The RatLaps? (CTX-I) is based upon the competitive binding of a polyclonal antibody to soluble RatLaps antigens EKSQDGGR or to immobilized RatLaps antigens. Briefly, the polyclonal antibody is raised against a synthetic peptide having a sequence (EKSQDGGR) specific for a part of the C-terminal telopeptide α1 chain of rat type I collagen. For standardization of the RatLaps? (CTX-I) a synthetic peptide (EKSQDGGR), which is specific for the C-terminal telopeptide α1 chain of type I collagen in rats has been used. During the pre-incubation step, biotinylated EKSQDGGR is immobilized by binding to the streptavidin-coated microtitre wells. The wells are emptied and washed. Standards, control, or unknown samples (culture supernatant or rat/mouse serum or urine) are pipette into appropriate wells, followed by a solution of a primary antibody (polyclonal rabbit). Following the primary-incubation step the wells are emptied and washed. In the secondary-incubation step a solution of a Goat anti-Rabbit antibody conjugated with peroxidase (secondary antibody) is added and binds to the polyclonal rabbit antibody. After the third washing step a chromogenic substrate (TMB) is added and the color reaction is stopped with sulfuric acid. Finally, the absorbence at 450 nm is measured with 650 nm as reference if possible. The absorbence level is inversely related to concentration of RatLaps antigens in the sample.
Size
96 wells
Concentration
n/a
Applications
colorimetric ELISA or EIA
Other Names
fragments of type I collagen kit, assay, immunoassay