Question

PO PULLIC313 TUVUULICHCLUJ STUDY QUESTIONS Answer the following study questions in your laboratory notebook or on a separate worksheet. 1. Why is there a 37°C incubation step after the addition of the sub- strate? 2. What is the effect of not including the antigen or the primary anti- body in the ELISA reaction? Why is it important to wash all the wells between the additions of the various components ? 4. Can nucleic acids be detected by the ELISA format

Answer 1

1) in each reaction, after addition of substrate 37 degree incubation is required. Because optimum temperature of enzyme activity is 37 degrees. So it is necessary to incubate a reaction at 37 degree after addition of substrate.

2) in absence of antigen or in absence of primary antibody in ELISA. Antigen antibody interactions will not occur, so secondary antibody coupled with enzyme HRP will not able to bind. So after addition of substrate there will be mo colour production in ELISA test. Because if no primary antibody, no secondary antibody will bind . So in absent of secondary antibody, no HRP and no colour.

3) washing in between step is important in ELISA. After reaction, there is additional antigen or antibody present in well or that is in excess . So to make sure specificity, means colour production is due to only antigen antibody interactions, excess material which does not participate in reaction is washed off.

4) ELISA is used for detection of proteins. So it can not be used for nucleic acids.