Radioimmunoassay of beta lipoprotein—protein of rat serum

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Abstract

A double antibody radioimmunoassay for rat serum beta lipoprotein-protein (beta Lp-protein) is described. The protein was purified by ultracentrifugation, selective heparin-manganous precipitation, and gel filtration on Sephadex G-200. Antiserum was prepared in rabbits by biweekly immunization and absorbed with nonbeta lipoprotein containing rat serum. Iodination with 125I and purification by gel filtration provided a radiolabeled protein which was > 98% displaced by purified beta lipoprotein in the immunoassay. The radioimmunoassay was sensitive to beta Lp-protein concentrations from 0.1 to 1.5 μg. Specificity of the immunoassay for beta Lp-protein was established by comparison of the displacement curves obtained with serum very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and density (d) > 1.21 fractions and with the beta and alpha migrating lipoproteins eluted from paper electrophoretograms. Suitability of the assay for measuring beta Lp-protein in serum was established by demonstrating 100% recovery of beta lipoprotein added to whole serum and by the absence of immunoreactive beta Lp-protein in serum of orotic acid-treated rats. Examination of sera from six other vertebrates species revealed partial cross-reactivity. Normal rat serum was found to contain 0.25±0.01 mg/ml of beta Lp-protein and hepatic production by an isolated perfused rat liver system was determined as 0.145 mg/hr.

Authors

R. Philip Eaton, David M. Kipnis