A specific and sensitive ELISA for laminin 5

Abstract

A sandwich ELISA for laminin 5 was developed by using two monoclonal antibodies specific for human laminin 5, basement membrane (BM)165 and 6F12, which were raised against the laminin alpha3 chain and the laminin beta3 chain, respectively. Laminin 5 was purified from squamous carcinoma cell (SCC) 25-conditioned medium, using 6F12-conjugated Sepharose. This preparation was used as the standard for the ELISA. This sandwich ELISA was sensitive enough to detect reproducibly as little as 125 pg of laminin 5. This assay could detect laminin 5 produced by human keratinocytes but not other laminins produced by human fibroblasts. A portion of the laminin 5 that was secreted by the keratinocytes was deposited around the cells, and the rest was released into the medium. To measure total synthesized laminin 5, the intracellular and deposited laminin 5 was solubilized by treatment with a mixture of several kinds of detergents that did not interfere with the sandwich ELISA, and the laminin in both the culture medium and the solubilized cell layer was quantified. The amount of laminin 5 synthesis by keratinocytes depended on the cell number, the duration of culture, and the extracellular matrix proteins on which keratinocytes were plated.