REGION-SPECIFIC IMMUNOASSAYS FOR PARATHYROID HORMONE

Abstract

SUMMARY Immunoassays specific for limited regions of bovine parathyroid hormone were developed in four ways. With the heterogeneous antisera produced by immunizing with intact bovine parathyroid hormone (BPTH 1–84), the specificity of radioimmunoassays could be enhanced by presaturating either with an amino-terminal (BPTH 1–34) or carboxy-terminal (BPTH 53–84) fragment. Then, the antibodies which had not been neutralized reacted exclusively with the opposite end of the molecule, even using [125I]BPTH 1–84 as tracer. With some antisera, the appropriate fragment and intact hormone reacted identically. However, with other antisera, the fragment reacted less well than the intact hormone, possibly because these antisera contain antibodies reacting with the middle of the molecule. Using the labelled fragment ([125I]BPTH 1–34) as tracer, with heterogeneous antisera, radioimmunoassays specific for the amino-terminal region were obtained. With one antiserum, BPTH 1–34 reacted identically with the intact hormone, but with another antiserum, the fragment was more reactive than the intact molecule. A region-specific radioimmunoassay was also developed using antibodies produced by immunization with a fragment of the hormone. An antiserum raised against BPTH 1–34 had high affinity for the amino-terminal fragment, but reacted less well with the intact hormone. Immunoradiometric assays, specific for the amino- or carboxy-terminal regions, were developed by using immunoadsorbents consisting of a fragment (either BPTH 1–34 or BPTH 53–84) coupled to cellulose. These were used to fractionate 125I-labelled antibodies. With some of these selected antibodies, the appropriate fragment was of lower reactivity than the intact hormone. This may have been due to the presence of an incomplete antigenic site on the fragment, or to conformational differences between the fragment and the corresponding region of the intact hormone. With other selected antibodies the fragment and the intact molecule reacted identically. Careful selection of antisera and of technique is necessary to obtain an assay in which a fragment and the intact hormone behave identically.