Characterization of monoclonal antibodies recognizing human merosin and their use in affinity purification of native merosin.
Two monoclonal antibodies (MAbs) to human placenta laminin (pl-LAM), 1D8 (IgG1) and 6G5 (IgG2b) were generated and shown by ELISA and immunoblot analysis to recognize only native pl-LAM, but not denatured, reduced pl-LAM or mouse EHS laminin. Intact pl-LAM was easily isolated and purified in large scale from human placenta by 1D8-conjugated affinity chromatography. Electrophoretic analysis of the purified pl-LAM revealed the presence of a major 750-kDa component composed of 320-, 220-, and 200-kDa polypeptides and a minor 800-kDa component composed of 320-, 240-, and 220-kDa polypeptides. Neither molecule had a 400-kDa component corresponding to the A chain. It has already been shown that the 320-kDa polypeptide is identical to the M chain of human merosin (Hori et al. J. Biochem. 1994;116:1212-1219). Electron microscopy revealed that isolated merosin was composed of three short arms and one long arm. By immunohistochemistry, MAbs showed positive staining in human adult kidney and liver. These results indicate that these MAbs recognize only native merosin and can be used to study merosin structure and function by rapid purification of native merosin and by immunohistochemical analysis.
Mizuta
T
T
Department of Internal Medicine, Saga Medical School, Japan.
Shimada
H
H
Arai
K
K
Hori
H
H
Hattori
S
S
Yamamoto
K
K
Sakai
T
T
Nagai
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D018604:Epitope Mapping; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D007797:Laminin