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Trouble Shootings

1.  How to avoid the routine problems in running ELISA:

   FOLLOW THE PROTOCOL EXACTLY:  In order to get satisfactory result, it is very  important to run the ELISA to following the exact protocol provided by , even if you have done other ELISA many times before.

   PRECAUTION IS KEY TO ELISA SUCCESS:  Carefully check all components needed for the test, and make sure everything is ready before starting to pipette. If it is possible, avoid interruptions during the pipette process.

   CAREFULLY PREPARE THE SAMPLE:  Select symptomatic and/or infective plant tissues for the test when it is possible. Grind sample thoroughly to release maximum antigen from the tissues for reliable test result. 

   AVOIDING CONTAMINATION:  Because of the sensitivity of ELISA, any small contamination may cause the test to fail. Change pipette tips for every sample tested, as well as all the reagents used in the test.  

    REDUCING THE OUTER-ROW / EDGE-EFFECT:  Make sure the plates are tightly covered using parafilm,  rubber sheets, and/or covers. Place plates in moist chamber during incubation.

   CAREFUL WASHING IS IMPORTANT:  There are commercial ELISA washers if needed. However, small number of plates can be washed with a squeeze bottle. Empty the wells by a quickly flipping motion without mixing the contents among the wells. Filling wells with washing buffer gently and quickly, and then quickly emptying them again.  Repeat washing 7-8 times.

   SOAKING PLATE TO REDUCE BACKGROUND:  If high background frequently appears on negative control wells, soaking may help to reduce the background.  After last washing, fill the wells with washing buffer and keep soaking for 2-3 min before emptying.

   DO NOT LEAVE THE PLATE EMPTY FOR TOO LONG:  Either keep the wells filled with washing buffer or place it upside down on a wet towel.  Keep the plate partially covered during prolonged work.


2.  A test might not work for the following reasons:

    If you obtained a no color reaction or too low OD values:
        Procedure was not followed
        Wrong buffer, pH, old buffer, contaminated buffer
        Wrong concentration of reagents
        No positive control
        Wells were dried up between steps
        Wrong microtiter plate
        Malfunctioned ELISA reader
        Faulty reagents

    If you obtained irregular color or non-specific reaction:
Incomplete washing and/or spillage between wells
        Wrong concentration of reagents
        Mistake in application of reagents (e.g. conjugate instead of coating)
        Edge effect due to incubation conditions
        Wells have dried up between steps
        Old substrate
        A faulty reagents (e.g. contamination of coating antibody with conjugate).


3.  Frequently Asked Questions: