RESUMEN
@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.