Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen

TLDR

Type I collagen is the most abundant collagen in soft tissues and the sole collagen type present in mineralized bone. The study aimed to develop a rapid equilibrium radioimmunoassay for the carboxy‑terminal propeptide of human type I procollagen (PICP) as a marker of type I collagen synthesis. To create the assay, the authors isolated type I procollagen from primary human skin fibroblast cultures, digested it with bacterial collagenase, purified PICP by lectin‑affinity chromatography, gel filtration, and ion‑exchange HPLC, verified purity by SDS‑PAGE and N‑terminal sequencing, and then generated a polyclonal rabbit‑antibody‑based radioimmunoassay. The assay detected a single, antigenically identical form of PICP in human serum with intra‑ and interassay CVs of 3 % and 5 %, established reference intervals of 38–202 µg/L for men and 50–170 µg/L for women (men’s levels inversely related to age), and confirmed serum antigen stability during storage and repeated thawing.

Abstract

Type I collagen is the most abundant collagen type in soft tissues and the only type found in mineralized bone. We established a rapid equilibrium radioimmunoassay for the carboxyterminal propeptide of human type I procollagen (PICP), to be used as an indicator of the synthesis of type I collagen. We isolated type I procollagen from the medium of primary cultures of human skin fibroblasts, digested the protein with highly purified bacterial collagenase, and purified PICP by lectin-affinity chromatography, gel filtration, and ion-exchange separation on HPLC. The purity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N-terminal amino acid sequencing of its component chains. The final radioimmunoassay was established with polyclonal rabbit antibodies. Material antigenically related to PICP is readily detected in human serum. There is only one form of the serum antigen, its molecular size and affinity to the antibodies being similar to those of the isolated propeptide. Intra- and interassay CVs are 3% and 5%, respectively. Preliminary reference intervals for healthy adults (18 to 61 years of age) are 38-202 micrograms/L for men and 50-170 micrograms/L for women: in men the concentration is inversely related to age. The serum antigen is stable during storage and after repeated thawing.