Enzyme-linked immunosorbent Assay (ELISA)

Materials

Nunc Maxisorp flat-bottom clear 96-well microplate (Fisher # 10547781)
Wash buffer (phosphate buffered saline [PBS] pH 7.4 with 0.05% w/v Tween 20)
Capture antibody solution
Biotinylated detection antibody solution
Avidin-conjugated horseradish peroxidase (Av-HRP) solution
Calibrator solutions
Sample solutions
Carbonate buffer (pH 9.4)
Bovine serum albumin (BSA; Sigma # A7030)
Citrate buffer (pH 5.0)
o-Phenylenediamine (OPD) solution

Procedure

The typical sandwich ELISA protocol comprises (assuming 96-well microplate format):

Coat plate with 100 μL capture antibody solution per well. Incubate at 4°C overnight, then wash.
Block plate with 200 μL blocking buffer per well. Incubate at room temperature for 2 hours (or 4°C overnight), then wash.
Add 100 μL calibrator or sample solution per well. Incubate for 1-2 hours at room temperature, then wash.
Add 100 μL detection antibody solution per well. Incubate for 1-2 hours room temperature, then wash.
Add 100 μL enzyme (e.g. Av-HRP) solution per well. Incubate for 1 hour at room temperature, then wash extensively.
Add 100 μL substrate (e.g. OPD) solution per well. Incubate at room temperature, usually for 20–30 minutes but optimal incubation time may vary from one assay to another. It is best to monitor the reaction during this time.
Read absorbance.

Here’s a playlist of video tutorials on ELISA. I did not create these videos. Credits go to the respective content creators.

Solutions

Carbonate buffer: Dissolve 152 mg Na₂CO₃ and 300 mg NaHCO₃ in 50 mL deionised water. Final pH should be 9.6.
Citrate buffer: Empty the content of 1 capsule (Sigma # P4922) into 100 mL of deionised water. Stir to dissolve. Final pH should be 5.0.
Wash buffer: Dissolve 1 PBS tablet (Sigma # P4417) in 200 mL of deionised water. Final pH should be 7.4. Add 100 mg of Tween 20 and mix well. Final concentration of Tween 20 is 0.05% w/v.
Blocking buffer: 5% w/v BSA in wash buffer.
Diluent for protein/peptide reagents: 1% w/v BSA in wash buffer.
OPD solution: Dissolve 1 tablet (Sigma # P5412) in 12.5 mL of citrate buffer immediately before use. Final OPD concentration is 0.4 mg/mL.

Notes

1% w/v = 10 mg/mL
Deionised water should be at least 10 MΩ∙cm (15 MΩ∙cm water is available from the dispenser in room H415).
Use the reverse-pipetting technique when dispensing solutions into the wells to avoid foam formation.
Cover plate with plastic seal or cling film at each incubation step.
Suppliers usually recommend a range of working concentrations for the reagents they supply. When using such reagents for the first time, start with the median concentration and optimise as necessary. If the supplier has not recommended a concentration range, start with 2 μg/mL and optimise as necessary.
Capture antibody must be free of BSA or other ‘carrier proteins’, and diluted with carbonate buffer. Other antibodies, calibrators and enzyme solutions should be diluted with 1% w/v BSA solution. OPD substrate should be diluted in citrate buffer.
Washing should be done using the wash buffer. To wash, fill each well with the wash buffer and discard, then pat the plate dry on paper towel. Repeat 2–5 times. The volume of wash buffer per well should exceed the volume of any other reagent ever loaded into the wells during the experiment.
Av-HRP solution from eBioscience (# 18-4100) is diluted 500 times. Final concentration is 2.5 μg/mL.
You only need to use a small volume of the carbonate and citrate buffers per assay. If you prepare fresh stock, a large proportion of it will probably not be used on that occasion. You may save the buffers for future use by freezing at -20°C to maintain pH, in which case just defrost and warm them to room temperature before use.
Always include an excess of solutions in your calculations, e.g. if you need 100 μL of coating antibody solution per well, and you have 32 wells to fill, calculate for 40 wells and prepare 4 mL (40 × 100 μL = 4000 μL). If you intend to use a multichannel pipettor and a reagent reservoir, include 1-2 mL excess.

Calculations

Here’s a worked example in 5 easy steps:

We need to fill 32 wells with a reagent, at 100 μL per well. We have decided that the working concentration will be 2 μg/mL, but the stock concentration (as supplied) is 500 μg/mL.
We know that the stock solution is 250 times (500 ÷ 2 = 250) more concentrated than the working solution that we want to achieve. Therefore we need to dilute it down 250 times.
We have decided that we need 32 × 100 μL = 3200 μL of the working solution, but at this point we have not included any excess in our calculation. A straightforward solution is to prepare 4000 μL (i.e. 4 mL) to give us 800 μL of excess solution. This should be sufficient to allow us to perform reverse-pipetting using a single-channel pipettor.
We already know that we need to dilute the stock solution down 250 times. In other words, 1/250 of the 4000 μL will come from our stock solution. Therefore, the volume of stock solution that we need is 4000 ÷ 250 = 16 μL.
In principle, we would subtract 16 μL from the total volume of 4000 μL, to give us the volume of diluent that we need (4000 – 16 = 3984 μL). In practice, the effect of this correction is probably negligible, and it would be difficult to measure this volume accurately anyway, so this correction may be regarded as optional.

Why not just use the equation c₁v₁ = c₂v₂, I hear you ask.

I do not usually recommend this equation to junior researchers (e.g. students), as in my experience, they often apply the equation blindly without an understanding of the underlying principles. For this reason, it is not uncommon for some of them to substitute the wrong values for the variables. I much prefer laying out the solution logically using first principles.

If you are competent and comfortable with using the equation, by all means, use it. But in case you are wondering why the equation is the way it is, it stems from the very simple relationship between solute concentration (c), solute mass (m) and solution volume (v):

c = m ÷ v

Thus,

m = c × v

When you dilute a solution, you increase its volume (v) and reduce the concentration of a given solute (c). The mass of the solute (m) remains unchanged. Therefore, if the subscript 1 denotes quantities before dilution, and the subscript 2 denotes quantities after dilution, you have:

m = c₁v₁ = c₂v₂