Indirect ELISA

Coating antigen to microplate

Dilute the antigen to a final concentration of 20 µg/ml in PBS or other carbonate buffer.

Coat the wells of a PVC microtiter plate with the antigen by pipetting 100 µl of the antigen dilution

Cover the plate with an adhesive plastic and incubate for 2 hr at room temperature, or 4°C overnight.

Remove the coating solution, wash the plate one time with ddH2O and three times by filling the wells with 200 µl PBS or TBST. 

Blocking

​Block the remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer, 5% non-fat dry milk or 5% serum in PBS, per well. 

Cover the plate with an adhesive plastic and incubate for at least 15 min to 2 hr at room temperature or, if more convenient, overnight at 4°C.

Wash the plate once with ddH2O then twice with PBS.

Incubation with primary antibody

Add 100 µl of diluted primary antibody to each well.

For hybridoma screening, add10 or 25 ul conditioned medium from hybridoma to 75~90 ul PBST in each well

Cover the plate with an adhesive plastic or plate cover and incubate for 2 hr at room temperature.

Note: This incubation time may require optimization. Although 2 hr is usually enough to obtain a strong signal, if a weak signal is obtained, stronger staining will often be observed when incubated overnight at 4°C.

Wash the plate once with ddH2O then 4 times with 200 ul PBST.

Incubation with  secondary antibody

Add 100 µl of conjugated secondary antibody (diluted at the optimal concentration, for anti-mouse HRP use 1:20000 dilution,i.e., add 0.5 µl to 10 ml PBST) in PBST buffer immediately before use.

Cover the plate with an adhesive plastic or plate cover and incubate for 30 min to 1 hr at room temperature.

Wash the plate 4 times with PBST.

Detection

TMB (3,3',5,5'-tetramethylbenzidine)

Add  50 µl or 100 µl TMB solution to each well

Incubate for 15-30 min

Add equal volume of stopping solution (2 M H2SO4 or 0.5 N HCl)

Read the optical density at 450 nm 

Buffer

Bicarbonate/carbonate coating buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:
3.03 g Na2CO3
6.0 g NaHCO3
1000 ml distilled water
pH 9.6

PBS
1.16 g Na2HPO4,
0.1 g KCl
0.1 g K3PO4,
4.0 g NaCl (500 ml distilled water) pH 7.4.

Add NaCl to 0.3 M to  avoid unspecific reactions

Blocking solution
Commonly used blocking agents are 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.

Wash solution
Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

Antibody dilution buffer
Primary and secondary antibody should be diluted in 1x blocking solution to reduce non specific binding.

0.05 M Phosphate-Citrate Buffer

Dissolve one Phosphate-Citrate Buffer Tablet (Product No. P 4809) in 100 ml of deionized water with stirring to yield a 0.05 M phosphate-citrate buffer, pH 5.0.
OR
Add 25.7 ml of 0.2 M dibasic sodium phosphate (Product No. S 0876), 24.3 ml of 0.1 M citric acid (Product No. C 7129), and 50 ml deionized water. Adjust pH to 5.0, if necessary.

TMB Substrate Solution

Dissolve one 3,3’,5,5’–tetra- methylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M Phosphate-Citrate Buffer, pH 5.0. Add 2 μl of fresh 30% hydrogen peroxide (Product No. H 1009) per 10 ml of substrate buffer solution, immediately prior to use.

OR

Dissolve one 3,3’,5,5’–tetramethylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M phosphate- citrate buffer, pH 5.0, containing 0.03% sodium perborate (capsules, Product No. P 4922).

Stop Solution 

Reaction may be stopped by the addition of 50 μl of 2 M H2SO4 per 200 μl of reaction mixture, or equal vol of 0.5 N HCl.

Home-Made TMB Solution

Working Protocol (receipe 1)

Solution A:

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 0.564 g Urea hydrogen peroxide while continuous stirring.

Solution B

Add 100 mg TMB to 15 ml  N,N-Dimethylacetamide. Store in a dark bottle at -20 oC.

Working Solution

Just before use, add 50 ul solution B to 5 ml Solution A, then add 1 drop of 30% H2O2.

Receipe 2

Phosphate-Citrate Buffer 0.1 M at pH 4.6 ± 0.2 plus perborate
Mix 24 ml of 0.2 M dibasic sodium phosphate (dissolve 1.78 g Na2HPO4-2H2O or 3.58 g of Na2HPO4•12H2O in dH2O and fill up to 50 ml) with 26 ml 0.1 M citric acid (2.10 g citric acid monohydrate in dH2O and make to 100 ml with dH2O), check pH and dissolve 0.02 g sodium perborate. Store in polyethylene bottle at 4 °C stable 1 year.

TMB substrate 100 mM 

Dissolve 240 mg 3,3’,5,5’–tetramethylbenzidine (m.w. 240) in 10 ml of DMSO. Store in opaque polyethylene bottle at 4 °C stable 1 year.

Working TMB chromogen 
Mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C.

Receipe 3

from Patent

https://patents.google.com/patent/US5206150

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 1.422 g Bacitracin  with stirring until it dissolved.

Add 0.564 g   Urea hydrogen peroxide while  continuous stirring.

Add  300 mg 3,3',5,5'-Tetramethylbenzidine  (TMB)  to 50 ml of dimethyl sulfoxide

Add  TMB solution aqueous  solution.

Mixing in an amber bottle

Store at 4° C.

The final concentrations of each of the above components is: bacitracin, 1.0 mM; urea hydrogen peroxide, 6 mM; TMB, 1.25 mM; DMSO, 5%; citrate, 25 mM and the final pH is 3.4.