Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome

Background. Alport's syndrome can be diagnosed by staining the alpha 5 chain of type IV collagen in kidney biopsy specimens with a monoclonal antibody. Because antibodies already established against the alpha 5 chain require denaturation treatment of cryostal sections to expose their epitopes. To save time and effort for staining, a new epitope-defined monoclonal antibody whose epitope is initially exposed on the surface of the molecule was established. Methods. Two monoclonal antibodies against the triple-helical domains of the type IV collagen alpha 2 and alpha 5 chains were established with synthetic peptides as immunogens by the rat lymph node method. Their epitope were EAIQP at the positions of 675-479 of the alpha 2 chain, and IDVEF at the positions of 251-255 of the alpha 5 chain, respectively. They were purified with synthetic peptide-coupled affinity columns, and then conjugated with Texas red acid FITC, respectively. Results. The mixture of fluorochrome-conjugated 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 of the sections. Conclusions. The direct double immunofluorescence staining of kidney and skin cryostat sections with the fluorochrome-conjugated antibodies is useful, reliable, and convenient for diagnosis of Alport's syndrome.