Characterization of a monoclonal antibody against human placenta type IV collagen by immunoelectroblotting, antibody-coupled affinity chromatography, and immunohistochemical localization.
We have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260,000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D003094:Collagen / Q000032:analysis / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008854:Microscopy, Electron / Q000379:methods; D010920:Placenta / Q000032:analysis
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D003094:Collagen