How to make the ELISA system more stable--Shanghai Yuanmu Biological Detailed! -Huaqiang Electronic Network

How to make the ELISA system more stable

The ELISA detection system is the most widely used and sensitive technology for the application of immunological reactions to scientific research. However, there are always big or small problems in the operation of the new veteran. I have faced many difficulties when I started the ELISA. Let me give you a glimpse of the experience of doing ELISA.

1. The nature of the original coating is very important. Whether the protein concentration is degraded or not depends on whether the antibody you made can be recognized by it. Therefore, it is very important to preserve the antigen. When I am doing recombinant protein, my brothers strictly warn me that I must be The slow melting under the ice bath is the truth. There are also some coatings that may not be proteins. For biotin and lipids or small molecules, we have to modify and then apply them. I will briefly list the methods as follows: 1 Avidin Biotin: First Avidin is first coated with a carrier, and biotinylated DNA is added. This coating method is uniform and firm, and has been expanded to be used for quantitative determination of various antigenic substances. 2 lipid substances: can be dissolved in an organic solvent (such as ethanol), added to the ELISA plate wells, open the lid to the refrigerator overnight or cold air to dry, after the alcohol is evaporated, let the lipids naturally dry on the solid surface. 3 small molecules must be coupled to a large protein carrier to be immobilized on a solid support.

2, the choice of coating solution, generally choose ph9.6 carbonate buffer. However, sometimes due to the needs of the test, the specificity of the original coating may also be packaged with a neutral buffer solution. Attention should be paid to the following principle: Since the protein and the polystyrene solid phase carrier are bound by physical adsorption, the interaction between the hydrophobic group on the structure of the protein molecule and the hydrophobic group on the surface of the solid phase carrier is Non-specific, affected by the molecular weight, isoelectric point, concentration, etc. of the protein, the small molecular protein of the macromolecular protein usually contains more hydrophobic groups, so it is more easily adsorbed onto the surface of the solid support. The specific test must be practiced in addition to a solid theory. If you look at it, you can finally apply it to your own experiments. Commonly used coating solutions include pH 7.2 phosphate buffer and pH 7-8 Tris-HCL buffer, etc., in addition to the pH 9.6 carbonate buffer just mentioned.

3. Blocking: The process of recoating with a high concentration of unrelated protein solution after coating. Blocking is the filling of these voids with a large number of unrelated proteins, thereby re-absorbing the re-adsorption of interfering substances in the steps following the ELISA. Commonly used blocking agents are: 0.05%-0.5% A; 10% calf serum or 1% gelatin; skim milk powder, relatively inexpensive, can be used at high concentration (5%-10%); there are some rare useful Various animal serums (mainly to rule out similar protein interference) and casein and the like. But what to choose in the end, according to the specific test of practice.

4, washing plate: It can be said that in the ELISA operation, washing is the most important key technology. Because the adsorption of proteins by plastics such as polystyrene is universal, in order to achieve separation of free and bound enzyme labels, the free substances remaining in the pores of the plates, as well as non-specifically adsorbed interfering substances, are washed. This non-specifically adsorbed interfering substance should be washed away. Therefore, there will be some errors in the washing of the plate, the human factor is very large (except of the conditional use of the washing machine), the washing is not complete or stringed holes, which is not a small impact on such a sensitive ELISA system.

5, add antibody specimens (and secondary antibodies): pay attention to the change of the gun head when changing the gun head. Specimen dilution can generally be diluted, or diluted with blocking solution. If you need to add a secondary antibody, you should also pay attention to the working concentration of the secondary antibody, too high waste, too low to light color.

6, color: a lot of color system, we must choose the appropriate color system when we start. Note the preservation of the enzyme-active substrate of the chromogenic system. The HRP conjugate plus the sulphuric willow pump, the AP conjugate can be added with sodium azide.

7, every time do as much as possible to do the three comparisons of yin and yang, if there is a problem, analyze it.

related information:

1. Q: The indirect Elisa method was used to test the IgE antibody in the serum of mice. The blank control and the negative control were used. The dilution ratio of the positive serum was 5,000, 10,000, 100,000, 200,000, and the biotin label was used. The goat anti-mouse IgE antibody, and the avidin horseradish peroxidase. However, except for the blank control hole, there is no color, and the other holes are all colored, and the OD values ​​are not much different. Why?

Answer: The possible reasons are as follows:

(1) Water quality is contaminated by metal ions; prevent fresh water or distilled water as much as possible.

(2) Insufficient washing, residual components or residual enzymes in the sample; prevention method Washing solution should be filled with micropores and washed thoroughly.

(3) The pipetting head is reused, not washed or incompletely disinfected; the preventive method is preferably used once.

(4) The sensitivity of the plate is too high.

(5) Control of the color development time of the primary antibody, etc., pay attention to each step of the ELISA.

2. Small error prevention experience when ELISA is added

(1) Use a piece of paper full of words. The more words, the better, such as newspapers, padded underneath, so that the small holes that have been added to the specimen will look smaller and the words below will become smaller. distinguish.

(2) It can be distinguished by liquid level reflection and no added hole

(3) After the specimen is added, the gun is pressed down to make the liquid surface produce small bubbles, which can also be distinguished.

3. The operation method of the checkerboard test:

(1) After the antigen is diluted at a certain gradient ratio, it is coated in the order of ELISA from top to bottom (or from left to right).

(2) The antibody was diluted at a certain gradient and then added from left to right or top to bottom according to ELISA.

(3) The dilution factor of the secondary antibody is constant, then the color is developed and the value is read.

When measuring the OD value, the wavelength depends on the color developer. Generally, everyone uses TMB color development and 450 nm reading. According to the results of the reading, the appropriate antigen and antibody titer can be selected. This is the purpose of the experiment. If the antigen coating amount is known and the working concentration of the secondary antibody is uncertain, the primary antibody and the secondary antibody are tested.