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ELISA is always unsuccessful, what is wrong?

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ELISA has always been widely used clinically because of its high sensitivity and good specificity. As a classic immunoassay method, although it seems ordinary, it is not easy to do the experiment well. The reading of the ELISA plate will always be unsatisfactory. It can be seen that each link in the actual operation of the experiment is not easy. The detection effect has a great influence, if not paying attention, it will produce some bad experimental results.

Question 1
Low signal strength

If the ELISA reading is lower than the lower limit of absorbance (<000) and the target sample concentration is outside the standard curve range, the sample concentration for analysis may be too low, or the best conditions for sample detection that the instrument can analyze may not be met. In this case, the detection signal generated by the sample should be cascaded and amplified.


  1. Increase the incubation time of the antibody. If the experimental results after incubating your antibody for 2 hours at room temperature according to the protocol are not satisfactory, you can try incubating overnight at 4°C. In this way, the antigen and antibody can be allowed to bind to the greatest extent, and the reaction signal in ELISA can be expanded.
  2. Increase the concentration of the secondary antibody-enzyme conjugate. The E in ELISA stands for enzyme (usually horseradish peroxidase, HRP), which reacts with the substrate (usually TMB) to produce a bright blue color. Therefore, increasing the HRP concentration by 50% or doubling it will produce stronger colors that are easier to detect.
  3. Fully avoid light to protect the TMB substrate from the influence of light, and to ensure that it can maximize its performance. Considering that TMB is very sensitive to light, the color reaction of the ELISA plate in the dark will produce a stronger signal.

Question 2
High background

A blank well on an ELISA plate is usually used as a negative control. When the reading of any well in the group is significantly greater than 0, it means that there is a problem with the experimental background.


  1. Too high room temperature may lead to high experimental results. Therefore, if you find that the readings of all the experimental holes are too high, please pay attention to control the temperature of the incubator, and stabilize it at about 37°C as much as possible.
  2. The incomplete cleaning of the plate wells results in residues of enzymes or substrates, which will also produce high background. At this time, the ELISA plate needs to be cleaned in accordance with the protocol, and the volume of the plate washing solution and the number of plate washings are not arbitrarily reduced. At the same time, it is also necessary to operate as required, and appropriately extend the time for the washing solution to soak the wells.
  3. Contamination of negative wells was caused when samples were added or enzymes were added. At this time, you need to replace the tip in time to avoid contamination, and don’t get lucky.
  4. The reagent is out of date or contaminated. Do not use expired reagents during the experimental operation, and prevent component contamination. If you use the container, you should pay attention to the cleanness of the container.

Question 3
Reproducibility is not good

There is a significant difference in the readings of each replicate of the same sample.


  1. The sample volume varies, and the operating time varies. When repeating the same sample, the sample addition amount and sample addition time should be the same, and attention should be paid to the calibration of the pipette. After adding the sample, shake the ELISA plate slightly to mix the reaction solution thoroughly.
  2. The samples should be consistent, non-contaminated, and should be operated by the same operator.
  3. The precision measurement method is not standard, and the correct method should be used at this time.

Question 4

Interfering substances such as cleaning agents or NADH (present in serum) may affect the absorbance reading of the ELISA plate and cause the experimental results to deviate from the true results.


  1. Perform experimental operations strictly in accordance with the protocol. Usually the protocol will point out the chemical substances that will interfere with the experiment, and will tell the experimenter how to avoid them. For example, in the serum determination of hemoglobin interference, it is usually recommended not to use hemolyzed serum samples.
  2. Sometimes you may have no choice but to use samples containing interfering substances. If the concentration of the antigen you are interested in is higher, you can use a sample diluent with a lower concentration but still easy to detect, which can dilute the interfering substances in the sample and reduce its interference to the experiment.

Question 5
Positive and negative cannot reach the ratio of 2.1

The negative color is too dark, or the positive color is too light.


  1. When the negative color is too dark, the negative control (that is, the other components of the positive control except the target antigen) may have a certain component and the primary antibody. At this time, the interfering components can be removed by purifying the antigen.
  2. When the positive color is too light, there may be three reasons: the titer of the primary antibody is not good, switch to the primary antibody or increase the dosage of the primary antibody; if the antigen is too thin, increase the antigen concentration; if the blocking time is too long, the blocking time should be shortened. The closing time is generally 37 degrees Celsius for 2 hours, or 4 degrees Celsius overnight.

Question 6
Increasing the antigen concentration, the primary antibody concentration remains unchanged, the color reaction does not show the expected gradient

The ratio of primary antibody to antigen is saturated.


Increase the dosage of the primary antibody; or reduce the antigen concentration to make a gradient while the dosage of the primary antibody remains the same.

Question 7
Increasing the concentration of the primary antibody, the antigen concentration remains unchanged, and the color reaction does not show the desired gradient

The ratio of primary antibody to antigen is saturated.


Increase the amount of antigen; or reduce the concentration of the primary antibody without changing the amount of antigen to make a gradient.

Question 8
The color gradient is obvious after adding TMB for color development, but after adding sulfuric acid to terminate the reaction, the color gradient is not so obvious.

Sulfuric acid may have charred other ingredients


Add a little less sulfuric acid, reference value: 50 μl TMB+35 μl sulfuric acid; or use 1M HCL as the stop solution, 50ul TMB+50ul 1M HCL.

Reference: Troubleshooting a Faulty ELISA

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