LH ELISA

The US LH ELISA is a quantitative three-step sandwich type immunoassay. In the first step Calibrators, Controls and Unknown are added to LH antibody coated microtiter wells and incubated. After the first incubation and washing, the wells are incubated with biotinylated LH antibody solution. After the second incubation and washing, the wells are incubated with streptavidin horseradish peroxidase conjugate (SHRP) solution. After the third incubation and washing step, the wells are incubated with substrate solution (TMB) followed by an acidic stopping solution. In principle, the antibody-biotin conjugate binds to the solid phase antibody-antigen complex which in turn binds to the streptavidin-enzyme conjugate. The antibody-antigen-biotin conjugate-SHRP complex bound to the well is detected by 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 LH in the samples and calibrators.