Determination of serum luteinizing hormone (LH) level

Standards or samples are incubated in monoclonal anti-LHβ antibody coated wells to capture LH. After 2 hours’ incubation and washing, biotinylated anti-LHα antibody is added and incubated further for 1 hour to bind with captured LH. After washing, HRP (horseradish peroxidase)-conjugated streptavidin is added and incubated for 30 minutes. After washing, HRP-complex remaining in wells is reacted with a chromogen (TMB) for 20 minutes, and reaction is stopped by addition of acidic solution, and absorbance of yellow product is measured spectrophotometrically at 450 nm. The absorbance is proportional to LH concentration. The standard curve is prepared by plotting absorbance against standard LH concentrations (Pakarainen et al., 2007).