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Sandwich ELISA Protocol


Introduction of sandwich ELISA protocol

ELISA is a highly sensitive test technique based on immunological reactions that combine the specific reaction of antigens and antibodies with the efficient catalytic action of enzymes on substrates. Since the reaction of the antigen and the antibody is carried out in the well of a solid phase carrier-polystyrene microtiter plate, after each reagent is added, the excess free reactant can be removed by washing to ensure the specificity and stability of the test result. In ELISA, the sandwich ELISA is one of the most useful immunoassay formats and it is designed for detection of soluble antigens. This type of assay is useful where a single species antiserum is available and where antigen does not attach well to plates.

These systems are useful when antigens are in a crude form (contaminated with other proteins) or at low concentration.

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Reagent

  • Coating antibody (1-10 µg/ml in coating buffer)
  • Samples, standards and controls
  • Primary antibody (unlabeled or biotinylated)
  • Enzyme labeled secondary antibody
  • Substrate
  • Coating buffer (pH 9.6 0.05M carbonate buffer): Na2CO3 1.59g, NaHCO3 2.93g, add distilled water to 1000ml. Adjust pH to 9.6 with hydrochloric acid
  • Phosphate Buffered Saline (PBS), pH7.4: 0.086 M disodium hydrogen phosphate (Na2HPO4), 0.020 M monopotassium phosphate (KH2PO4), 3.08 M sodium chloride
  • Blocking buffer: PBS, 1% BSA, 500 ml PBS, 5 g BSA
  • Wash solution(pH 7.4 0.15M PBS): KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.05% 0.5ml, add distilled water to 1000ml.
  • Dilution buffer: Bovine serum albumin (BSA) 0.1g, add washing buffer to 100ml or use serum of sheep serum, rabbit serum and other washing liquid to prepare 5-10%.
  • PBS, 0.1%BSA: 500 ml PBS, 0.5 g BSA
  • Stop Solution: 2 % oxalic Acid
  • TMB (tetramethylbenzidine) solution: TMB (10mg/5ml absolute ethanol) 0.5ml, Substrate Buffer (pH 5.5) 10ml, 0.75% H2O2 32μl.

Equipment:

  • Polystyrene plastic plate (referred to as microplate) 40 holes or 96 holes.
  • ELISA detector.
  • Incubators.

Procedures:

  • Coating with capture antibody

Coat the wells of a PVC microtiter plate with the capture antibody at 1–10 μg/mL concentration in carbonate/bicarbonate buffer (pH 9.6).

Unpurified antibodies (eg ascites fluid or antiserum) may require increased concentration of the sample protein (try 10 μg/mL) to compensate for the lower concentration of specific antibody.

Cover the plate with adhesive plastic and incubate overnight at 4°C.

Remove the coating solution and wash the plate thrice by filling the wells with 200 μL PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

  • Blocking and adding samples

Block the remaining protein-binding sites in the coated wells by adding 200 μL blocking buffer (5% non-fat dry milk or BSA/PBS) per well.

Cover the plate with adhesive plastic and incubate for at least 2 h at room temperature or overnight at 4°C.

Wash the plate thrice with 200 µL PBS. Add 100 μL of diluted samples to each well. Always compare signal of unknown samples against those of a standard curve. Run standards (duplicates or triplicates) and blank with each plate. Incubate for 90 min at 37°C.

Ensure concentration of standards spans the most dynamic detection range of antibody binding. You may need to optimize the concentration range to obtain a suitable standard curve. Always run samples and standards in duplicate or triplicate.

Remove samples and wash the plate twice with 200 μL PBS.

  • Incubation with detection and secondary antibody

Add 100 μL of diluted detection antibody to each well.

Check that the detection antibody recognizes a different epitope on the target protein to the capture antibody. This prevents interference with antibody binding. Use a tested matched pair whenever possible.

Cover the plate with adhesive plastic and incubate for 2 h at room temperature.

Wash the plate four times with PBS.

Add 100 μL of conjugated secondary antibody, diluted in blocking buffer immediately before use.

Cover the plate with adhesive plastic and incubate for 2 h at room temperature.

Wash the plate four times with PBS.

  • Detection

Add 100 µL of TMB Microwell Substrate solution to each well (substrate solution should be at RT prior to use).

The estimated incubation times for the enzyme-substrate reaction range from 20 to 30 minutes. Add 50 μl of stop solution. After stopping the enzymatic reaction the plate should be read at 450 nm.

  • Analysis of data

Prepare a standard curve from the data produced from the serial dilutions with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Read the concentration of the sample from this standard curve by the OD of sample.

Note: Do not use sodium azide in any buffers or solutions as sodium azide inactivates the horseradish-peroxidase enzyme. All solutions should be at ambient temperature prior to use.

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