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Common problems and solutions in the ELISA

Jun 01, 2016

The following analysis of Elisa test operation reason that may influence the results, and gives the corresponding solution

一、selective reagent

Choose detection reagent of excellent quality , reagent in strict accordance with the specifications, reagent balance 30 to 60 minutes at room temperature before operating .

二、Adding sample

possible cause:

1) in serum or plasma samples separation is not good that add sample;2) the manual operation, plus sample from excess and sample after waiting for a long time before put into the incubator (especially when indoor temperature higher);3) enzyme splash out of hole when add the specimen and add enzyme reagent. 


1) specimens of serum: it is best to first natural blood stored 1 to 2 hours, with 3000 RMP centrifugal 15 minutes;Specimens for plasma: must use collection tube of anticoagulant blood specimen , after the blood must be immediately reverse vascular mixed 5-10 times, placed after a period of time, 3000 RPM centrifugal 15 minutes;If testing in a few days, can put in 2-8 ℃ refrigerator, if you want to store, under - 20 ℃ low temperature refrigerator.2) after add sample in a timely manner in the incubator.3) after the enzymatic reagent with blotting paper gently dry blot in enzyme label plate surface.4) if use the AT or other automatic sample, had better choose FAME or other post-processing equipment with enzyme reagent.5) specimen is large, please batch operation.


possible cause:

1) when the incubation without seal or capping, make specimens or diluent evaporation, adsorption on hole wall, difficult to wash thoroughly;2) artificial incubation time prolonged, which leads to the nonspecific combined tightly attached around the reaction hole, it is difficult to clean thoroughly.


1)sealingor capping; 2)strictly control the operation time according to instruction steps

四、 Washing the plate

possible cause:

1) using manual washing board, liquid cross between hole and hole.2) using semi-automatic washing machine to wash plate, insufficient lotion, lead to wash the plate is not completely;Wash the plate needle blocking, suction is not completely;Wash the plate not smooth, lead to wash plate effect is poor.3) reaction plate nimiety causes washing plate wait too long.


1) ensure the lotion fill each hole, washing plate needle smooth , after washing a plate in the best absorbent paper (select clean, no or less dust suction material) and gently pat dry;2) reasonable arrangement, or use more washing machines.


possible cause:

1) storage time is too long after compound chromogenic agent or use expired chromogenic agent;2) when adding chromogenic agent splash out of hole caused liquid backflow.


1) the chromogenic agent in preparation before use as far as possible, stick to need not expired chromogenic agent, need to macroscopic light-blue TMB chromogenic agent;2)keep chromogenic agent not outflow when add sample ;3) A and B should avoid touch with the metal equipment.


Possible reasons for such as generating more bubbles when add terminated liquid, leads to false positives increased.So it should avoid to generate bubbles When add terminated liquid .

七、reading plate

Such as plate bottom is not clean when reading plate.It should guarantee the enzyme plate clean.So in the whole operation process to ensure enzyme plate does not touch hypochlorous acid;As far as possible to realize the automatic ELISA test standard, effectively improve the quality of detection.

In practice, in addition to choose excellent reagents, must be in strict accordance with the operation steps, at the same time, make indoor quality control, room between qualitative evaluation, with the rigorous work style to detect each specimens, to ensure the quality of detection.Now has a number of domestic units with automatic enzyme standard instrument, For the implementation the standardization of ELISA detection, improve the quality of detection plays an important role.

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