Every step of ELISA Kit experiment is particularly important, and the details can not be ignored. It is the key to the success of the experiment; In order to better complete the experiment, the following analysis and summary are made on the taboos in the operation steps of highly sensitive ELISA Kit:

1. It is not easy to absorb liquid too fast to avoid bubbles, and the amount absorbed is not accurate enough.

2. When washing the plate manually, add the washing solution every time. It should stand for 15 ~ 30s, and do not splash the washing solution in one enzyme label l into another enzyme label hole to prevent cross contamination. After throwing away the washing solution, put the enzyme label plate on a towel or absorbent paper and pat it dry.

3. The substrate is TMB and avoid contact with skin. The concentrated sulfuric acid with 2tool as the termination solution is corrosive, and contact with the skin should be avoided as far as possible.

4. After adding samples, put them into the incubator in time. When there are many samples, operate them in batches. Strictly control the operation time according to the described steps to prevent the artificial extension of incubation time, resulting in non-specific binding tightly around the reaction hole, which is difficult to clean thoroughly.

5. Wear gloves and work clothes when working, and strictly improve and implement the disinfection and isolation system.

6. The remaining samples and wastes shall be sterilized by 121 ℃ high-pressure steam for 30 minutes, or treated with 5.0g/l sodium hypochlorite and other disinfectants for 30 minutes.

7. Reagent components of the same batch number shall not be mixed.

8. When washing, each hole shall be filled with liquid to prevent the hole from being washed with free enzyme.

9. One hole is reserved for each experiment as a blank zero adjustment hole, which does not add any reagent, but finally adds substrate solution and 2n H2SO4. Use this hole to adjust the OD value to zero during measurement.

10. To ensure the accuracy of the micro sampler, distilled water and electronic balance can be used for determination (since the distilled water does not contain impurities, when the temperature environment is about 20%, LML of water can be regarded as LG and 200 L of water can be regarded as 0.2g). The highest and lowest ranges of each micro sampler should be determined.

11. When using the micro sampler, replace the gun head after absorbing the liquid in different bottles, even if absorbing the standard solution.

12. The inspection technician shall have the basic quality and practical experience in laboratory operation. Be able to skillfully operate experimental instruments and instruments, have the ability to summarize and analyze problems, and timely and properly solve unexpected situations in the experiment.

13. Read the manual carefully before operation and carry out standardized operation in strict accordance with the requirements of the manual. Reagents of different batches cannot be mixed. The areas worthy of improvement can only be improved after repeated tests are established, such as mastering the number of plate washing, etc.

14. It is very important to evaluate the practicability and stability of the reagent for the accuracy. Before the reagent is used, the negative and positive control and repeated comparison tests of samples shall be carried out to determine that the reagent meets the requirements before use.

15. Add samples accurately and quickly. Inaccurate sample addition and uncertain amount of enzyme products directly affect the color development results. In addition, the depth of color development and the determination of a value are related to the amount of color developer and termination solution, so sampling should be careful. For experiments requiring the completion of sample addition within a certain time, if the sample addition is slow, there will be errors, and the reagent will be exposed for a long time, especially when the room temperature is too high, the storage period will be shortened or even invalid.

16. Use calibrated micropipette to eliminate natural errors. Whether the pipette is accurate or not is particularly important for quantitative detection

17. Strictly control the color development time. If the color development time is too short, the amount of substrate conjugate is too small after adding the termination solution and the reaction is terminated, which is prone to false negative. If the color is developed after the required time for color development, it shall be judged as false positive, which may be related to the reagent itself. Color development shall be carried out immediately after adding color developing agent, and no positive result shall be reported, which may be the result of background color development.

18. If the washing is complete and the washing plate is not complete, the background color of enzyme conjugate will show false positive. In addition, the washing liquid should be fresh and ready for use. The background of old washing liquid will increase, and false positive may also occur.

19. Strictly control the reaction time. If the reaction time is too long, the enzyme will be inactivated; The reaction time is too short, the enzyme conjugate can not fully bind with the microbial antigen antibody in the serum, and the structure of the product is loose and unstable, which is easy to wash off, which may cause false negative.

20. The operation steps of ELISA are complex, and there are many factors that may affect the determination results, which are distributed in each step of the determination operation. Therefore, the quality control of each link must be strengthened in order to obtain accurate and reliable detection results.