Binding of prolactin and monoclonal antibody to prolactin receptors immobilized on a nitrocellulose membrane filter

Abstract

Mammary prolaction (PRL) receptors in 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps) extract were immobilized on a nitrocellulose (NC) filter, and a binding assay using the filter-bound receptors was carried out in the absence of detergent. PRL binding to the receptors was dependent on the quantity of the receptors and the PRL added to the filters. The filter-bound receptors retained the specificity of binding to peptide hormones. Scatchard analysis showed that the number of PRL receptors and the dissociation constant for PRL binding are essentially unchanged after immobilization on a NC filter, indicating that the PRL binding site does not participate in the binding to the NC filter and is equally available for PRL binding. The monoclonal antibody (MAb) against the PRL receptor was able to bind specifically to the Chaps-solubilized and filter-bound PRL receptors, as shown by curvilinear Scatchard plots. Immobilization on NC filters permits direct detection and characterization of the soluble PRL receptor using labeled PRL or MAb.