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Indirect ELISA procedure

2021-08-03
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Indirect ELISA procedure-Experimental materials

1. 96-well microplate

2. TMB

Experimental reagents

1. Encapsulation solution (pH9.6) : Na2CO3 0.17g, NaHCO3 0.28g, add distilled water to 100ml.4 ℃.

2. Washing solution (PBST): KH2PO4 0.2g, KCl 0.2g, NaCl 8.0g, Na2HPO4·12H2O 2.9g, Tween-20 0.5mL, add deionized water to 1000mL, adjust pH to 7.2-7.4.

3. Substrate reaction liquid:

(1) Substrate buffer: 0.51g citric acid, 1.84g Na2HPO4·12H2O, add distilled water to 100ml, fully dissolve it, and store at 4°C for later use.

(2) Substrate reaction liquid: A. OPD color: say 5mg OPD, add 10ml substrate buffer in the dark, and generally match and use, using 5ml+8ul 30%H2O2.B. TMB color: Pre-configured 10mg TMB/1ml DMSO solution 5mL, protected from light at 4℃, used 9.9 mL substrate buffer +100ul TMB/DMSO solution +10ul 3%H2O2 to avoid light, available) 4.Sheep anti-mouse igg-hrp

Experimental steps

(1) Coating: Dilute the antigen with the coating solution to a suitable coating concentration, calculate the amount of coating solution required according to the required wells, add 100ul to each well, act at 37°C for 1 hour and then 4°C overnight. This step is to link the specific antigen with the solid phase carrier.

(2) Blocking: the next day, take out the microtiter plate and fill each well with PBST, wash 3 times for 5 minutes each time, and pat dry each time. Then add 200ul of blocking solution (1% BSA or 5% skimmed milk) to each well, incubate at 37°C for 2 hours, and wash 3 times with PBST.

(3) Add primary antibody: dilute the corresponding primary antibody in a certain proportion and add 100ul of diluent to each well.Negative control and blank control were set, treated at 37℃ for 1h, and washed with PBST for 3 times.This step is to dilute the tested serum, so that the specific antibody in the serum and solid phase antigen binding, the formation of solid phase antigen antibody complex, after washing, solid phase carrier only left specific antibody, other immunoglobulin and impurities in the serum can not be combined with solid phase antigen, in the washing process is washed away.

(4) Add secondary antibody: dilute the corresponding secondary antibody at 1:500, 100ul per well, apply at 37℃ for 1h, and wash with PBST for 3 times.The purpose of this step is to add hrP-labeled anti-immunoglobulin (HRP-labeled anti-antibody), which binds to the primary antibody so that the antibody is indirectly labeled with the enzyme. After cleaning, the amount of enzyme on the solid phase carrier represents the amount of specific antibody.

(5) Add substrate: add 100ul of substrate reaction solution (for use in situ, away from light) to each well and leave it at 37℃ for 30min, then take it out and add 2mol/L H2SO4 to stop the reaction.The substrate was added for color, and the color depth represented the amount of antibody tested in the specimen.

(6) A450 was measured by microplate reader.

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