Detection of anti-LKM-1 antibody by recombinant fusion peptide in enzyme-linked immunosorbent assay:a preliminary study / 中国免疫学杂志

ABSTRACT

Objective:To detect anti-LKM-1 antibody with enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion peptide which comprises 257-351 amino acid fragment of CYP2D6 as antigen.Methods:We obtained CYP2D6 cDNA fragment by means of PCR,using total liver cDNA library as the template.The PCR products were ligated into pEGH expressing vector to construct the recombinant expressing vector with high efficiency in Saccharomyces Cerevisiae Y258.The positive clones were identified by PCR reaction and then induced by galactose.Glutathione-Sepharose 4B was used for purification of recombinant CYP2D6 protein.After affinity purification,the antigenicity was identified with Western blot.Serum samples from 26 patients who were positive for anti-LKM-1 antibody,20 patients with other connective tissue disorder(CTD) and 30 normal controls were retrospectively tested with ELISA.Results:A fusion peptide was expressed and purified.The antigenicity was confirmed with Western blot using standard of anti-LKM-1 antibody-positive serum.Of the 26 serum samples which are positive for anti-LKM-1 antibody,5 of 6 samples positive for anti-HCV antibody also recognized the recombinant fusion peptide with ELISA,only one serum sample which was showed positive anti-HCV antibody displayed a negative result in ELISA assay.All other 20 patients with positiv anti-LKM-1 antibody were shown positive in ELISA assay using this recombinant peptide.All the serum samples from patients with other CTD were negative in ELISA assay.Conclusion:The recombinant antigen fragment contains major epitope regions in natural CYP2D6 antigen.Detection of anti-LKM-1 antibody with ELISA using the recombinant peptide can improve the sensitivity and has a potential role in determining its clinical association.