Objective: To establish a more sensitive ELISA method for the determination of kanamycin residues and validate the methodologyfor the quality control of recombinant biological products. Methods: Using direct competitive ELISA method,the residual kanamycin in the test sample can compete with enzyme-linked kanamycin antigen to compete with the pre-coated antibody specific for kanamycin on the microplate. The sample was colored with 3, 3, 5, 5-tetramethylbenzidine(TMB) substrate. The absorbance value of the sample was negatively correlated with the content of residual kanamycin. The sample was detected by enzyme-labelled instrument and analyzed by Ridasoft Win analysis software. Results: The ELISA method has higher sensitivity and good spline linearity in the concentration range of 0.2-3.0 ng·mL-1, the relative standard deviation for parallel double wells was less than 10%. The assay had good precision,and the recoveries of the standard addition were between 80% and 120%. This method was used for determination of kanamycin residues in a batch of TMP-Fc products for injection. The results showed that the residues of kanamycin in each bottle of TMP-Fc (250 μg) were less than 0.2 ng. Conclusion: The method has the characteristics of high sensitivity,convenient operation and rapid detection, and can be used for the quality control of kanamycin residues in recombinant biological products.
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