Enzyme-linked immunosorbent assay ELISA for color development and colorimetry

Enzyme-linked immunosorbent assay ELISA for color development and colorimetry

The colorimetric determination of ELISA is performed by a microplate reader. Some colleagues may think that since it is carried out by a microplate reader, everything can be done at this time, but it is not the case, because the more advanced microplate readers in the contemporary age have more functions and are improperly used, which is difficult to understand the result of. For example, after the colorimetric measurement using a microplate reader, there are a lot of negative determinations, the absorbance value of L is negative; or the false positive rate is greatly increased. This is mainly caused by not understanding and using the microplate reader correctly.

In the current commercial ELISA kits that use HRP as a labeling enzyme, if TMB is used as the substrate, the provided substrates are two bottles of application solution A and B; if OPD is used as the substrate, the kit provides OPD tablets Or powder, prepared before use. The color reaction conditions of the general commercial kits are 37 ℃ or room temperature for 15 ~ 30min. Theoretically speaking, the substrate catalytic reaction of HRP can be completed only at 37 ℃ for 30min, although most of the catalytic reactions can be completed within the first 10min. Therefore, in order to allow the weak positive sample wells to have sufficient color development, it is recommended to terminate the colorimetric measurement of the reaction after the reaction at 37 ° C for 25 to 30 minutes.

In addition, before adding the substrate to start the color reaction, it is best to check the effectiveness of the substrate solution, that is, add 1 drop of each of the two solutions A and B to the clean empty plate hole or eppendorf tube and observe Whether there is color development, if there is, it means that the substrate has deteriorated. With OPD as the substrate, it should be five-color after preparation, otherwise it cannot be used. With TMB as the substrate, the entire color reaction process does not need to be protected from light, while OPD is used as the substrate, which needs to be protected from light. About TMB and OPD's color reaction characteristics and points of attention, please refer to the previous chapters. After the color reaction is completed, acid is added to terminate the reaction. After shaking and mixing, the following colorimetric measurement or visual judgment can be performed.

The colorimetric determination of ELISA is performed by a microplate reader. Some colleagues may think that since it is carried out by a microplate reader, everything can be done at this time, but it is not the case, because the more advanced microplate readers in the contemporary age have more functions and are improperly used, which is difficult to understand the result of. For example, after the colorimetric measurement using a microplate reader, there are a lot of negative determinations, the absorbance value of L is negative; or the false positive rate is greatly increased. This is mainly caused by not understanding and using the microplate reader correctly.

1. When measuring colorimetrically, we must pay attention to whether the wavelength of the microplate reader has been adjusted to the appropriate or the filter used is correct. Some clinical laboratories use TMB as a substrate and OPD as a substrate when performing ELISA measurements. The former has a colorimetric wavelength of 450nm and the latter has a colorimetric wavelength of 492nm. replace. Therefore, there is a problem that the filter is misused.

2. The problem of single wavelength or dual wavelength colorimetric selection. Microplate readers above the mid-range basically have both single-wavelength and dual-wavelength colorimetric functions. The so-called single-wavelength colorimetric is the colorimetric measurement of the wavelength with the largest absorption of color, such as 450nm or 492nm; the dual-wavelength colorimetric is the sensitivity of the microplate reader at sensitive wavelengths such as 450nm and non-sensitive wavelengths such as 630nm Each measurement is performed once, and the measured absorbance at the sensitive wavelength is the sum of the absorbance specific to the color of the sample measured by the enzyme reaction and the absorbance caused by fingerprints, scratches, dust and other dirt on the plate hole; the measurement at the non-sensitive wavelength changes the wavelength To a certain value, 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 non-sensitive wavelength. Therefore, the dual-wavelength colorimetric measurement has the advantage that it can eliminate the influence of the non-specific absorption, fingerprints, scratches, dust, etc. of the microtiter plate itself and the specimen in the plate hole on the specific color measurement of the absorbance, and it is generally unnecessary to provide a blank hole. If the dual-wavelength colorimetry is used, the blank hole is still set, which may cause the aforementioned phenomenon that the absorbance of the measurement hole is negative. Because the non-specific absorption of a single blank well in the ELISA measurement has a certain degree of uncertainty, that is to say, each measurement or the same measurement of the blank hole at different positions may obtain different absorbance measurement values, so when ELISA measurement colorimetric It is best to use dual wavelength colorimetry.