Editing 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&nbsp;three times by filling the wells with 200 µl PBS or TBST.&nbsp;

== 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.&nbsp;

Cover the plate with an adhesive plastic and incubate for at least '''15 min to&nbsp;'''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&nbsp;antibody ==

Add 100 µl of diluted primary antibody&nbsp;to each well.

For hybridoma screening, add10 or 25 ul conditioned medium from hybridoma to&nbsp;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&nbsp;once with ddH2O then&nbsp;4 times with 200 ul PBST.

== Incubation with&nbsp; secondary antibody ==

Add 100 µl of conjugated secondary antibody (diluted at the optimal concentration, for anti-mouse HRP use '''1:20000''' dilution,''i.e.,''&nbsp;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&nbsp;hr at room temperature.

Wash the plate 4 times with PBST.

== Detection ==

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

Add&nbsp; 50 '''µl&nbsp;'''or 100 '''µl&nbsp;'''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&nbsp;

== Buffer ==

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

'''PBS'''<br/>1.16 g Na2HPO4,<br/>0.1 g KCl<br/>0.1 g K3PO4,<br/>4.0 g NaCl (500 ml distilled water) pH 7.4.

Add NaCl to 0.3 M to&nbsp;&nbsp;avoid unspecific reactions

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

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

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

'''0.05 M Phosphate-Citrate Buffe'''r

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.<br/>OR<br/>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&nbsp;'''

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&nbsp;&nbsp;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 '''<br/>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.

<br/>'''TMB substrate 100 mM&nbsp;'''

<br/>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.

<br/>'''Working TMB chromogen&nbsp;'''<br/>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 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&nbsp; with stirring until it dissolved.

Add 0.564 g&nbsp; &nbsp;Urea hydrogen peroxide while&nbsp; continuous stirring.

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

Add&nbsp; TMB solution aqueous&nbsp; 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.
[[Category:Antibodies]]