We then proceed to the point of attention to the various operational steps of the plate-type ELISA.
In the indirect and sandwich method ELSIA, the positive hole is darker than the negative hole.
In the competition law ELISA, in contrast, the negative pore color is darker than the positive hole.
The results of the two types of reactions are different and are described below.
(1) indirect method and sandwich method
The qualitative results of such reactions can be judged by the naked eye.
Visual specimens were also colorless or close to colorless and were negative, with clear and clear positive.
However, in ELSIA, normal serum reaction can often appear in a color background, the depth of this background is different from the composition of the reagent and the conditions of the experiment, so the negative control must be tested in the experiment.
The composition of the negative control should be the normal serum or analogue without the subject matter.
When judging the result with the naked eye, it is more appropriate to use the color depth in negative control as the indicator of positive specimen.
The visual approach is straightforward, but subjective.
Under the conditions permit, a colorimeter should be used to determine the absorption value so that the objective data can be obtained.
Read the sample (S), positive control (P), and negative control (N), then calculate.
There are many kinds of calculation methods, which can be divided into two categories: positive judgment value method and specimen and negative control ratio.
A.Positive judgment value
The positive judgment value (cut-off value) is generally negative and A value plus A specific constant as the standard for positive or negative results.
Asking experimental conditions are constant, with this method the judgment reagent preparation should be standardized, the positive and negative reference substance should comply with the specifications, with sophisticated equipment, operating strictly in accordance with regulations.
The constant in the positive value formula is obtained by the test of a large number of specimens in this particular system.
For example, an example of HBsAg is tested.
The negative control in the kit was found to be free of HBsAg and the content of HBsAg was indicated as P = 9 + 2ng/ml.
Two positive controls and three negative controls were established for each test.
Calculate A measured value and the negative contrast A value, the average number of X (NC) and positive control A value of the average (PCX), two of the average difference (P - N) must be greater than A certain value (0.400), the test is valid.
Three negative control A values should be greater than 0.5 x NCX, and less than 1.5 x NCX, if one of them exceeded this range, then the NCX was recomputed with two negative controls;
If two negative control A values exceed the above range, the subexperiment is invalid.
The positive judgment value is calculated according to the following formula:
Positive value = NCX + 0.05
The value of the sample A was positive for > positive and negative for the positive value.
It should be noted that the constant of 0.05 in the formula is suitable for this particular condition, not for all kinds of reagents.
According to the above description, it can be seen that negative control and positive control in this method also play a role in the quality control of the test, and the reagent deterioration and improper operation will result in the result of "invalid test".
B. Specimen/negative control ratio
This method is suitable in the case that the experimental conditions (including reagents) are not constant.
After the sample (S) and negative control (N) are obtained, the S/N value is calculated.
There is also writing P/N, where P is not positive, but patient's abbreviation, should not be misunderstood.
To avoid confusion, S/N is more appropriate.
In the early indirect method ELISA, some authors have established the S/N as the positive standard, and now many of them are used in various measurements.
In fact, each measurement system should use the experiment to find the respective S/N threshold.
It should be noted that the negative control represented by N is the serum of the person who does not contain the substance.
The negative control in some of the kit contains a buffer that contains no protein or protein content, so that the underlying background may be much lower than that of normal serum.
Therefore, this type of kit, such as N < 0.05 < > (or other numerical value), is calculated at 0.05, otherwise false positive results will be presented.
(2) competition law
In the competition method ELISA, the negative pore is darker than the positive hole.
The intensity of negative color is determined by the concentration of the enzyme binding in the reaction and the amount of the competition suppressor. Generally, the absorbance of negative control is between 1.0 and 1.5, and the reaction is most sensitive.
It is not easy to judge the results of the competition law ELISA, because it is difficult for the naked eye to discern the difference between the weak positive reaction and the negative control, and the absorbance value of S, P and N is usually measured by the colorimeter.
There are two main kinds of calculation methods: positive judgment value method and inhibition rate method.
A. Positive judgment value method
The positive value method in the indirect method and sandwich method is basically the same, but the positive control A value is introduced in the calculation formula, and some test kits for testing anti-hbc are used as an example.
The negative control in the kit was not included in the plasma of the anti-hbc compound calcium, and the anti-hbc content in the positive control was 125 + or 100u/ml.
Two positive controls and three negative controls were established for each test.
Calculate A measured value and the negative contrast A average value of X (NC) and positive control A value of the average (PCX), two of the average difference (N - P) must be greater than A certain value (e.g., 0.300), the test is valid.
The three negative control A values should be less than 2.000, and should be greater than 0.5 x NCX and less than 1.5 x NCX. If one of them is out of this range, it will be discarded, and the other two negative control will be calculated.
If two negative control A exceeds the above range, the subexperiment shall be invalid.
The positive judgment value is calculated according to the following formula:
Negative judgment value = 0.4xncx + 0.6 * PCX
The response of the sample A was positive and the reaction of A > positive was negative.
B. Inhibitory rate method
The inhibition rate indicates the degree of inhibition of the color of the specimen in the competition in combination with the negative reaction, and the following calculation:
Inhibition rate (%) = (negative control A value - specimen A) x 100% / negative control A
In general, the inhibition rate is more than 50% positive, < 50% negative.
The quantitative determination of
ELSIA procedure complex, the factors affecting reaction is more, especially in the solid phase carrier bag be killed in a disaster to achieve consistent among individuals, therefore, in the quantitative determination of each batch test must be made with a series of different concentrations of reference standard under the condition of same production standard curve.
Clip art ELISA, large molecular weight materials are generally wide range of standard curve, the curve peak absorbance can be closer to 2.0, draw semi-log paper, often used to detect the concentration of content for the abscissa and ordinate for absorbance, connect the value of each concentration point by point, the general in an s-shaped curve shape, its head and tail curve tends to smooth, the straight part of the detection area is the most ideal.
Determination of the common competition law of small molecular weight substances, the absorbance of the standard curve is negatively correlated with the concentration of the tested substance.
The shape of the standard curve varies slightly depending on the pattern of the kit.
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