An in vitro enzyme immunoassay kit for the quantitative determination of human procollagen type I C-peptide (PIP) in plasma, serum, cultured cell extracts, cell culture supernatants, and other biological fluids

Description

Procollagen Type I C-Peptide (PIP)
Collagens (types I, II, III, IV and V) are synthesized as precursor molecules called procollagens. These contain additional peptide sequences, usually called “propeptides”, at both the amino-terminal and the carboxy-terminal ends. The function of these propeptides is to facilitate the winding of procollagen molecules into a triple-helical conformation within the endoplasmic reticulum. The propeptides are cleaved off from the collagen triple helix molecule during its secretion, after which, the triple helix collagens polymerize into extracellular collagen fibrils. Thus, the amount of the free propeptides reflects stoichiometrically the amount of collagen molecules synthesized (a relationship analogous to that between the carboxy-terminal peptide of proinsulin and the endogenously produced insulin). Quantitative detection of collagen synthesis was first reported by Taubman et al.¹⁾ who performed competitive radioimmunoassays for procollagen type I carboxy-terminal peptide (PIP) using polyclonal antibodies. Similar methods using PIP as reference have been used in studying the correlation of collagen levels with certain health disorders, ie. bone diseases²⁾, alcoholic liver diseases³⁾, liver cirrhosis⁴⁾ and scirrhous (Borrmann type IV) adenocarcinoma of the stomach^{5, 6)}.

Storage

4°C

Kit components

1.	Antibody Coated Microtiterplate	1 plate (8 well × 12 strips)
2.	Antibody-POD Conjugate	1 vial (for 11 ml × 1)
3.	Standard	1 vial (for 1 ml × 1)
4.	Sample Diluent	2 vials (11 ml × 2)
5.	Substrate Solution	1 vial (12 ml × 1)

Performance

Range of assay:	10 - 640 ng/ml
Sensitivity:	10 ng/ml
Assay duration:	3 hours and a half for assay protocol
Specificity:	This kit specifically measures human PIP with no detectable cross reaction with fibronectin, vitronectin, laminin, collagen type I, or collagen type III. This kit can be also used to measure bovine, dog, monkey and horse PIP, but not mouse PIP. Applications for quantitative detection of PIP from other sources have not been tested.
Test specimen type:	Serum or plasma of human, bovine, dog, monkey and horse, culture supernatants (serum-free), cell extracts; If each test sample is run in duplicate, about 50 µl (20 µl per assay well plus approx. 10 µl for waste per transfer) is required.

Monoclonal antibodies

This kit uses clone PC8-7(Cat.# M012) as solid antibody and clone PC5-5 (Cat.# M011) as labeled antibody. These monoclonal antibody clones were raised against purified carboxy-terminal propeptides of human procollagen type I.

Note

The measurement with this kit may be disturbed, when it is performed with samples which include animal serum such as Fetal Bovine Serum and House Serum. It is recommended to perform the measurement under the serumfree condition.

Typical standard curve (Do not use to calculate unknowns)