Elisa experiment: Elisa curve problem

In ELISA experiments, if there are problems with the curve, it is mostly due to inadequate standards, procedures, or equipment. Core conclusion: The problem with calibration is usually caused by the handling of standard samples, sample addition operations, or incubation conditions. By focusing on these steps, it is possible to quickly locate them.

1、 Common reasons for poor standard curves

Improper dissolution and dilution of standard samples

The standard powder has not been fully dissolved or diluted evenly, resulting in inaccurate concentration.

Suggestion: Use the specified diluent according to the instructions, dissolve and centrifuge briefly. When diluting in a gradient, use a new nozzle, blow or vortex to mix well.

Inaccurate sample addition operation

The pipette is not calibrated, or the sampling speed and angle are inconsistent, resulting in significant differences in pore volume.

Suggestion: Regularly calibrate the pipette, add samples vertically and steadily, keep the tip of the pipette close to the liquid level but not touching the bottom, and avoid bubbles.

The incubation conditions are not constant

Lack of sealing film or inadequate covering, resulting in edge effects; The temperature and time are unstable.

Suggestion: Use high-quality sealing film, place the enzyme-linked immunosorbent assay (ELISA) plate in a constant temperature incubator, and avoid natural incubation.

2、 Other common curve problems and countermeasures

Overall low

Possible reasons: expired reagents, standard samples not dissolved, failure to test immediately after termination solution, mixed reagents.

Suggestion: Store the reagents according to the instructions, centrifuge the standard solution before dissolution, add diluent, let it stand and mix well, and immediately test after terminating the solution.

No signal

Possible reasons: expired reagents, HRP enzyme contamination, operational errors (such as incorrect sample addition sequence).

Suggestion: Check the reagent label, prepare a buffer solution with fresh distilled water, and let the reagent equilibrate at room temperature for 30 minutes.

Overall high

Possible reasons: The enzyme-linked immunosorbent assay (ELISA) instrument has a wide range of optical density, high concentration of working solution, long incubation time/high temperature.

Suggestion: Set the optical density range to 0-3.5, prepare and use the working solution immediately, and control the incubator temperature at 37 ± 0.5 ℃.

3、 Optimization suggestions

Standard processing: Centrifuge before dissolution, add diluent and let it stand and mix well.

Sample addition operation: calibrate the pipette, add the sample vertically and steadily, and avoid bubbles.

Incubation conditions: Seal with a sealing plate membrane and place the enzyme-linked immunosorbent assay (ELISA) plate in a constant temperature incubator.

Equipment inspection: Regularly calibrate the pipette and set the enzyme-linked immunosorbent assay (ELISA) reader to the correct optical density range.

Note: The above is for reference only. This product is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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