Introduction to common problems and solutions for ELISA experiments

Temperature is an important factor in the ELISA binding response. In order for all samples to react at a consistent temperature, all reagents must be equilibrated to room temperature prior to the experiment, including test samples. Avoid inaccurate ELISA results due to differences in temperature kinetic responses.

Store the standard as recommended; centrifuge briefly to completely collect the powder before dissolving the standard; ensure that the standard is completely dissolved and mixed (approximately 10 min), followed by a subsequent series of dilution steps to ensure that each step is thoroughly mixed and precise Pipetting; proper termination of color development; selecting a suitable mathematical fitting equation to draw a standard curve.

In order to obtain more accurate experimental results, it is strongly recommended that the standards and samples be tested for duplicate wells.
Because the duplicate hole detection can:
★ Calculate the average to ensure more accurate results;
★ Solve the phenomenon of jumping holes caused by misoperation in the experiment;
★ Calculate the CV value and evaluate the operation of the experiment and the precision of the kit.

Incubation with shaking allowed the reaction to be more complete, and the shaking wash made the background cleaner. A 96-well microplate shaker dedicated to the enzyme label is recommended.
Oscillation during the incubation process can accelerate and enhance the collision of antigen-antibody molecules, making the reaction process more complete, and the OD value is usually higher than that of the non-oscillation incubation; the washing process oscillates, which makes the washing plate cleaner and large. The degree of background can be reduced and the sensitivity of the kit detection can be improved.

The ELISA experiment finally needs to be carried out by enzyme-catalyzed substrate color reaction, and the termination of the reaction at the optimal time is an important factor for the success of the ELISA experiment. In the HRP-TMB enzymatic reaction system, when the color of the highest concentration standard is no longer deep, and the countdown 2-3 concentration standards begin to have a light blue color, the reaction needs to be terminated; or it can be based on the highest concentration standard. The pores are determined by the OD value at 620 nm and can be terminated when OD620 = 0.9-0.95.

First of all, the microplate reader must be preheated for 10-15 minutes before use, which makes the test results more stable. Secondly, the ELISA test uses two wavelengths to measure the absorbance, which can eliminate the measurement interference (concentration of the specimen, interference color, etc.) at the time of single wavelength detection. Generally, the maximum wavelength is used as the reference wavelength. At the reference wavelength, the absorbance of the detection object is the smallest, and the difference between the absorption wavelength of the detection wavelength and the reference wavelength can eliminate non-specific absorption; therefore, the dual-wavelength measurement can eliminate fingerprints and impurities to the utmost extent. And the error caused by the opaque material on the microplate reader reading to ensure the accuracy of the experimental data.

ELISA is the gold standard for quantitative protein detection, and the requirements for experimental operation are extremely strict. If you want accurate, stable results and want the perfect data analysis, please choose Lianke Bio, we will provide you with high quality testing services under standardized operating procedures.

For details, please refer to the instruction manual or call Lianke Bio (400-6721-600).