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).