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 cryostat 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 675-679 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 and 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.