Key points for drawing ELISA standard curve

Many reagent tests involve the problem of the standard curve. The good or bad of the standard curve will directly affect the results of the experiment, or even the success or failure of the experiment. So how do you draw or make a standard curve?

First, there are several problems that need to be paid attention to when testing standard curve samples.

1. The concentration of the sample and other indicators are calculated according to the standard curve, so it is important to consider the standard curve as a more important thing than the formal experiment. Otherwise, the results of the latter experiment cannot be discussed.

2. Set the standard concentration range of the standard curve sample to have a relatively large span, and to cover the concentration of the test sample you want to test, that is, the concentration of the sample should be within the concentration range of the standard curve, including the upper and lower limits. For the standard curve of S type, try to make the concentration of the experimental sample in the steepest section of the middle slope, that is, the curve is almost in a straight line.

3. It is better to prepare the standard sample concentration in the standard curve by the double dilution method, so as to ensure that the concentration of the standard sample does not deviate greatly.

4. When testing standard samples, they should be carried out in increasing order of concentration to reduce the effect of high concentration on low concentration and improve accuracy.

5. The number of samples in the standard curve is generally 7 points, but at least 5 points must be guaranteed.

6. The correlation coefficient of the standard curve made varies according to the experimental requirements, but in general, the correlation coefficient R is at least greater than 0.98. For some experiments, at least 0.99 or even 0.999.

Second, choose what equation to fit

The so-called "standard curve" used for immunoassay is actually called a fitting curve. The standard point of immunoassay (may be diluted or not). If the concentration and the corresponding absorbance (OD) value can be linear, it is of course ideal. At this time, it can be easily obtained by EXELL. The curve, in turn, derives the concentration value of the sample. However, we do not have the ideal situation for immunoassay. The concentration of the target and the corresponding OD value are often the relationship of the "S" type curve. At this time, the straight line fitting method cannot be used, and the fitting method is used. Making a choice is necessary.

Regarding the fitting method of the standard curve, straight lines, quadratic curves, cubic curves, indices, logarithms, etc. can be used for curve fitting in ELISA and other biological reactions, but they are only applicable to a part of the curve, and some are applicable. In the first half, some apply to the second half, some apply to the middle section, and the Logistic curve has good applicability to all of the curves. Of course, if used for quantification, it is better in the middle. Although none of these methods can be used universally, they can all be used. The key is which part of the S shape your calibration curve does, and which part of the curve you want to test. The low concentration part of the S curve can be well fitted by the power equation, the linear equation can be used for the middle and low concentration parts, the logarithmic equation can be used for the middle part, and the four parameters can be used for the middle and rear sections.

At present, the most popular immunological detection fitting method in the world is “four-parameter logistic fitting”. This kind of fitting method can often accurately compare the relationship between concentration and absorbance, so as to further obtain the correct test substance in the sample. Concentration value.

In fact, within a long interval, Logistic should be able to fit well. But it is not that it is omnipotent. In fact, not only ELISA, but many other biological reactions are sigmoidal curves, and can also be fitted with Logistic curves. But building a model is one thing, and using it to quantify is another matter. If used for quantification, the middle section of the S-shaped curve (shorter) is better, and the flat part of the two ends will have a large calculation error, sometimes even large.

Shanghai Hengyuan Biotechnology Co., Ltd. is a professional manufacturer, supplier and agent of ELSIA kits. The total number of ELSIA kits produced by our company has exceeded 10,000. It has been tested for major universities, hospitals and scientific research units. The people's praise, the customer's trust in our products, gave us a positive and enterprising momentum, we will better do a good job, better provide technical services, better serve China's scientific research.

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