We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

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.

We have developed quantitative immunoassays for the intact, trimeric amino-terminal propeptide of human type I procollagen (PINP) and its Col1 domain. Intact PINP was isolated from the pleural fluids of cancer patients by a combination of ion-exchange, gel-filtration, and reversed-phase chromatographies. The amino-terminal Col1 domain of PINP was isolated after bacterial collagenase treatment of the heat-denatured trimeric propeptide. For the intact PINP assay we used a polyclonal antibody with only 1.2% cross-reaction with the monomeric Col1 domain. In human serum, this assay detects only one peak of PINP antigenicity that has the size of known intact PINP. Under similar conditions, an assay for the Coll domain of PINP recognized two circulating antigens. The biological relevance was further verified in wound fluid. Interassay and intraassay CVs were 3.1-9.3% for values within the reference intervals (mean +/- 2SD) for intact PINP in serum, which were 19-84 microg/L for women and 20-76 microg/L for men.

This is an equilibrium-type radioimmunoassay for the amino-terminal propeptide of type III procollagen (PIIINP), which overcomes the problem of nonparallelism between the standard and human serum samples encountered with earlier assays. Proper selection of antiserum and reaction conditions diminishes interference from degradation products of the propeptide in serum. Because a rapid solid-phase-bound second-antibody step is included, the assay takes only 3 h. The intra-assay and the interassay CVs are both about 5%. In infants and children the concentration of PIIINP in serum closely parallels the growth-velocity curve. For 88 presumably healthy adults, the PIIINP concentration was 1.7-4.2 micrograms/L, about a third that measured with the previously available commercial assay. This is because of lack of inhibition by small Col 1 domain-related degradation products.

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