Hydroxyapatite - a reagent for the separation of free and antibody-bound steroid during the radioimmunoassay.
The use of hydroxyapatite to absorb antibody-bound steroid and thus separate free and antibody-bound steroid during radioimmunoassay has been examined using three steroid antisera (to testosterone, to 17-hydroxyprogesterone and to estradiol-17beta). For all three antisera studied the separation was shown to be independent of length of time in contact with hydroxyapatite (up to 1h); temperature variations from 4 degrees -37 degrees and pH over the range 4.9-8.0. The presence of protein affected the absorption of antibody-bound steroid but this effect could be overcome by the addition of increasing amounts of hydroxyapatite. Further increase in the amount of hydroxyapatite added had no effect on the separation of free and bound steroid. Sodium phosphate buffers of molarity greater than 0.01M eluted antibody-bount steroid from hydroxyapatite, but Tris-HC1 buffers up to molarities of 0.1 M had no effect. Hydroxyapatite when used as a dry powder had the same effects as suspensions. No effect on the cross-reactivities of the antisera used could be demonstrated when hydroxyapatite was used and plasma testosterone assays on 22 plasma samples using hydroxyapatite gave essentially the same results as assays on the plasma using a coated-tube assay. Hydroxyapatite can also be successfully pumped along small bore plastic tubing without settling and can thus be used in automated immunoassay systems.
Hydroxyapatite - a reagent for the separation of free and antibody-bound steroid during the radioimmunoassay. The use of hydroxyapatite to absorb antibody-bound steroid and thus separate free and antibody-bound steroid during radioimmunoassay has been examined using three steroid antisera (to testosterone, to 17-hydroxyprogesterone and to estradiol-17beta). For all three antisera studied the separation was shown to be independent of length of time in contact with hydroxyapatite (up to 1h); temperature variations from 4 degrees -37 degrees and pH over the range 4.9-8.0. The presence of protein affected the absorption of antibody-bound steroid but this effect could be overcome by the addition of increasing amounts of hydroxyapatite. Further increase in the amount of hydroxyapatite added had no effect on the separation of free and bound steroid. Sodium phosphate buffers of molarity greater than 0.01M eluted antibody-bount steroid from hydroxyapatite, but Tris-HC1 buffers up to molarities of 0.1 M had no effect. Hydroxyapatite when used as a dry powder had the same effects as suspensions. No effect on the cross-reactivities of the antisera used could be demonstrated when hydroxyapatite was used and plasma testosterone assays on 22 plasma samples using hydroxyapatite gave essentially the same results as assays on the plasma using a coated-tube assay. Hydroxyapatite can also be successfully pumped along small bore plastic tubing without settling and can thus be used in automated immunoassay systems.