Sandwich Enzyme Immuno-Assay (EIA) for analyte detection and/or quantitation

Background

Sandwich ELISA’s are the tool of choice for measuring small quantities of antigen in complex biological mixtures. The principle involves ‘capturing’ an antigen/analyte between two ‘layers’ of antibodies which target separate epitopes on the target molecule (i.e. capture and detection antibody). The target molecule must of necessity contain more than one epitope for a sandwich ELISA to be performed. Small molecule’s or haptens should be measured in a competitive ELISA (to be covered in a separate application note.

Either monoclonal or polyclonal antibodies can be utilized as the capture and/or detection antibodies in Sandwich ELISA systems. Monoclonal antibodies inherently only recognise a single epitope and are more often used as capture antibodies. Polyclonal antibodies may be used to capture but since much of a poyclonal is non-antigen specific in nature, an immunoaffinity purified polyclonal should be used when ever possible

Warning

Sandwich ELISA procedures are often difficult to optimise especially when testing biological fluids (serum, plasma etc) and when ever possible, proven ‘match pair’ antibodies should be used.

Procedure Outline

Coating with Capture antibody
1. Coat the wells of a high binding microtiter plate with the capture antibody at a concentration of 1-10 μg/ml in either dI water or PBS (pH 7.4), 100 ul /well.
2. Cover the plate with an adhesive plastic plate cover or parafilm and incubate overnight at 4°C.
3. Remove the coating solution and wash the plate x3 by filling the wells with 200 μl PBS (the addition of a non-ionic detergent often required for complete washing). 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 or other absorbent material.

Blocking the plates
4. Block any remaining protein-binding sites in the wells to be utilized by adding 200 μl blocking buffer / well, 1-5% non fat dry milk or fatty acid free BSA in PBS.
5. Cover the plate with an adhesive plastic plate cover or parafilm and incubate 2-4 hours at RT or overnight at 4°C.

Samples
6. Add 100 μl of appropriately diluted samples to each well diluted in PBS BSA or other suitable diluent. For quantitative results, a standard curve should be constructed using an antigen of known concentration. Compare unknowns to the standard curve and perform regression analysis to calculate values for unknowns. Samples and standards should always be run in duplicate. Standards and blanks must be run with each plate to ensure accuracy. Incubate for 1-4 hours at RT, 4°C or 37°C (this should be determined empirically and will be different for each antibody pair and antigen.
You will need to optimize the concentration ranges to ensure a suitable standard curve and appropriate detection sensitivity. 
7. After incubation, wash the plate x3 by filling the wells with 200 μl PBS (the addition of a non-ionic detergent often required for complete washing).

Detection antibody and Secondary antibody 
8. Add 100 μl of diluted detection antibody to each well.
Ensure the secondary detection antibody recognizes a different epitope on the target protein than the coating antibody. Use proven matched pairs when possible.
9. Cover the plate with an adhesive plastic plate cover or parafilm and incubate for 1-4 h at room temperature.
10. Wash the plate as before.
11. Add 100 μl of conjugated (HRP, AP etc) secondary antibody, diluted at the optimal concentration (empirically determined) in blocking buffer immediately before use.
12. Cover the plate with an adhesive plastic plate cover or parafilm and incubate for 1-2 h at room temperature.
13. Wash the plate as before.

Detection
14. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multichannel pipette or a multipipet.

There are numerous detection systems available. The choice of system will depend on sensitivity requirements and the type of reader/systems available in your laboratory. The main types of enzymes used for detection in ELISA are horse radish peroxidase (HRP) and alkaline phosphatase (ALP), with HRP being the more sensitive of the two.
For ALP, pNPP (p-Nitrophenyl-phosphate) is the most commonly used and readily available substrate. The yellow colour of nitrophenol can be measured at 405 nm after 30 min incubation/develoment at room temperature. This reaction can be terminated by adding equal reaction volume of 0.75 M NaOH.
NOTE: Shorter incubations often yield inconsistent results between the beginning of the assay plate and the end of the assay – front-to-back variability. Longer incubations generally only allow background to ‘creep’ up and lower overall sensitivity. If your assay develops too rapidly for a 30 min substrate incubation – optimization of primary and secondary antibodies is required.
The substrate for HRP is TMB (3,3’,5,5’-tetramethylbenzidine). Exalpha provides a ready to use liquid format TMB solution that offers excellent sensitivity while minimizing background.
TMB incubation should be for 30 min followed by addition of add equal volume of stopping solution (2 M H2SO4) and the assay is then read at OD 450 nm. NOTE: Shorter incubations often yield inconsistent results between the beginning of the assay plate and the end of the assay – front-to-back variability. Longer incubations generally only allow background to ‘creep’ up and lower overall sensitivity. If your assay develops too rapidly for a 30 min substrate incubation – optimization of primary and secondary antibodies is required.

Data Analysis

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