We then proceed to the point of attention to the various operational steps of the plate-type ELISA
Heat preservation
In ELISA, there are usually two antigen antibody responses, which are the combination of the specimen and the enzyme.
The completion of antigen antibody response requires a certain amount of temperature and time, which is called incubation, which is called incubation.
ELISA is a solid phase immunoassay, and the binding of antigens and antibodies only occurs on the solid phase surface.
Antibody package is clip art, for example, to join the board hole in the specimen, the antigen is not have equal and solid phase combination of opportunity, only the most close to the hole wall antigen direct contact with the antibodies in a layer of solution.
This is a gradual balancing process, so it needs to be spread to reach the end of the reaction.
The same is true for the binding of the enzyme marker antibody and the solid antigen.
That's why ELISA always takes a certain amount of time to warm up.
Incubate commonly used temperature 43 ℃ and 37 ℃ and room temperature and 4 ℃ (refrigerator temperature), etc.
37 ℃ is the commonly used thermal insulation temperature in laboratory, and most of the antigen antibody combined with appropriate temperature.
When establishing ELISA methods reaction kinetics research, two antigen-antibody reaction in 37 ℃ after 1 to 2 hours, the generation of product can reach the top.
To accelerate the reaction, can raise the temperature of the reaction, some experiment was carried out in 43 ℃, but should not be used at higher temperatures.
Antigen-antibody reaction 4 ℃ more thoroughly, more make reaction in radiation immunoassay in the refrigerator overnight, in order to form the most precipitation.
But because it takes too long, it is generally not adopted in ELISA.
In addition to some ELISA instruments with special electric heating blocks, the thermal insulation can be used in water bath. The ELISA board can be placed in the water bath box, and the base of the ELISA board should be attached to the water surface, so that the temperature can be quickly balanced.
In order to avoid evaporation, the board should be covered with cover, or plastic cover paper or plastic film cover plate hole, which can let the reaction board float on the water.
When using the insulated box, the ELISA plate should be placed in the wet box. The wet box should be used for the heat transfer of good materials such as metal, the wet gauze at the bottom of the box, and finally the ELISA board on the wet gauze.
The wet box should be placed in the incubator to warm to the specified temperature, especially when the temperature is low.
In order to avoid the error of reading errors caused by moisture or abrasion at the bottom of the enzyme plate, it is generally used to keep warm air oven insulation. This process must be put on the top of the plate to avoid evaporation.
Whether it is water bath or wet box, the reaction plate should not be stacked to ensure that the temperature of each plate can be balanced rapidly.
Room [) reaction, operating room temperature should be strictly limited within the prescribed scope, standard at room temperature is 20 to 25 ℃, but specific operation is controlled according to the requirement of the specifications to incubate.
When the room is warm and warm, the ELISA board is only flat on the operating table.
It should be noted that the temperature and time of the temperature should be accurate according to the regulations.
In order to ensure the accuracy of time, one should not operate more than two boards at one time.
washing
Washing is not a step in the process of ELISA, but it also determines the success or failure of the experiment.
ELSIA is the purpose of separating free and binding enzyme markers by washing.
In the process of washing, the removal of substances that have not been able to bind to solid antigen or antibody in the plate hole, as well as the interfering substances that are specifically adsorbed to the solid carrier during the reaction process.
The adsorption of polystyrene and other plastics on protein is universal, and the non-specific adsorption of interference material should be washed in the wash.
It can be said that in the ELISA operation, washing is the most important key technology, should arouse the operator's high attention, the operator should be strictly according to the request, not sloppiness.
In addition to some ELISA instruments, some ELISA instruments are equipped with special automatic washing machines. There are two kinds of manual operation: soaking and running water. The process is as follows:
(1) immersion type:
A. Dry or dry pore reaction fluid;
B. Rinse the liquid with detergent (after filling the plate with the washing liquid);
C. Soak, the washing liquid will be filled with plate holes, placed 1-2min, intermittent shaking, and the soaking time should not be shortened.
D. Dry pore fluid.
Suction dry should be thoroughly, can be used water pump or vacuum pump suction, also can dump the liquid to pat dry on clean towel or absorbent paper;
E. Repeat operation c and d, washing 3-4 times (as specified).
In indirect method, if the background is higher, it can increase the number of washing times or lengthen the soaking time.
The trace titration board adopts immersion washing method.
Liquid detergent is a neutral buffer containing non-ionic detergent.
Polystyrene carrier with the combination of the protein is hydrophobic, nonionic detergents contain hydrophobic groups, both also contain hydrophilic group, the hydrophobic groups and protein hydrophobic groups by hydrophobic bond, thus weakening the protein and the combination of the solid phase carrier, and by using the combination of hydrophilic group and water molecules, make the protein to return to the aqueous solution, which from the solid phase carrier.
The non-ionic detergent in washing liquid is usually twenty20, and its concentration can be between 0.05% and 0.2%, which is higher than 0.2%, which can reduce the sensitivity of the test by desorption of antigen or antibody in the solid phase.
(2) running flush: water flushing method is originally used for the washing of small beads, and the cleaning solution is only distilled water and even available running water.
A special device is attached to the washing, which will keep the beads rolling and rinsing continuously under the impact of flowing water. After continuous washing for 2min, soak dry liquid and soak in distilled water for 2min.
Immerse is like bath, running water is like shower, its washing effect is more thorough, and also simple, quick.
The experimental results show that the washing process of water flow is also applicable to trace titration plate.
When washing, try to increase the water flow or increase water pressure, let flow impact plate hole surface, wash effect better (this method kit is less used).
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