The Method Of Colorimetry And Colorimetry By ELISA

(1) color rendering

Chromogenic ELISA is the last step in the incubation reaction, at this time the enzyme catalyzed colorless substrate to produce colored products. The reaction temperature and time are still the factors that affect the color development. In a certain period of time, the negative hole can remain colorless, and the positive hole with the extension of time and color enhancement. Appropriate increase of temperature is helpful to speed up color development. In quantitative determination, the reaction temperature and time after adding the substrate should be accurate according to the regulations. Qualitatively determined color can be carried out at room temperature, the time generally does not need to be strictly controlled, sometimes according to the positive control well and negative control well color situation can be appropriate to shorten or extend the reaction time, timely judgment.

The color development of OPD substrate is generally not deepened after 20-30 minutes of reaction at outdoor temperature or 37℃, and then the reaction time can be extended to increase the background value. OPD substrate liquid will automatically change color when exposed to light. The color reaction should be carried out in the dark. When the color reaction is over, a termination solution is added to terminate the reaction. After OPD product is terminated with sulfuric acid, the color changes from orange yellow to brown yellow.

TMB is not affected by light and can be placed on the operating table at room temperature to observe the results by side reaction. However, in order to ensure the stability of the experimental results, it is advisable to read the results at the appropriate time. After HRP treatment, the color of TMB reached about 40 minutes, then gradually weakened, and completely faded to colorless after 2 hours. TMB terminates in a variety of solutions. Enzyme inhibitors such as sodium azide and sodium dodecyl sulfate (SDS) can terminate the reaction. This kind of terminator can also make the blue color maintain for a long time (12-24 hours), is a good terminator for visual judgment. In addition, various acidic terminators will change the blue to yellow, when the specific wavelength (450nm) can be used to read the absorption value.

(2) colorimetric

Before colorimetry, clean absorbent paper should be used to dry the liquid attached to the bottom of the plate, and then the plate should be properly placed in the colorimetric frame of the enzyme standard colorimeter. In the test with soft plate as carrier, the plate should be placed in the standard 96-hole frame before color comparison can be carried out. Before adding the substrate solution for color, cut the edge of the soft plate clean, so that the plate can be completely seated in the seat frame.

When colorimetry, the zero point should be adjusted with distilled water first, and the substrate hole (the hole with substrate solution added without any reaction) and the blank hole (the hole with normal saline or diluent instead of the specimen as the whole process) should be measured and read to record the reagent status of this test. Then blank holes can be used to calibrate the zero point with distilled water, and the absorbance of the above holes should be subtracted from the absorbance of the blank holes, and then calculated.

The expression of the colorimetric result is oplical density (OD) in the past, but absorbence (A) is used according to the regulations, which have the same meaning. The usual expression method is to write the absorption wavelength in the lower right corner of the letter A. For example, the absorption wavelength of OPD is 492nm, and the expression method is "A492nm" or "OD492nm".

(3) enzyme standard colorimeter

The enzyme standard colorimeter is referred to as the enzyme standard instrument, usually refers to the photometer of measuring the absorbance of ELISA results. According to the different forms of solid carrier, there are special designs for plate, bead and small test tube. Many reagent companies provide matching enzyme label instrument. The main performance indexes of the enzyme plate instrument are: reading speed, reading accuracy, repeatability, degree and measurable range, linearity and so on. The reading of a good microplate meter is generally 0.001, with accuracy ±1% and repeatability up to 0.5%. For example, if the A value of A hole is 1.083, then the true A value of the hole relative to the air should be 1.083±0.01 (1.073~1.093). Repeated measurement several times, the A value should be 1.083±0.05 (1.078~1.088). The measuring range of the enzyme plate analyzer varies according to the performance of the enzyme plate analyzer. The limit of the common enzyme plate analyzer is 0.000~2.000, but the limit of the new enzyme plate analyzer is widened to 2.900, or even higher. A value that exceeds the upper limit of measurement is often indicated by "*" or "over" or other symbols. Attention should be paid to the difference between the measurable range and the linear range. The linear range is often less than the measurable range. For example, the measurable range of a certain enzyme plate instrument is 0.000~2.900, while its linear range is only 0.000~2.000, which should be paid attention to when making the standard curve in quantitative ELISA.

The enzyme label instrument should not be placed under sunlight or strong light irradiation. The room temperature should be 15~30℃ during operation. The instrument should be preheated for 15 to 30 minutes before use, so that the reading results are more stable.

When measuring the A value, the sensitive absorption peak of the product should be selected. For example, OPD uses the wavelength of 492nm. The first time is at the optimum wavelength (W1) and the second time is at the insensitive wavelength (W2). The position of the ELISA plate is not moved between the two measurements. For example, OPD uses 492nm as W1 and 630nm as W2, and the final measured A value is the difference between the two (W1-W2). Dual wavelength reading can reduce the light interference caused by scratches or finger marks on the container.