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J. Biochem, 1988, Vol. 103, No. 5 829-835
© 1988 Japanese Biochemical Society

research-article

Characterization of a Monoclonal Antibody against Human Placenta Type IV Collagen by Immunoelectroblotting, Antibody-Coupled Affinity Chromatography, and Immunohistochemical Localization

Jun Kino*,1, Eijiro Adachi**, Tsuyoshi Yoshida*, Keisuke Nakajima* and Toshihiko Hayashi***

* Shiseido Basic Research Laboratories Kouhoku-ku, Yokohama, Kanagawa 223
** Department of Anatomy, Osaka University Medical School Kita-ku, Osaka, Osaka 530
*** Department of Chemistry, College of Arts and Sciences, The University of Tokyo Meguro-ku, Tokyo 153

1To whom correspondence should be addressed.

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.


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