In ELISA experiments, the most worrying outcome is that the desired indicator cannot be detected in the sample, or the absorbance of the indicator falls outside the detection limit. What should be done in this case?

1、 Exceeding the detection limit

This situation is more common for highly expressed proteins, which remain outside the standard curve even after dilution by a factor of 100. In this case, you need to refer to relevant literature and adjust according to the dilution factor in the literature. However, due to the different samples, the dilution factor in the literature may not be suitable for your sample. If it still exceeds the upper limit, you can continue to dilute it.

2、 Below the detection limit - signal too weak

Often, it is due to the low concentration of the target protein, which may be due to a high dilution factor or a relatively low content of the target protein itself. This situation is mainly common in the detection of inflammatory factors in healthy samples, where the expression levels are often extremely low.

Solution:

① Firstly, reduce the dilution factor until effective absorbance can be detected.

② Increase the incubation time of antibodies. If the instructions state that the antibody needs to be incubated at room temperature for 2 hours and the results are not good, try incubating at room temperature for an additional 4 ° C overnight to maximize antibody binding or increase the amount of antibody. The instructions only recommend the amount, and some samples may require more antibodies to improve the detection signal.

③ Increase the concentration of HRP by 50%, or double it, to produce a stronger color when reacting with TMB chromogenic substrates.

④ If the ELISA box is not ready for immediate experimentation, it should be stored away from light to maximize the performance of the TMB substrate. When incubating TMB, the ELISA plate needs to be placed in the dark because TMB is very sensitive to light.

⑤ If the dilution factor has been reduced to the sample solution and experimental optimization has been carried out, but still cannot be detected, it is recommended to add a positive control well in the experimental group setting and use a clearly expressed sample as the positive control. If the standard curve and positive sample can measure normal apparent concentrations, it indicates that the experiment is successful. The expression levels of the remaining test samples are below the detection limit and can be recorded as 0 or marked as below the detection limit in the table.

When using ELISA to detect IL-17 in pleural effusion and serum of patients with tuberculous pleurisy (experimental group) and non infectious pleural effusion (control group), Cheng Bizhen et al. [1] found that the IL-17 level in pleural effusion of the control group was lower than the detection limit and the concentration could not be measured, but it could be clearly detected in the experimental group. Therefore, the control group was marked as △ and marked as below the detection limit.

If you still want to detect or need to make a comparison, you can choose a reagent kit with higher sensitivity and lower detection limit, which has a greater possibility of detecting the target protein.

If the normal expression concentration cannot be measured in the positive control wells, the problem may be the sample itself, whether it is not suitable for ELISA experiments. ELISA samples are generally selected from culture supernatant, serum, and plasma, and other sample forms may not be suitable. At this time, it is necessary to consider changing the sample or testing method.

Summary of Absin High Sensitivity ELISA Kit:：

References:

[1] Cheng Bizhen, Lin Shuyong, Zhang Qiaoxin, Yang Li, Cai Yingmu The role of IL-17 and IL-23 immune axis in the immunopathogenic mechanism of tuberculous pleurisy [J]. Chinese School Medical Journal, 2014, 28 (05): 353-355

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