ELISA kit experimental technical points! -Huaqiang Electronic Network

Elisa kit experimental technical points!

The results of the ELISA kit were divided into qualitative and quantitative. In the indirect method and the sandwich ELSIA, the positive holes were darker than the negative ones. In the competition ELISA, the negative hole was darker than the positive well. So how do you judge the two types of results? Far from the biological professionals to explain for you:

(1) Indirect method and sandwich method

The qualitative results of such reactions can be judged by the naked eye. The visual specimens were also colorless or nearly colorless, and those with clear color were positive. However, in ELSIA, the background of the coloration often appears after normal human serum reaction. The depth of the background varies depending on the composition of the reagent and the experimental conditions. Therefore, a negative control must be added in the experiment. The composition of the negative control should be normal serum or the like that does not contain the test substance. When judging the results with the naked eye, it is more preferable to use a color developed deeper than the negative control as an indicator of positive samples.

The visual method is simple and clear, but quite subjective. When conditions permit, the absorbance should be measured with a colorimeter so that objective data can be obtained. The absorbance values ​​of the sample (S, S), the positive control (P), and the negative control (N) are read first, and then calculated. There are many calculation methods, which can be roughly divided into positive judgment value method and specimen and negative control ratio method.

a. Positive judgment value

The cut-off value is generally the negative control A value plus a specific constant as a criterion for positive or negative judgment.

Judging the results by this method requires that the experimental conditions are very constant, the preparation of the reagents must be standardized, and the positive and negative control materials should meet certain specifications, and must be equipped with precision instruments and strictly in accordance with the regulations. The constants in the positive decision value formula are obtained by experimental testing of a large number of specimens in this particular system. Here is an example of a kit for detecting HBsAg. The negative control in the kit was recalcified human plasma without HBsAg, and the content of the positive control HBsAg was indicated as P=9±2 ng/ml. Two positive controls and three negative controls were set up for each test. After measuring the A value, first calculate the mean of the negative control A value (NCX) and the positive control A value (PCX), the difference between the two averages (PN) must be greater than a specific value (example 0.400), The test is effective. The values ​​of the three negative control A should be ≥0.5×NCX and ≤1.5×NCX. If one of them exceeds this range, discard it, and the other two negative controls recalculate NCX; if there are two negative controls, the A value exceeds The above range is invalid for this experiment. The positive judgment value is calculated by the following formula: positive judgment value = NCX + 0.05, specimen A value > positive judgment value is positive, and less than positive judgment value is negative. It should be noted that 0.05 is the constant of the kit and is only suitable for the specific conditions, and is not universal for various reagents.

As can be seen from the above description, in this method, the negative control and the positive control also play a quality control role in the test, and the deterioration of the reagent and the improper operation may result in "invalidation of the test".

b. Specimen/negative control ratio

In the case where the experimental conditions (including reagents) are difficult to ensure constant, the ELISA kit is more suitable. After the A values ​​of the specimen (S) and the negative control (N) are obtained, the S/N value is calculated. There is also writing P/N. The ELISA kit here does not represent positive, but is an abbreviation of the patient and should not be misunderstood. To avoid confusion, it is better to use S/N. In the early indirect ELISA, some authors determined that S/N was a positive standard and are now used in various assays. In fact, each measurement system should experimentally determine the respective S/N threshold. It should be noted that the negative control represented by N is human serum containing no test substance. In some kits, the negative control is a buffer containing no protein or protein content, so that the background generated after the reaction may be much lower than the background of normal human serum. Therefore, such kit rules, such as N < 0.05 (or other values), are calculated as 0.05, otherwise false positive results will occur.

(2) Competition law

In the competition ELISA, the negative wells were darker than the positive wells. The intensity of the negative coloration depends on the concentration of the enzyme conjugate in the reaction and the amount of the competitive inhibitor added. The absorbance of the negative control is generally adjusted between 1.0 and 1.5, at which time the reaction is most sensitive.

The competitive method ELISA is not easy to judge by self-viewing. It is difficult for the naked eye to distinguish the difference between the weak positive reaction and the negative control. Generally, the colorimetric values ​​of S, P and N are read by a colorimeter. There are two main calculation methods, namely the positive judgment value method and the inhibition rate method.

Positive judgment value method

It is basically the same as the positive judgment value method in the indirect method and the sandwich method. The ELISA kit introduces the positive control A value in the calculation formula, and some kits for detecting anti-HBc are exemplified. The negative control in the kit was recalcified human plasma without anti-HBc, and the anti-HBc content in the positive control was 125 ± 100 u/ml. Two positive controls and three negative controls were set up for each test. After the A value is measured, the average of the negative control A value (NCX) and the positive control A value (PCX) are calculated first, and the difference between the two averages (NP) must be greater than a specific value (for example, 0.300). The test is effective. The values ​​of the three negative control A should be less than 2.000, and should be ≥0.5×NCX and ≤1.5×NCX. If one of them exceeds this range, discard it and recalculate ×NCX with the other 2 negative controls; if there are 2 If the negative control A exceeds the above range, the experiment is invalid. The positive judgment value was calculated by the following formula: the negative judgment value = 0.4 × NCX + 0.6 × PCX, the reaction of the specimen A value ≤ the positive judgment value was positive, and the reaction of the A> positive judgment value was negative.

b. inhibition rate method

The inhibition rate indicates the degree of inhibition of the coloration of the negative reaction by the specimen in the competitive binding, and the ELISA kit is calculated as follows:

Inhibition rate (%) = (negative control A value - specimen A value) × 100% / negative control A value, generally specified inhibition rate ≥ 50% positive, < 50% negative.