Monika Casaretto

Homologous radioimmunoassay for human mid-regional parathyroid hormone

A radioimmunoassay, selective for the clinically important mid region of human parathyroid hormone (hPTH), is reported. The synthetic44–68 amino acid sequence (h44–68PTH) was used as both the standard and tracer material. This eliminated many of the undesirable characteristics associated with PTH assays that employ hormone of biological origin. These components allowed the detection of mid regional fragments present in both intact and fragmented hPTH, with a working range between 50 and 2500 pg/ml h44–68PTH. There was no interference from serum proteins and no significant cross reactivity to a range of N-terminal, C-terminal and other mid regional hPTH peptides. The assay proved to be rapid (total time 24 h) and was extremely reproducible, with an intraassay and interassay variation of 2.8% and 5.6% respectively (expressed as percentage SE of mean). The plasma concentration of normal subjects was established as 129 ± 6 pg/ml (h44–68PTH) with a range of 50–300 pg/ml (n = 42). This assay, using fully synthetic hPTH peptides, was able to distinguish between euparathyroid and hyperparathyroid subjects, which suggests that the assay is of diagnostic value.

CHARACTERISATION OF THE BINDING SITES OF ANTI-PARATHYROID HORMONE ANTISERA USING SYNTHETIC PARATHYROID HORMONE PEPTIDES

Four antisera raised against partly purified PTH preparations all showed a wide range of specificities when reacting with radioiodinated PTH peptides representing several different portions of the intact hormone sequence. In contrast, antisera raised against individual peptides were only able to cross-react with other peptides that contained all or part of their amino acid sequence in common. Cross-reacting peptides were seen to contain one or more amino acid residues having high interspecies variability in common. We have explained the antigenicity and cross-reactivity of the peptides on the basis of these common highly variable amino acid sequences. We have concluded that the selection of hormonal material in radioimmunoassays for PTH should be made on the basis of the highly variable amino acid residue content. This will allow a narrowing of the assay specificities and permit detection of a desired region of the PTH hormone.

Preliminary Results on the Use of an Antiserum to Human Parathyrin in a Homologous Radioimmunoassay

A new antiserum (Ab Giselle) raised in sheep against extracted human parathyrin was evaluated and compared with an established antiserum (Ab S-478 VI) under several test conditions. The assay system contained an extracted 1--84 human parathyrin standard and a 1--84 bovine parathyrin tracer. The total assay time was 24 h and the main assay characteristics as follows: B0/T 0.28 +/- 0.02 and 50% intercept 553 +/- 47 U . 1(-1). The corresponding data for Ab S-478 VI were: B0/T 0.23 +/- 0.02 and 50% intercept 890 +/- 142 U . 1(-1). The normal range in 152 normocalcaemic volunteers (age range 16--67 years) was 10.6--423 U . 1(-1) (where 1 vial MRC reference preparation 75/549 for human parathyrin = 25 U), compared with 0--300 U . 1(-1) for Ab S-478 VI. With the new antiserum, differentiation between hypoparathyroid patients and those with normal function was often possible, and differentiation between normal and elevated levels, as in hyperparathyrinaemia, was very good. Correlation between Ab Giselle and Ab S-478 VI in 80 normal volunteers was positive (r = 0.450, p = 0.01) although the regression line showed that the antisera had different specificities (data for the regression line y = a + bx, a = 0.13, b = 0.55). Under the assay conditions, the association constant for Ab Giselle was 0.41 +/- 10(14) l . mol-1 in contrast to Ab S-478 VI which had a Ka for 0.53 x 10(10) l . mol-1 under identical conditions. Assays using Ab Giselle could be performed at room temperature, whereas those using Ab S-478 VI performed best at 0 degrees C. Preliminary results suggest that Ab Giselle is better for the routine assay of human parathyrin in serum than Ab S-478 VI, especially in the case of hypoparathyroid patients.

[Synthesis of two median segments of human parathyrin (author's transl)].

Human parathyrin-(32-43)-dodecapeptide (H-His-Asn-Phe-Val-Ala-Leu-Gly-Ala-Pro-Leu-Ala-Pro-OH) and tyrosyl-[human parathyrin-(43-55)-tridecapeptide] (H-Tyr-Pro-Arg-Asp-Ala-Gly-Ser-Gln-Arg-Pro-Arg-Lys-Lys-Glu-OH) were prepared, using the strategy of segment condensation in combination with acid-labile protecting groups. The peptides will be utilized for radioimmunoassay and receptor-binding studies.

Characterization of Two Anti-Human Parathyrin Antisera

Two anti-human parathyrin antisera were raised in sheep. These were characterized by radioimmunoassay using two commercially available bovine parathyrin preparations and one synthetic human parathyrin fragment (sequence 42-55 (42-Tyr)) for radioiodination. In addition, four synthetic human parathyrin fragments (sequences 1-34, 32-43, 434-68, 53-84), one bovine parathyrin peptide (sequence 28-48) and a human parathyrin standard from a tissue culture containing the intact hormone were utilized in a competitive inhibition assay against the two radiolabelled bovine parathyrin preparations. On column chromatography, both tracers revealed a difference in molecular weight, which is believed to be related to the extraction technique. The sequence fragment 44-68 of human parathyrin had the highest affinity for the two antisera when using the smaller molecular weight tracer and there were no qualitative changes observed in the presence of plasma. Using the higher molecular weight tracer, the addition of plasma to one of the antisera resulted in a higher affinity for the sequence fragment 1-34 of human parathyrin compared to that of intact parathyrin. Antiserum from a second sheep remained specific only for the mid-region (sequence 44-68) of the parathyrin molecule independent of the tracer used. Due to the antiserums constant characteristic, it revealed a high reliability for the discrimination between plasma parathyrin levels in normal probands and in patients with hyperparathyroidism. Our data demonstrate that the specificity of the radioimmunoassay for human parathyrin is not exclusively dependent on the antibody source, but also on the tracer preparation and the protein content of the incubation medium.