Development of an assay for the quantification of type I collagen synthesis in the guinea pig.
There is a need for a reliable assay for the quantification of collagen type I synthesis in the guinea pig, an important model for many connective tissue diseases. Procollagen type I C-terminal propeptide (PICP) is the established marker of type I collagen synthesis but, to date, no assay has been developed to measure PICP in guinea pig tissue extracts. A monoclonal antibody, known to cross-react with intact guinea pig procollagen type I (anti-PICP), was tested for its ability to bind soluble guinea pig PICP in crude skin extracts using a biosensor. Anti-PICP was immobilised to the surface of a sensor chip and antibody-antigen binding was detected using the phenomenon of surface plasmon resonance (SPR). The binding component in the SPR-immunoassay was identified as PICP by purification and N-terminal sequencing. Guinea pig PICP was purified from skin by gel filtration, ion exchange chromatography and lectin affinity chromatography. Purified PICP was then biotinylated and used with anti-PICP to develop a competition ELISA that was able to selectively and sensitively measure PICP in extracts of guinea pig connective tissue.
Quasnichka
Helen L
HL
Matrix Biology Research Group, University of Bristol, Langford, Bristol BS40 7DY, UK. helen.quasnichka@bristol.ac.uk
Tarlton
John F
JF
Anderson-Mackenzie
Janet M
JM
Billingham
Michael E J
ME
Bailey
Allen J
AJ
Pickford
Andrew R
AR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D024042:Collagen Type I / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011347:Procollagen / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D024042:Collagen Type I; D010446:Peptide Fragments; D011347:Procollagen; C042813:procollagen type I carboxy terminal peptide