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  • Development of an assay for the quantification of type I collagen synthesis in the guinea pig.

Development of an assay for the quantification of type I collagen synthesis in the guinea pig.

Journal of immunological methods (2005-03-22)
Helen L Quasnichka, John F Tarlton, Janet M Anderson-Mackenzie, Michael E J Billingham, Allen J Bailey, Andrew R Pickford
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IgG1 Antibody, Mouse, 1.95 mg/mL, Chemicon®