ELISA FAQ

Frequently Asked Questions of ELISA (Shanghai Lianshuo Biological Technology Co., Ltd., one of the major suppliers of domestic immunology products)

The ELISA method is widely used in the determination of various antigens and antibodies. However, there are many influencing factors in ELISA measurement, and there are certain technical requirements in its operation. In addition to the normal reaction in the detection process, sometimes some wrong results are common. The main causes of ELISA measurement error results are: â‘  specimen factor; â‘¡ reagent factor; â‘¢ operation factor. The following is an analysis of some common problems in the operation of ELISA.

1. Sample dilution
ELISA is a highly sensitive reaction. If the serum is not diluted, it will inevitably produce a strong non-specific reaction and false positives. Therefore, the kits for detecting serum antibodies in foreign countries all require that the serum be diluted to an appropriate multiple to reduce non-specific reactions, so that specific antigen-antibody reactions can be fully reflected. The sample dilution ratio of general products is determined through a large number of tests to ensure the sensitivity and specificity of the test. However, some users fail to strictly follow the instructions. 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 stained on the nozzle and the precision of the micropipette Insufficient, so the dilution factor of the sample is not accurate, and the test results are problematic.

2. Kit balance
All reagents and slats in the kit should be equilibrated to room temperature (approximately 25 ° C) before the test, and generally need to be left at room temperature for more than 20-30 minutes. If the equilibration time is too short, the reagent mixing will not be enough, the sample incubation time will be relatively shortened, and the ELISA reaction will be insufficient. In winter when the room temperature is low, the kit can be placed in a 37 ° C incubator for 20 minutes.

3. Sample and reagent mixing
The samples before and after dilution must be thoroughly mixed, and all reagents must be shaken before loading to ensure the uniformity of the test.

4. Loading
In the current ELISA commercial kit, there must be a step of adding a sample using a micro-sampler. The key point to note is that the sample should not be added too fast, and should not be added to the upper part of the hole wall. The sample addition is too fast to guarantee the accuracy and uniformity of the micro-sample addition. The non-coating area added on the upper part of the hole wall may easily lead to non-specific adsorption. Splashes can contaminate adjacent holes. When bubbles appear, there is a difference in the reaction liquid interface. Therefore, sometimes a specimen is tested positive with the same kit this time, and the next test is negative, which is often caused by the above sample addition and reagent errors.

5. Incubation
Incubation is the most critical factor affecting the success or failure of the assay in ELISA. ELISA is a solid-phase immunoassay. The antigen-antibody binding reaction is performed on the solid phase. To make the antigen or antibody in the liquid phase completely bind to the specific antibody or antigen on the solid phase, it must be reacted under certain temperature conditions. time. The time required for incubation is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperature is 37 ℃ and room temperature, followed by 43 ℃ and 2 ~ 8 ℃. Some operators change the manual operation without permission and use their favorite incubation time and incubation temperature, which causes unnecessary trouble. Because different kits have different choices of incubation time and incubation temperature, arbitrarily changing the incubation time or incubation temperature will lead to deviations in the test results.

6. Wash board
The solid-phase immunoassay technique is a heterogeneous immunoassay technique. It is necessary to separate the antigens or antibodies specifically bound to the solid phase from the non-specific components adsorbed during the reaction incubation with a washing operation to ensure the specificity of ELISA measurement Sex. Plate washing is also an extremely critical step for ELISA determination. Do not spill the lotion out of the hole as much as possible; let it stand for 1 minute after adding the lotion. After removing the lotion from the plate hole, be sure to pat it dry; replace the absorbent paper in time, especially the absorbent paper that has been taken with enzyme markers. Discard, otherwise it may affect the test results.

7. Edge effect
In ELISA assays using 96-well plates, "edge effects" are often found, that is, the peripheral wells are deeper than the central wells. Studies have confirmed that the thermodynamic gradient in incubation may be the root cause. Polystyrene itself is a poor thermal conductor. In the routine ELISA measurement in the laboratory, the plate is placed in a 37 ° C incubator from room temperature (usually around 25 ° C). When the plate is also heated, it may be between the outer peripheral hole and the central hole There is a thermodynamic gradient. Therefore, using a water bath or heating the plate and solution to the incubation temperature (such as 37 ° C) when adding the reaction solution to the wells of the plate, the "edge effect" can be easily eliminated and the repeatability of the measurement can be improved.

8. Color rendering
The color development must be controlled by time, according to the instructions of the kit. Generally speaking, if the color rendering time is too short, the result is low; if the color rendering time is too long, the blank increases or the non-specific color rendering increases.

