By comparing the signals from both plates, you can determine the fraction of the free antibody that is retained on the first plate (by comparing the slopes of absorbance vs. Special care is taken that all the incubation and detection steps are the same for both plates. After incubation, antibody solutions are transferred to the second plate. For (b), two plates are coated with Ag and a series of Ab solutions with different concentrations is added to the wells. An initial experiment has to be performed beforehand to ascertain that: (a) the antibody concentration used is in the linear range of the ELISA response (so that the absorbance is proportional to the Ab concentration) (b) only a small fraction of the total free antibody in solution is retained on the plate (so that the measurement does not significantly affect the Ab-Ag equillibrium in solution). Basically, indirect ELISA is used to determine concentration of the free antibody in a series of solutions, pre-incubated with varying concentrations of the antigen.
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