EPITOPE-DEFINED MONOCLONAL-ANTIBODIES AGAINST TYPE-IV COLLAGEN FOR DIAGNOSIS OF ALPORTS-SYNDROME

Background. Alport's syndrome can be diagnosed by staining the alpha 5 chain of type IV collagen in kidney biopsy specimens with a monoclona l antibody. Because antibodies already established against the alpha 5 chain require denaturation treatment of cryostal sections to expose t heir epitopes. To save time and effort for staining, a new epitope-def ined monoclonal antibody whose epitope is initially exposed on the sur face of the molecule was established. Methods. Two monoclonal antibodi es against the triple-helical domains of the type IV collagen alpha 2 and alpha 5 chains were established with synthetic peptides as immunog ens by the rat lymph node method. Their epitope were EAIQP at the posi tions of 675-479 of the alpha 2 chain, and IDVEF at the positions of 2 51-255 of the alpha 5 chain, respectively. They were purified with syn thetic peptide-coupled affinity columns, and then conjugated with Texa s red acid FITC, respectively. Results. The mixture of fluorochrome-co njugated antibodies was able to detect the distribution of the alpha 2 and alpha 5 chains in the normal and Alport kidney and skin by direct immunofluorescence staining with and without denaturation treatment o f the sections. Conclusions. The direct double immunofluorescence stai ning of kidney and skin cryostat sections with the fluorochrome-conjug ated antibodies is useful, reliable, and convenient for diagnosis of A lport's syndrome.