IGFBP-5 ELISA

The IGFBP-5 ELISA is a quantitative two-step sandwich type immunoassay. In the first step, Calibrators, Controls and unknown diluted samples are added to IGFBP-5 antibody coated microtiter wells and incubated. After the first incubation and washing step, the wells are incubated with horseradish peroxidase labelled antibody conjugate. After a second incubation and washing step, the wells are incubated with substrate solution (TMB). After TMB incubation, an acidic stopping solution is added. In principle, the antibody-HRP conjugate binds to the solid phase antibody-antigen complex. Finally, the antibody-antigen and conjugate complex bound to the well is detected by addition of enzyme-substrate reaction. The degree of enzymatic turnover of the substrate is determined by dual wavelength absorbance measurement at 450 nm as primary test filter and 630 nm as reference filter. The absorbance measured is directly proportional to the concentration of IGFBP-5 in the samples and calibrators.

References:
1. HHS Publication, 5th ed., 2007. Biosafety in Microbiological and Biomedical Laboratories. Available http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5
2. DHHS (NIOSH) Publication No. 78€“127, August 1976. Current Intelligence Bulletin 13 - Explosive Azide Hazard. Available http:// www.cdc.gov/niosh.
3. Kricka L. Interferences in immunoassays €“ still a threat. Clin Chem 2000; 46: 1037€“1038.