ELISA method and its answer

ELISA methods are widely used in various antigen and antibody assays. However, there are many influencing factors in the ELISA assay, and there are certain technical requirements in the operation. In addition to the normal reaction, sometimes some wrong results are often found in the detection process. The main causes of ELISA error results are: 1 specimen factor; 2 reagent factor; 3 operational factors. The following are some of the problems in the common ELISA process.

1. Sample dilution enzyme-linked immunoreactivity is a highly sensitive response. If the serum is not diluted, a strong non-specific reaction will inevitably occur and a false positive will occur. Therefore, the kit for detecting serum antibodies abroad requires that the serum be diluted to an appropriate multiple to reduce the non-specific reaction, so that the specific antigen-antibody reaction is fully reflected. The dilution factor of the sample of the general product is determined by a large number of tests to ensure the sensitivity and specificity of the test. However, some users fail to follow the instructions carefully. For example, some products should be diluted with 10μl samples, and individual users should take 5μl or even 1μl samples for dilution, because the sample is inevitably contaminated with the sample and the accuracy of the micropipette. Not enough, so the sample dilution factor is not accurate, and the test results are problematic.

2. All reagents and strips in the kit balance kit should be equilibrated to room temperature (about 25 ° C) before the test, generally at room temperature for 20 to 30 minutes. Too short a balance time will result in insufficient reagent mixing, a relatively short incubation time, and insufficient ELISA. The room temperature is low in winter, and the kit can be placed in a 37 ° C incubator for 20 minutes.

3. Samples and reagents should be thoroughly mixed before and after dilution. All reagents should be shaken before loading to ensure uniformity of the test.

4. Loading In the current ELISA commercial kit, there is necessarily a step of adding a sample using a micropipette. The key point of attention is: do not add too fast, avoid adding to the upper part of the hole wall, do not spill and create bubbles. The loading is too fast and the accuracy and uniformity of the micro-dosing cannot be guaranteed. The non-coated area applied to the upper part of the pore wall is liable to cause non-specific adsorption. Splashing can contaminate adjacent holes. When bubbles appear, there is a difference in the interface of the reaction solution. Therefore, sometimes a specimen is tested positive with the same kit, and the next measurement is negative, which is often caused by the above-mentioned errors in loading and reagents.

5. Incubation incubation is one of the most critical factors affecting the success or failure of an assay in an ELISA assay. ELISA as a solid phase immunoassay, the antigen-antibody binding reaction is carried out on a solid phase. In order to completely bind the antigen or antibody in the liquid phase to a specific antibody or antigen on the solid phase, it must react under certain temperature conditions. time. The time required for incubation is inversely proportional to temperature, ie the higher the temperature, the shorter the time required. The most commonly used incubation temperatures are 37 ° C and room temperature, followed by 43 ° C and 2 to 8 ° C. Some operators, arbitrarily change the manual operation, use their favorite incubation time and incubation temperature, which causes unnecessary trouble. Because different kits have different incubation times and incubation temperature options, arbitrarily changing the incubation time or incubation temperature will result in bias in the test results.

6. The plate washing solid phase immunoassay technique is a heterogeneous immunoassay technique in which the antigen or antibody specifically bound to the solid phase is separated from the non-specific components adsorbed during the reaction incubation by a washing operation to ensure the ELISA assay. Specificity. Washing the plate is also an extremely critical step for ELISA assays. Do not spill the washing liquid as much as possible; after standing for the washing liquid, let it stand for 1 minute. After washing the liquid in the plate hole, be sure to shoot it vigorously; replace the absorbent paper in time, especially the absorbent paper that has taken the enzyme label. Discard, otherwise it may affect the test results.

7. Edge effect In the ELISA assay using a 96-well plate, an "edge effect" is often found, ie, the peripheral pores are darker than the central pores. Studies have shown that thermodynamic gradients in incubation may be the root cause. Polystyrene itself is a poor thermal conductor. In the laboratory routine ELISA, the plate is placed in a 37 ° C incubator from room temperature (usually around 25 ° C). When the plate is also warmed, it may be between the peripheral hole and the center hole. There is a thermodynamic gradient. Therefore, when a water bath is used or when the reaction solution is added to the pores of the plate, both the plate and the solution are heated to an incubation temperature (e.g., 37 ° C), the "edge effect" can be easily eliminated, and the repeatability of the measurement can be improved.

8. Color development must be controlled by time, according to the kit instructions. In general, the color development time is too short, and the result is low; the color development time is too long, the blank is increased, or the non-specific color development is increased.

9. Colorimetric colorimetry should pay attention to the choice of wavelength. The TMB is used as a substrate and the OPD is used as a substrate. The former has a colorimetric wavelength of 450 nm and the latter has a wavelength of 492 nm. The filter needs to be replaced at any time according to requirements. Therefore, the problem of misuse of the filter is apt to occur.
Second, the problem of single-wavelength or dual-wavelength colorimetric selection. The so-called single-wavelength colorimetric is usually measured by a colorimetric method such as 450 nm or 492 nm which has the maximum absorption for color development; and the dual-wavelength two-color is measured by a microplate reader at a sensitive wavelength such as 450 nm and a non-sensitive wavelength such as 630 nm. Once, the absorbance measurement of the upper and lower sides of the sensitive wave is the sum of the absorbance of the specific color of the enzyme reaction and the absorbance caused by the fingerprints, scratches, dust and the like on the plate hole; the wavelength is changed to a certain value when the non-sensitive wavelength is measured. The absorbance value of the sample for measuring the specific color of the enzyme reaction is zero, and the absorbance measured at this time is the absorbance value of the dirt. Finally, the value given by the microplate reader is the difference between the absorbance value at the sensitive wavelength and the absorbance value at the extreme sense wavelength. Therefore, the dual-wavelength colorimetric measurement has the advantage of eliminating the influence of the non-specific absorption of the microtiter plate itself, the non-specific absorption of the specimen in the plate hole, fingerprints, scratches, dust, etc. on the specific colorimetric measurement of the absorbance. Due to the degree of uncertainty in the non-specific absorption of individual blank wells in the ELISA assay, that is, it is possible to obtain different absorbance measurements for each measurement or the same position of the blank wells. Therefore, the colorimetric assay is performed in ELISA. It is best to use dual wavelength colorimetry.

In summary, although the procedure of the ELISA assay is very simple, there are many factors that may affect the assay results, which are distributed in the various steps of the assay, especially for loading, incubating and washing. In order to help you analyze the possible causes of problems in the search, the common problems and causes are summarized in the following table.

Possible problems and solutions during operation