How to choose the appropriate positive control for ELISA kit?
The basic composition of the positive control should be as close as possible to the composition of the test sample. Generally, the positive control mostly uses a buffer containing a protein protective agent as a matrix, and a certain amount of the substance to be tested is added. For example, for the determination of human serum as a specimen, a positive control usually uses a certain patient serum or a calcium supplemented human plasma as a raw material to add a certain amount of standard. How to choose a suitable negative control? The basic composition of the negative control should also be as consistent as possible with the composition of the test sample. It is best to test first to make sure that it does not contain the substance to be tested. For example, when testing ELISA kits for human serum specimens, the negative control should select normal human sera; when detecting antibody titers in the sera of immunized animals, the negative controls should select the pre-immune sera of the animals.
How to choose the optimal coating conditions?
1. Selection of coating antigen
The coating antigen can be divided into three categories: natural protein, recombinant protein and small molecule antigen. Natural proteins need to be purified before they can be coated by direct adsorption. Indirect capture methods can be used for antigens with a lot of impurities (firstly, substances that can react with the target antigen, such as antibodies, are directly adsorbed on the enzyme plate, and then passed specific The sexual reaction solidifies the antigen). The purified recombinant protein can generally be coated directly. Small molecule antigens such as peptides and some small molecule organic compounds, because of their small molecular weight, are often difficult to adsorb directly on the enzyme plate. Generally, they are first coupled to unrelated proteins such as BSA and the conjugate is adsorbed on a solid support .
2. The choice of coating solution is commonly used carbonate buffer pH9.6, if the coated antigen is unstable under alkaline conditions, you can also use phosphate buffer pH7.2.
3. The choice of coating temperature is usually 4-8 degrees overnight or 37 degrees for 2 hours, we strongly recommend coating under 4-8 degrees, which is conducive to the maintenance of protein activity.
4. Selection of coating concentration The optimal concentration of coating varies with the properties of the solid phase carrier and the coating. Generally, the coating concentration of protein is 1-5ug / ml, and the optimal coating for a specific coating antigen should be clearly defined The concentration needs to be determined by experiment. Do I need to block after coating? What kind of blocking system should I choose? Whether blocking is necessary depends on the ELISA mode and the specific experimental conditions. Generally speaking, the double antibody sandwich method, as long as the enzyme label is highly active, is washed thoroughly during operation, and satisfactory results can be obtained without blocking. In the indirect method of measurement, closure is generally essential. Commonly used blocking agents are 0.05% -0.5% BSA, 10% calf serum, 1% gelatin, 5% skimmed milk powder, AbMART recommends the use of 5% skimmed milk powder, cheap and strong sealing ability, but 5% skimmed milk powder It is only suitable for short-term use, not for long-term storage, so it is rarely used in ELISA kits.
How to use enzyme conjugate correctly in ELISA kit?
1. Diluting solution of enzyme conjugate A high concentration of irrelevant protein (such as 1% BSA or 5% skimmed milk powder) is often added to the diluent to inhibit the non-specific adsorption of the enzyme conjugate on the solid phase carrier through competition. Generally, non-ionic surfactants that can inhibit protein adsorption on the plastic surface are also added, such as Tween 20 (0.05% concentration is more suitable).
2. Correctly dilute the enzyme conjugate The proper working concentration of the enzyme conjugate needs to be determined through preliminary experiments. Too high a concentration will easily lead to a high background, while too low a concentration will lead to a reduction in the positive signal. The enzyme conjugate is best diluted before use. The diluted enzyme conjugate is not suitable for long-term storage, because the low concentration of enzyme conjugate is easily inactivated.
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