Can make your ELISA kit better than expected - Database & Sql Blog Articles

Probe domestic double-head probe 038-BB-5.7L The shape of the two ends of the needle is outside the pointed needle

EL-C1600N100013-B

3 ways to make your elisa kit better than expected

1, reagent preparation

Prepare the reagents required for the experiment as required by the ELISA kit instructions. Distilled or deionized water used in ELISA, including for washing, should be fresh and of high quality. The self-contained buffer is measured using a pH meter. The test reagent taken out of the refrigerator should be used after the temperature and room temperature are balanced. The parts of the kit that are not needed for this test should be returned to the refrigerator in time for storage.

2, washing

Washing is not a reaction step in the ELISA process, but it also determines the success or failure of the experiment. The ELISA kit relies on washing to achieve separation of 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 most important key technology, which should be highly valued by the operator. The operator should wash it strictly according to requirements, and should not be sloppy.

Washing method In addition to some special ELISA instruments equipped with special automatic washing instruments, there are two types of manual operation: immersion and running water. The process is as follows:

(1) Immersion type a. Drain or dry the reaction solution in the well; b. Wash once with the washing liquid (after the washing liquid is filled into the plate hole, it is smashed); c. Soak, the washing liquid is filled Hole, place for 1-2 minutes, intermittent shaking, soaking time can not be shortened arbitrarily; d. suck the liquid in the hole. Drain dry thoroughly, pump with a water pump or vacuum pump, or pat dry on a clean towel or absorbent paper after removing the liquid; e. Repeat operations c and d, wash 3-4 times (or as specified). In the indirect method, if the background is higher, the number of washings or the soaking time can be increased.

Microtiter plates are mostly immersed. The washing solution is mostly a neutral buffer containing a nonionic detergent. 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 the concentration thereof may be between 0.05% and 0.2%. When the temperature is higher than 0.2%, the antigen or antibody coated on the solid phase may be desorbed to reduce the test. Sensitivity.

(2) The running water rinse type water washing method is originally used for the washing of the bead carrier, and the washing liquid is only distilled water or even tap water. A special device is attached during washing to continuously roll and wash the beads under the impact of running water. After rinsing for 2 minutes, the liquid is absorbed, and then immersed in distilled water for 2 minutes, and dried. The immersion is like a bath, and the running water is like a shower. The washing effect is more thorough and simple and fast. It has been shown in experiments that the flow rinse method is also suitable for the washing of microtiter plates. Try to increase the water flow or increase the water pressure during washing, so that the water flow impacts the surface of the plate hole, and the washing effect is better.

3, insulation

There are typically two antigen-antibody reactions in an ELISA, ie, after addition of the sample and addition of the enzyme. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation, which is called incubation and may not be appropriate in ELISA.

ELISA is a solid phase immunoassay in which antigen and antibody binding occurs only on the surface of a solid phase. Taking the sandwich method of antibody coating as an example, the specimen added to the well of the plate does not have an equal opportunity to bind to the solid phase, and only the antigen in the solution closest to the pore wall is directly in contact with the antibody. . This is a process of gradual equilibrium, so it needs to be diffused to reach the end of the reaction. The same applies to the binding of the enzyme-labeled antibody added thereafter to the solid phase antigen. This is why ELISA reactions always require a certain amount of incubation.

The temperatures commonly used for incubation are 43 ° C, 37 ° C, room temperature and 4 ° C (refrigerator temperature). 37 ° C is the usual incubation temperature in the laboratory, and is also the appropriate temperature for most antigen-antibody binding. When the ELISA kit was used to establish the kinetics of the reaction kinetics, the experiment showed that the two antigen-antibody reactions generally reached the peak at 37 ° C for 1-2 hours. In order to accelerate the reaction, the temperature of the reaction can be raised. Some tests are carried out at 43 ° C, but higher temperatures are not suitable. The antigen-antibody reaction was more thorough at 4 ° C, and the reaction was mostly allowed to stand overnight in the refrigerator in a radioimmunoassay to form the most precipitate. However, because the time required is too long, it is generally not used in ELISA.

Insulation method In addition to the special ELISA instrument with special electric heating block, the water bath is generally used. The ELISA plate can be placed in the water bath box. The bottom of the ELISA plate should be attached to the water surface to make the temperature balance quickly. In order to avoid evaporation, the board should be covered, or the plate hole can be covered with plastic sealing paper or plastic wrap. At this time, the reaction plate can float on the water surface. If an incubator is used, the ELISA plate should be placed in a wet box. The wet box should be made of a material with good heat transfer such as metal, a wet gauze at the bottom of the box, and finally the ELISA plate should be placed on the wet gauze. The wet box should be pre-warmed in the incubator to the specified temperature, especially at lower temperatures. Whether it is water bath or wet box incubation, the reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. For the reaction at room temperature, the room temperature during the operation should be strictly limited to the specified range. The standard room temperature refers to 20-25 ° C, but the specific conditions can be controlled according to the requirements of the instructions. When incubated at room temperature, the ELISA plate should be placed flat on the console. It should be noted that the temperature and time of incubation should be as accurate as required. In order to ensure this, one person should not measure more than two plates at the same time.