This protocol is used for estimation of serum IgM level in mice using indirect ELISA.
Method Article
Estimation of anti-OMV serum IgM level using indirect ELISA
https://doi.org/10.21203/rs.3.pex-1929/v1
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This protocol is used for estimation of serum IgM level in mice using indirect ELISA.
Indirect ELISA
Outer membrane vesicles
mice
This method uses the estimation of serum IgM level using single dilution indirect-ELISA.
1. Wash Buffer (PBS with 0.05% tween-20)
2. Coating buffer (0.05M sodium Carbonate buffer, pH9.5)
Na2CO3 : 0.159%
NaHCO3: 0.293%
3. Blocking solution: 2% bovine serum albumin.
4. Antibody dilution buffer (pH 7.0): 250 mg of BSA was dissolved in 100 ml of PBS and stored at 4°C.
5. Substrate buffer, 100ml
0.1 M Citric Acid solution: 24.3ml
0.2 M Dibasic Sodium phosphate solution: 25.7ml
Triple Glass Distilled Water: 50ml
Orthophenyl Diamine: 40mg
H2O2: 40µl
The solution is prepared fresh before use.
6. Stop Solution: 1.5N H2SO4
Microplate reader
The OMV was diluted in coating buffer up to the final concentration of 10 µg per 100 µl (0.1 mg per ml). The plate was sealed with parafilm and incubated at 4 °C for overnight followed by three times washing of the plates with washing solution. The 100 µl of blocking solution was added in each well. The plate was then incubated at room temperature for 1 hour followed by three times washing of the plates with washing solution and then tapping against blotting paper. The 100 µl of 1:100 diluted serum (in antibody dilution buffer) was added in each well. The plate was incubated for 2 hours at room temperature followed by three times washing of the plates with washing solution and then tapping against blotting paper. The 100 µl of anti-mouse HRP conjugated antibody (diluted 1:3000 times in antibody dilution buffer) was added in each well. The plate was then incubated for 1 hour at room temperature. The OPD substrate solution was prepared during the time of incubation (24.3 ml of 0.1 M citric acid, 25.7 ml of 0.2 M dibasic sodium phosphate solution, 40 mg orthophenyl diamine, 0.04 ml of hydrogen peroxide and 50 ml of triple glass distilled water). The plate was washed thrice with washing solution followed by tapping against blotting paper. The 100 µl of OPD substrate solution was added in each well followed by incubation of the plate at room temperature in dark for 30 minutes. The 100 µl of 1.5 N NaOH or 1 N H2SO4 was added in each well. The OD was read at 492 nm in the ELISA plate reader.
The whole procedure takes about 6 hours.
The authors are indebted to DBT, India for providing the necessary funds
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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