Comparison of four different carboxylterminal tracers in a radioimmunoassay specific to the 68-84 region of human parathyroid hormone.
Two synthetic carboxylterminal fragments, [tyr52]hPTH(52-84) and [tyr63]hPTH(63-84), and purified bPTH(1-84) were iodinated with 125Iodine to be compared as tracers in a late carboxylterminal radioimmunoassay. Tracer 125I-bPTH(41-84) was generated in vitro by incubating 125I-bPTH(1-84) with plasma membranes of rat kidney cortex. Region specificity was achieved by saturating the unwanted middle component of our multivalent antiserum with a molar excess of hPTH(44-68). A charcoal-dextran separation was worked out for each tracer. The titer of the antiserum giving approximately equal to 30% specific binding of each tracer was used in all experiments. Displacement of each tracer with increasing molar concentration of hPTH(1-84), hPTH(53-84), hPTH(41-84) and of hPTH (64-84) was studied. hPTH(41-84) was also generated by incubating hPTH(1-84) with rat cortex kidney membranes and was calibrated against a commercial preparation of bPTH(37-84). A progressive increase in the titer of the antiserum was seen as the molecular weight of the tracers decreased from a titer of 1/20,000 with 125I-bPTH(1-84) to a titer of 1/50,000 with the two synthetic tracers. Similarly the so-called damage seen during the charcoal-dextran separation in absence of antibody was reduced from 16.0 +/- 6.2% (mean +/- SD) with 125I-bPTH(1-84) to 1.3 +/- .2 with the two synthetic tracers. 50% displacement of the 125I-bPTH(1-84) tracer was achieved at 13.2 +/- .8 fmol/tube for hPTH(1-84) and at 6.3 +/- 1.0 fmol/tube for hPTH(41-84), reflecting the greater reactivity of fragments in that system. With the two synthetic tracers, a concentration of 5.0 +/- .4 fmol/tube of hPTH(1-84) or of 3.5 +/- 1.2 fmol/tube of hPTH(41-84) was necessary to achieve the same goal. With 125I-bPTH(41-84) results were between the two extremes. These results indicated that an increase in antiserum titer, a decrease in assay damage, an improvement in assay sensitivity and in comparative molar reactivity of the various circulating forms of hPTH can be achieved by using synthetic carboxylterminal fragments as tracers in region specific radioimmunoassays of hPTH.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D006801:Humans; D008970:Molecular Weight; D010281:Parathyroid Hormone / Q000032:analysis* / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats
D000906:Antibodies; D010281:Parathyroid Hormone; D010446:Peptide Fragments; C044885:parathyroid hormone (68-84)