9. Colorimetric
Colorimetric should pay attention to the choice of wavelength. Both TMB as a substrate and OPD as a substrate are used. The colorimetric wavelength of the former is 450nm, and the latter is 492nm. The filter needs to be replaced at any time according to requirements. Therefore, there is a problem that the filter is misused.
Secondly, the problem of single wavelength or dual wavelength colorimetric selection. The so-called single-wavelength colorimetry is the colorimetric measurement of the wavelength with the maximum absorption of color development, such as 450nm or 492nm; while the dual-wavelength dual-color measurement is performed at a sensitive wavelength such as 450nm and a non-sensitive wavelength such as 630nm. Once, the absorbance measured above and below the sensitive wave is the sum of the absorbance specific to the color of the sample to measure the enzyme reaction and the absorbance caused by fingerprints, scratches, dust and other dirt on the plate holes; at non-sensitive wavelengths, the wavelength is changed to a certain value , So that the absorbance value of the sample to determine 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 very sensitive wavelength. Therefore, the dual-wavelength colorimetric measurement has the advantage that it can exclude the influence of the non-specific absorption, fingerprints, scratches, dust, etc. of the microtiter plate itself and the specimens in the plate well on the specific colorimetric measurement absorbance. Because there is a certain degree of uncertainty in the non-specific absorption of a single blank hole in ELISA measurement, that is to say, each measurement or the same measurement of the difference in the position of the blank hole may obtain different absorbance measurement values, so the colorimetric measurement in ELISA It is best to use dual wavelength colorimetry.

In summary, although the operation steps of ELISA measurement are very simple, there are many factors that may affect the measurement results, which are distributed in each step of the measurement operation, especially sample addition, incubation and plate washing. In order to help you analyze and find the possible causes of problems in the measurement, the common problems and causes are summarized in the following table.

Possible problems and solutions during operation

problem

possible reason

Solution

Light color development, low sensitivity

1. The kit time is too long during transportation and the temperature is too high

Try to shorten the transportation time, ice cubes should be used to cool down in summer

2. The kit is not fully balanced

After removing the kit from the 2 ～ 8 ℃ refrigerator, open the lid and equilibrate at room temperature for at least 20 minutes to ensure that all reagents have been equilibrated to room temperature (about 25 ℃).

3. The temperature of the incubator is less than 37 ℃

Pay attention to the temperature of the incubator. After placing the reaction plate, try to reduce the number of openings as much as possible to avoid affecting the temperature constant.

4. Insulation time is insufficient

Correct fixed clock accurate timing

5. The impact force during washing is too large, the soaking time is too long, and the number of washing times increases

Keep the washing time according to the instructions and remember the washing times accurately

6. Insufficient pipetting volume, too much water hanging on the inner wall of the nozzle or the inner wall is not clean

To calibrate the pipette, the nozzle should be matched, the nozzle should be tightly packed, and the inner wall of the nozzle should be clean, preferably one-time

7. The quality of distilled water is problematic

Use fresh qualified distilled water

8. Insufficient substrate action time

Accurate timing

The background is deep and all are colored,

1. Insufficient washing, not dried after washing, residual other components or enzyme markers in the sample

The concentrated washing liquid is accurately prepared; if there are crystals in the 10-fold concentrated washing liquid, the crystals should be completely dissolved at room temperature and then diluted. The filter paper for adding samples or adding enzyme to the plate should be discarded and not used repeatedly, otherwise it will easily cause pollution

2. Sample contamination

Samples should be collected fresh or stored at low temperature to prevent contamination

3. The temperature of the incubator exceeds 37 ℃ or the reaction time is too long

Adjust the temperature of the incubator for accurate timing

4. Repeated use of suction nozzle, not cleaned or disinfected thoroughly

The nozzle is used as much as possible

5. Distilled water is contaminated

Use fresh distilled water

6. Mixed use of enzymes and other reagents

Do not mix reagents of different batches

7. The amount of specimens in one experiment is too much, and the sample addition time is too long, which leads to the prolonged actual reaction time.

Reasonably arrange the experiment to avoid loading several enzyme plates simultaneously

Poor repeatability

1. The number of samples varies, and the sample addition time varies

When repeating a sample, the sample addition time should be as close as possible to the first time

2. Inconsistent holding time, inconsistent washing conditions, and inconsistent operators

Repeat the measurement of the specimen, the operating conditions, personnel, etc. should be as consistent as possible last time to exclude the possibility of inconsistency caused by these factors

3. Inconsistent sample volume

The sample should be mixed well before dilution. Use the same pipette as far as possible and install the nozzle tightly

A white board appears, the positive control does not develop color

Deterioration of color developing solution

Replace with new developer

Wrong preparation of washing solution

Please prepare according to the dilution factor shown in the manual

No enzyme conjugate added but considered added

Be careful not to miss

The stop solution is mistakenly diluted as a washing solution or used as a substrate solution

Always read the label before adding liquid