Analysis of common problems in experimental operation by ELISA kit(2)

(3) Impact of operational technology

Control of the operation process:

1 Operate in strict accordance with the reagent instructions. The reagent was equilibrated at room temperature for 30 to 60 minutes before the operation.

2 Put the incubator in time after adding the sample. When there are many specimens, it is necessary to operate in batches. According to the instructions, the operation time is strictly controlled to prevent the incubation time from being artificially prolonged, resulting in non-specific binding surrounding the   reaction well,   which is difficult to clean thoroughly.

When the 3 sheets are incubated, the holes must be sealed to prevent the liquid of the positive specimen from evaporating, resulting in peripheral phenomena leading to the appearance of “flower plates”.

4 When washing the board with the washing machine, ensure that the washing liquid fills the holes, and the washing needle is unblocked. After washing the board, the Zui is patted dry on the absorbent paper (choose the clean, no or little dust-absorbing material): To prevent fibrin or foreign matter in the needle mouth, these foreign materials are prone to towing during the process of washing the plate, resulting in the appearance of "flower board".

5 Reasonably arrange the amount of detection to avoid long waiting time for washing the plate.

6 After adding the enzyme reagent, use a blotting paper to gently blot and dry on the surface of the microplate.

7 The color developer should be prepared as far as possible before use. It is not necessary to use the expired color developer, and the light blue TMB color developer can be seen by the naked eye.

8 Keep the developer from flowing out when loading the sample; A and B should avoid contact with the metal device. Avoid air bubbles when adding stop solution.

9 should ensure that C is clean, and the enzyme label is not exposed to hypochlorous acid during the whole operation. The above measures can reduce the "flower board" to the low limit of Zui. Thereby improving the specificity of the detection. And get more accurate and reliable experimental results.

The cleanliness of the sample suction nozzle and the accuracy of the suction directly affect the test results. Due to the special construction of the nozzle, it is difficult to clean and increase the chance of cross-contamination. A disposable nozzle is recommended. The sampler should also be cleaned frequently and calibrated regularly. Add the added substance to the bottom of the ELISA plate hole when loading, avoid adding it to the upper part of the hole wall, and be careful not to spill.

The influence of the warm bath, when the ELISA method was established for the reaction kinetics study, the experiment showed that the two antigen-antibody reactions generally reached the peak at 37 ° C for 1-2 hours. In order to avoid evaporation, the board should be covered, and the plate holes can be covered with plastic sealing paper or plastic wrap. The reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. It should be noted that the temperature and time of incubation should be as accurate as required. Since the company's kit incubation is done in an air bath, using a water bath can result in high values or flower plates. In addition, there is an edge effect in the incubation, and the value on the side will be too high. It is recommended to put the quality control at the non-edge position for the objective judgment result. The 96-well microtiter plate has a special structure; it is easy to produce edge effect, antigen-antibody binding and enzymatic reaction have strict temperature requirements, and the temperature rise and fall of the inner and inner pores of the microplate are different, resulting in differences in peripheral and internal pore results; dry bath and water bath There are significant differences, using a water bath as much as possible, and requiring the solid phase plate to be placed in the water to reduce uneven heating, sealing the film and preventing dirt from entering.

Washing is not a reaction step in the ELISA process, but it determines the success or failure of the experiment. ELSIA relies on washing to achieve the goal of separating free and bound enzyme labels. The substance remaining in the pores of the plate which is not capable of binding to the solid phase antigen or antibody, and the interfering substance which is non-specifically adsorbed to the solid phase carrier during the reaction are removed by washing.

The adsorption of proteins by plastics such as polystyrene is universal, and such non-specifically adsorbed interfering substances   should be washed away during   washing. It can be said that in the ELISA operation, washing is the main key technology of Zui, which should be highly valued by the operator. The operator should wash it strictly according to requirements, and should not be sloppy.

The washing solution is mostly a neutral buffer containing a nonionic detergent, and the lotion of the various kits should not be mixed. The binding of the polystyrene carrier to the protein is hydrophobic. The nonionic detergent contains both a hydrophobic group and a hydrophilic group, and the hydrophobic group binds to the hydrophobic group of the protein through a hydrophobic bond, thereby weakening the protein and The solid phase carrier is combined, and by the combination of the hydrophilic group and the water molecule, the protein is returned to the aqueous solution state, thereby being separated from the solid phase carrier. The non-ionic detergent in the washing liquid is generally Tween 20, and its concentration may be between 0.05% and 0.2%. When the concentration is higher   than   0.2%, the antigen or antibody coated on the solid phase may be desorbed to reduce the test. Sensitivity. The lotion needs to be diluted and should be diluted as required. The preparation of the lotion uses fresh and high-quality purified water with a conductivity of less than 1.5 μs/cm. The washing solution should be   prepared   if it is crystallized. Hand washing   conditions are inconsistent and   have a great influence on the results,   preventing the washing liquid from   forming bubbles in   the pores. Improper use of semi-automatic and fully automatic boring machines will also affect the results.

Crystals remaining in the serum fibrin or washing liquid in the serum may cause the pores of the washing machine to be completely blocked or semi-blocked,   resulting in unbound labeling enzyme elution. Incomplete, causing the "flower board" to cause false positives or false negatives; therefore, during the operation of the washing machine, the patency of the washing liquid in the needle hole of the washing machine should be observed from time to time, and corrected in time, and the deionized water should be used when the washing machine is not used. Hey a few times.