Common problems in ELISA experiments

ELISA method is widely used in the determination of various antigens and antibodies. Besides the quality of the box itself, many factors in the operation affect the normal results of the test. The most common problems are light color development, low sensitivity, poor repeatability, color but no obvious gradient, etc. No matter what the results may be, it will not only waste customers' money, samples, and energy, but also make dangerous judgments on the clinical practice .Here we analyze the common causes of abnormal ELISA results and explore solutions to improve the quality of the tests.
First，Light color and low sensitivity
1. The temperature of the incubator is less than 37℃. The incubator temperature should be paid attention to. After putting into the reaction plate, minimize the opening times so as not to affect the constant temperature.
2. Insufficient insulation time;So do the experiment must pay attention to the importance of time, if you are afraid to forget the time, you can set the clock accurate timing.
3. Strong impact force, long soaking time and increased washing times during washing;Keep the washing time according to the instruction and remember the washing times accurately
4. Insufficient amount of pipette, too much water hanging on the inner wall of the suction nozzle or unclean inner wall;So to ensure that the calibration pipette, suction nozzle to set, suction nozzle to be tight, suction nozzle inner wall to clean, the best one-time use.
Second, the repeatability is poor, often the customer feedback that the value difference between two double holes is too big.Possible questions include:
1. The number of samples varies, and the sample adding time may be long or short;Therefore, when repeating a sample, the sample adding time should be as close as possible to the first time.
2. Inconsistent insulation time, washing conditions and operators;Repeated testing of specimens, operating conditions, personnel, etc. should be consistent with the last time as far as possible, so as to eliminate the possibility of inconsistency caused by these factors.
3. Inconsistent sample quantity;The sample should be thoroughly mixed before dilution. If possible, use the same pipette and install the suction nozzle tightly.
Third， After adding TMB, ELISA plate had color but no obvious gradient:
1. TMB was not placed in the shade as required, resulting in blue TMB before the experiment.
2. forget to change the straw tip, causing cross contamination.
3. low concentration or low activation coating antibody or detection antibody, sealing temperature, concentration time does not meet the requirements resulting in coating antibody instability, washing effect and coating plate problems.
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