Purification of natural antikeratin autoantibodies from normal human serumand their effect on human keratinocytes cultured in vitro

Background Antikeratin (AK) autoantibodies, circulating antibodies against epidermal keratins, have been detected in all normal human sera. However, d irect evidence on the biological significance of AK autoantibodies is still lacking. Objectives To purify AK autoantibodies from human serum and to make a preli minary study of their biological effects on human keratinocytes. Methods We first extracted keratin polypeptides from human stratum corneum and analysed their purity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, a keratin affinity column was prepared wi th the extracted keratins, and AK autoantibodies were purified from pooled normal human serum. Antibodies obtained were identified with SDS-PAGE, enzy me-linked immunosorbent assay, immunoperoxidase staining, immunoelectron mi croscopy and Western blotting. The biological effect of AK autoantibodies o n cultured human keratinocytes was studied using a DNA synthesis assay, 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetri c determination and cell cycle analysis. Results On average, 1.83 +/-0.24 mg of antibodies could be purified from 10 mL of pooled human serum. High-titre IgG (about 1:70) and low-titre IgM (a bout 1:30) AX autoantibodies were obtained. The DNA synthesis assay and MTT colorimetric determination demonstrated that AK autoantibodies have a sign ificant dose-dependent inhibitory effect on cultured keratinocytes. Correla tion coefficients in the two experiments were -0.583 and -0.797, respective ly. Cell cycle analysis indicated that a small dose of AK autoantibodies le ads to inhibition of proliferation of cultured keratinocytes, whereas a lar ge dose of AK autoantibodies causes a visible hypodiploid peak, suggesting apoptosis of keratinocytes. Conclusions The present research lays a solid foundation for further invest igation into the biological significance of natural AX autoantibodies.