A sloppy labortary worker prepares to perform an indirect EL

A sloppy labortary worker prepares to perform an indirect ELISA assay to test for the presence of an antibody. however, he inadvertently adds the secondary antibody before the sample, washes, and then adds the sample and washes again. when the tag stimulus is added, will his assay give a result that always accurate, always positive, always negative? why?

Solution

In indirect ELISA, The antigen is loaded in the sample wells, primary antibody is added and then the secondary antibody is added that is tagged to an enzyme or colored substrate. The amount of color produced is proportional tothe concentration of antigen. Washing in between addition of primary and secondary antibodies is must to washout unbound sample and excess antigen. The secondary antibody added prior to the addition of sample and washing resulys in always negative reaction. This is because, the primary antibody must be bound to antigen and washed to remove unbound excess antigen. The secondary antibody is then added and it will be in suitable concentration to the antigen - primar antibody complex. The tag stimulus then gives the actual concentration of antigen. When secondary antibody added prior to sample addition, it may not be bound to the sample and concentration of antigen cannot be determined.