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13 successful tips for ELISA troubleshooting (part two)

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7. The remaining kit materials should be properly preserved

After the standard product is packed, it can be stored according to the temperature required by the manual. This can avoid contamination. It can avoid repeated freezing and thawing of the standard products that require cryopreservation. The enzyme label plate is put back into the foil paper bag, desiccant is added, the sealing is sealed, and stored at 4 ° C. Do not completely balance the enzyme plate to room temperature, then put it back into the foil paper bag, because at this time due to the temperature difference, there will be water vapor on the microplate, which is not conducive to stable storage.

8. Add samples in a fast and accurate way, avoiding air bubbles

The loading time should be controlled within 15 minutes. Mix the sample before loading, especially the sample that is thawed after cryopreservation (the sample that is naturally melted will have stratification), and mix it upside down, but pay attention to the movement and avoid bubbles. If the frozen sample is precipitated, it can be centrifuged again. Care should be taken to avoid air bubbles throughout the loading process. Air bubbles affect the mutual binding of proteins and affect the reaction.

9. Ensure uniform temperature during incubation to prevent evaporation and drying

The sealing film is compressed during incubation. When multiple plates are tested together, lay the plates flat and do not stack them to avoid drift or edge effects due to uneven temperature.

10. Application of the plate washer

The use of a plate washer not only reduces labor intensity, but also helps to ensure the stability of the ELISA test. At present, the automatic plate washer not only has many practical washing actions, such as bottom flushing, two-point aspiration, vibration, and cross-priming; it can also adjust parameters such as flushing pressure, flushing distance, and flushing time as needed. By optimizing these parameters, a good washing effect can be ensured. The performance of the plate washer has a great influence on the accuracy of the results. A good plate washer should ensure that the residual liquid after washing is as small as possible, generally not exceeding 2 μL, and the artificial clapper paper is not wet after washing. The suction needle should be checked regularly for blockage.

11. Manual washing instruction

Add lotion quickly. The lotion should be freshly prepared to avoid contamination by other reagents or bacterial. Soak it for 30s-1min. The pouring of the seesaw should be quick and easy. After pouring, draw the board on the absorbent paper and use high-quality absorbent paper without dust or debris. It is necessary to force the plate so that there is no visible liquid hanging in the hole; but it should not be too heavy to allow the sample to dry for too long. Immediately do the subsequent dosing after the ELISA plate is patted.

12. Key points of color reaction

The ELISA assay is an enzymatic substrate color reaction, which increases with time and darkens, so choosing the optimal time to terminate the reaction is an important factor in obtaining an accurate standard curve and sample concentration. The termination process should be complete. When using the TMB substrate, the complete termination point is yellow, and there will be no blue or green color. If necessary, the 96-well plate can be oscillated if necessary during the termination process, or mixed with a pipette tip (the stop solution contains strong acid to avoid corrosion).

13. Data analysis

Perform curve fitting and analysis according to the data analysis method recommended in the manual. The standard curve in the specification is the result obtained by a very experienced operator in the manufacturer's laboratory. The customer's standard curve and the standard curve in the specification may differ, but it’s not means bad. Generally, the criteria for judging the standard curve are: background OD value <0.2; highest point OD value>1.0; correlation coefficient R2 is close to or equal to 1. As long as the standard curve that meets these conditions is available.


When you understand and master these unique tips for ELISA, you are very close to make an excellent ELISA experiment.

Creative Diagnostics provides a wide range of enzyme-linked immunosorbent assay (ELISA) kits for the detection of hundreds of different proteins and molecules, including cytokines, growth factors, markers for infectious diseases, diabetes and tumor, drugs and small molecules, etc. You can search for the ELISA kit you need by target name, species, etc. on their website. Should you need any assistance, the company’s experienced scientists are more than happy to provide optimized protocols and helpful technical support to accelerate your research.

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