Enzyme linked immunosorbant assay (ELISA)

SOP for testing of serum antibody titer in mice

1、 Principle

      The antigen-antibody-anti-species antibody method is used to indirectly determine the antibody potency in serum samples. First, the antigen is coated on the ELISA plate to form a solid phase antigen, and then the serum sample is added. The antibody in the serum can form a solid-phase antigen-antibody complex with the solid-phase antigen, and then add goat anti-mouse enzyme-labeled antibody. When the substrate is added, a color reaction occurs, and the OD value is measured at the appropriate wave length with a microplate reader.

2、 Preparation before the test

2.1 Coating solution

      0.01 mol/L PBS (add 2.9 g disodium hydrogen phosphate dodecahydrate, 0.297 g sodium dihydrogen phosphate dihydrate and 8.5g sodium chloride, then add purified water to 1000ml do not need to adjust pH, pH is about 7.2).

2.2 Blocking solution

      0.01 mol/L PBS containing 1% BSA (add 10g BSA, 2.9 g disodium hydrogen phosphate dodecahydrate, 0.297g sodium dihydrogen phosphate dehydrate and 8.5 g sodium chloride then add purified water to 1000ml. do not need to adjust pH).

2.3 Sample diluent

      0.01 mol/L PBS containing 1% BSA(add 10g BSA. 2.9 g disodium hydrogen phosphate dodecahydrate,0.297g sodium dihydrogen phosphate dehydrate and 8.5 g sodium chloride then add purified water to 1000ml, do not need to adjust pH).

2.4 Wash solution

      0.01 mol/L PBS containing 0.5% tween 20 (add 2.9 g disodium hydrogen phosphate dodecahydrate, 0.297 g sodium dihydrogen phosphate dehydrate, 8.5 g sodium chloride and 5ml tween 20. then add purified water to 1000ml).

2.5 Enzyme diluent

      Wash solution containing 10% new bovine serum, 1% casein and 0.1% proclin 300.

2.6 Stop solution

      2mol/L sulfuric acid.

2.7 Sheep anti-mouse labelled antibody

      Sheep anti-mouse labelled antibody was purchased (Supplier: KPL) the concentration shall be adjusted according to the different batches (general recommendation-1:10000-1:20000). Currently the concentration used is 1:15000 for dilution.

3、 Test procedure

3.1 Coating

      Dilute the coating antigen with coating solution (0.01 mol/L PBS) at a final protein concentration of 1µg/ml. Mix well and add to the 96-well microplate at 100µL/well, incubate overnight at 2-8℃(16-20 hours), wash the plate for three times, pat to dry.

3.2 Blocking

      Prepare 0.01 mol/L blocking solution containing 1% BSA 200uL/well. incubate at 37℃ for 60-120 minutes or 2-8℃ overnight, store at 2-8℃, use within 7 days.

3.3 Sample adding

     Use sample diluent to perform series dilution of serum sample, the initial dilution factor is 100 times, followed by doubling dilution, loading from 200 folds (can be adjusted,1 well per concentration, 100 µL well, add 1:800 mice serum (can be adjusted) as negative control, 8 wells in parallel, 100 µL/well. Use sample diluent as blank add in 8 wells in parallel incubate at 37℃ for 60-70 minutes. Use washing solution to wash for 3 times, pat to dry.

*The layout of the test product is as follows：(Adjust the initial loading dilution factor according to the situation).

3.4 Add in enzyme-labelled antibody

      Use enzyme diluent to dilute sheep anti-mice enzyme labelled antibody add 100 µL/well incubate at 37°C for 60-70 minutes, wash by washing solution for 5 times pat to dry.

3.5 Coloration

      Add chromogen A B.50 µL each into the microplate, incubate at room temperature for 10-15 min.

3.6 Termination

      Add 50 µL/well stop solution. read at 450nm (630nm as reference wavelength)

3.7 Test result judgment standard

      Use 2.1 times of the OD of Negative mice serum as Cut-Off. Take the highest dilution factor that Sample OD>Cut-Off as the antibody titer in the sample, when the mean value of OD of negative serum is lower than 0.05. take 0.05 for calculation.

3.8 Site clearance

      The remaining unused test reagents and solutions should be returned to the original place, and the remaining test samples should be dealt with accordingly according to the test results.

3.9 Additional remarks

      This SOP is also suitable for assaying the titer of antibody in other species, with the difference being the use of enzyme-labelled antibody.