In the Elisa kit test, the nature of the coating is important, the protein concentration, whether it is degraded, which is related to whether the antibody you make can be recognized by it, so it is important to retain the antigen. When doing recombinant protein, it is necessary to be in the ice. The slow melting under the bath is the reason. The blockade is to allow a large amount of unrelated protein to fill these sputum vacancies, thereby rejecting the re-adsorption of interfering substances in the Elas step. However, sometimes due to the needs of the test, the specificity of the original coating may also be packaged with a neutral buffer solution.
The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The method is suitable for the determination of samples in cell culture supernatants, serum, plasma and tissue fluids, and the level of cytokines (or receptors) per milligram of nanograms per milligram can be measured with little interference.
Operational attention:
1. The kit is stored at 2-8 ° C and equilibrated for 20 minutes at room temperature before use. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use.
2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.
3. Carry out the incubation operation in strict accordance with the time indicated in the instruction manual, the amount of liquid added and the order.
4. Shake well all liquid components before use.
Prepare the reagents required for the experiment as required by the kit instructions. Distilled or deionized water used in ELISA, including for washing, should be fresh and of high quality. The self-contained buffer is corrected 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.
Preparation of the kit: For each cytokine, the instructions should be read carefully, paying attention to the details of each batch. Immediately centrifuge the reagent bottle and recapture the cytokines as much as possible before using the cytokine. Lyophilized cytokines were reconstituted according to the details in each batch of instructions. Generally, the linear range of the antigen standard curve to be tested can be from 2000 pg/ml to 15 pg/ml by 8 times serial dilution of the standard. Sensitivity can be increased to a certain extent using standard ELISA procedures, amplification kits, reagents of the third type, or changes to the enzyme substrate system. To optimize sensitivity, it is recommended to incubate standards and specimens. If peroxidase is used as the color development system, it is strictly forbidden to add sodium azide to the washing solution and the diluent. The sodium azide can inhibit the activity of the enzyme of the ELISA kit.
The operation of the kit differs depending on the formation of the solid phase carrier, and domestic medical tests generally use plate points.
Washing method:
1 Wash the plate by hand, suck (not touch the wall) or pry off the liquid in the plate; pour a few layers of absorbent paper on the test bench, and take the plate with the plate down several times; the recommended washing buffer is at least 0.3 Mol was injected into the wells, soaked for 1-2 minutes, and the process was repeated several times as needed.
2 Automatic washing, if there is an automatic washing machine, it should be used in the formal experiment process after skilled use.
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