Abstract
High sensitivity two-site immunoradiometric and immunochemiluminometric assays were developed for the direct measurement of rat GHRH in perifusate of hypothalamic fragments and in hypophysial portal plasma. A solid phase immunoradiometric assay was developed initially using avidin-coated polystyrene beads and radioimmunoassay quality polyclonal antibodies to N-terminal and C-terminal fragments of GHRH. Immunoaffinity purification of the high affinity antibodies required a strong eluant, 1 M acetic acid with 10% dioxane. For high sensitivity, the affinity-purified C-terminal signal antibody was iodinated by the lactoperoxidase-glucose oxidase method and absorbed with avidin-BSA-IgG conjugated matrix. Conversion to an acridinium ester based immunochemiluminometric assay required substantial modifications to optimize performance. For both assays, the innovative modification of delayed addition of the bead enhanced assay performance by two-fold. By precision profile analysis the sensitivity of the immunochemiluminometric assay (0.035 pg/tube) was two-fold better than the sensitivity of the immunoradiometric assay (0.066 pg/tube). These assays, 4–7-fold more sensitive than a highly optimized radioimmunoassay (0.26 pg/tube), were validated for direct unextracted measurement of GHRH in perifusate and in hypophysial portal plasma. They have significant advantages for biological measurements, reducing the number of hypothalami per perifusion chamber and enabling measurement of low concentrations of plasma GHRH not previously possible. The avidin-coated bead systen can be flexibly used to develop high sensitivity two-site peptide assays using polyclonal antibodies. Further, this system has been extended to acridinium ester based immunochemiluminometric assays with improvement of assay performance.
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