISA

ObjectiveTo prepare rabbit polyclonal antibody against E.coli endotoxin and to establish indirect competitive enzyme-linked immunosorbent assay(icELISA)for the detection of endotoxin.MethodsPurified E.Coli endotoxin was used as the immunogen to inject rabbit with gradually increased doses.Then the antibody was used for ELISA detection of endotoxin and reacted with the cell-bound E.coli endotoxin.ResultsThe endotoxin antiserum titer gradually increased with successive increased number of immunization times.The serum titer was 1:6 400 after the last immunization.At the fully optionized antigen concentration of 100μg/mL and rabbit polyclonal antibody dilution of 1:500,the icELISA results showed that concentration of endotoxin was in inverse proportion to the value of A,with a linear regression equation of y=-0.1ln(x)+0.765,correlation coefficient R² of 0.982,and the detection limit of 50 ng/mL.The titer of reaction with cell-bound E.coli O157:H7 endotoxin was 1:16 000.ConclusionThe rabbit polyclonal antibody against endotoxin was prepared successfully and could react with cell-bound E.coli endotoxin.