Washing: Wash 4-5 times with PBS + 1% Tween 20.
Secondary Antibody: Prepare appropriate dilution of secondary antibody and then disperse in 50µl per well.
Incubate at 370C for 45 mins.
Washing: Wash 4-5 times with PBS + 1% Tween 20.
Development: Dispense 1mg/ml OPD + H2O2 in citrate buffer (50mM citrate pH 5.6). Stop the reaction by 7.5% H2SO4 and take absorbance at 460nm. A typical result is given in the Figure 30.3.
Figure 30.3: The typical results expected from the indirect ELISA
Lab Experiment 30.2: Quantitative measurement of cytokines using sandwich ELISA
Background Information: The typical setup for detection of antigen from the sample is given in the Figure 30.4. In this method, a capture antibody is used to collect the antigen from the sample. Afterwards, second antibody is used to detect antigen bound to the capture antibody. Second antibody is directed against the antigen using a unique distinct epitope. The antibody is linked to the biotin and that can be recognized by the avidin/streptavidin-HRP complex. In the last step, peroxidase substrate is used to get a readout.
Figure 30.4: sandwich ELISA and use of different components.