Cat Cross-linked Carboxy-terminal telopeptide of type I collagen (CTX-I) ELISA Kit

 

The carboxyterminal telopeptide of type I collagen (ICTP) is an indicator of degradation of type I collagen. ICTP antigen consists of a trivalent collagen cross-link joining three polypeptide chains of which two are α1 chains of one collagen molecule while the third is derived from either an α1 or an α2 chain of the helical region of another molecule (Fig. 1) (Risteli et al. 1993). The major determinant for antigeneity must contain two phenylalanine-rich regions, which is only possible if the cross-link is trivalent in nature (Sassi et al. 2000). In osteoclasts, catepsin K cleaves the trivalently cross-linked ICTP structure at two sites between the phenylalanine-rich region and the cross-link, thereby destroying any reactivity with ICTP antibodies (Sassi et al. 2000). Therefore degradation of type I collagen in bone is not measurable by the present ICTP-test. ICTP is cleared from circulation by the kidneys and has a molecular mass of about 12 000-20 000 (Risteli et al. 1993).
 

Reagents	Quantity	Reagents	Quantity
Assay plate (96 Wells)	1	Instruction manual	1
Standard (lyophilized)	2	Sample Diluent	1 x 20 mL
Biotin-Conjugate (concentrate 100 x)	1 x 120 μL	Biotin-Conjugate Diluent	1 x 12 mL
Streptavidin-HRP  (concentrate 100 x)	1 x 120 μL	Streptavidin-HRP Diluent	1 x 12 mL
Wash Buffer (concentrate 25 x)	1 x 20 mL	Substrate Solution	1 x 10 mL
Stop Solution	1 x 6 mL	Adhesive Films	4

 

This assay has high sensitivity and excellent specificity for detection of Human CTX-I. No significant cross-reactivity or interference between Human CTX-I and analogues was observed.
 

Matrices listed below were spiked with certain level of recombinant Human CTX-I and the recovery rates were calculated by comparing the measured value to the expected amount of Human CTX-I in samples.

Sample Type	Number	Recovery range (%)	Average(%)
Serum	10	90-101	96
EDTA plasma	10	89-97	93
Heparin plasma	10	91-99	95

 

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
 

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CTX-I and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample Type	1：2	1：4	1：8	1：16
Serum	78-89%	81-99%	92-103%	95-105%
EDTA plasma	91-101%	90-98%	93-101%	91-98%
Heparin plasma	92-103%	93-102%	92-99%	91-101%

 

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
 

 

Store at 2-8°C. Please refer to Instruction Manual.