Standardisation of a two-site PTH immunoradiometric assay using various solid phase formats

Estimation of parathyroid hormone (PTH) levels is important in the management of metabolic bone disorders. Here we describe a simple, sensitive and specific second generation immunoradiometric assay (IRMA) to detect intact PTH levels using different solid phase matrices. Different methods for immobilization of antibodies have also been evaluated.

Experiments were carried out with physical adsorption of antibodies, covalent coupling using 2 per cent glutaraldehyde and N,N`carbonyldiimidazole. In all cases, antibodies raised against C-terminal were used as solid phase agent. Detector antibodies were N terminal antibodies that were radio-iodinated with [125] I followed by gel purification. Several of the antibodies coupled to various solid phase matrices were incubated with PTH standards and the detector antibody as well as the commercially available tracer from DiaSorin kit to identify a suitable match pair.

The best pair was polyclonal C-terminal PTH antibody along with the kit tracer from DiaSorin with regards to antibody coated to magnetic cellulose particles. Among the various antibodies and the solid phases evaluated, the best assay was obtained with the matched pair of antibodies (70×G67 and 70×G68) from Fitzgerald immobilized on polystyrene tubes. The polyclonal antibody against C-terminal PTH was chosen as the capture antibody and [125] I labelled polyclonal antibody against N-terminal PTH as the tracer. The sample values obtained in the antibody coated tubes were comparable to those obtained using a commercial kit.

The results indicated the feasibility of adopting this system for further development into a PTH IRMA for regular production as there is no indigenous kit available for intact PTH.