Every ELISA format detects an antigen with an antibody and an enzyme, but they suit different targets. Here is how each works, which one a quantitative kit is likely to use, and how to read the results.
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An enzyme-linked immunosorbent assay (ELISA) fixes one partner of an antibody-antigen pair to the wells of a polystyrene plate, lets the other partner bind, washes away everything that did not bind, and then reports what stayed with an enzyme. The enzyme, most often horseradish peroxidase (HRP), converts a colourless substrate into a coloured product. With TMB as the substrate the wells turn blue, then yellow when acid stops the reaction, and a plate reader measures absorbance at 450 nm.
The four formats differ in what is coated on the plate and how the enzyme is attached. Those choices decide sensitivity, specificity, how many antibodies you need and which targets the format can measure.
The antigen is coated on the plate and an enzyme-labelled primary antibody binds it. It has the fewest steps and no secondary antibody to cross-react, but there is no signal amplification, and every primary antibody has to be labelled. It is used mainly to screen antibodies or check that a coated antigen is present.
The antigen is coated, an unlabelled primary antibody binds it, and an enzyme-labelled secondary antibody binds the primary. Several secondaries can bind each primary, which amplifies the signal, and one labelled secondary works with any primary from the same host. Indirect ELISA is the classic format for measuring antibodies in serum, for example antibody titres after immunisation.
In a sandwich ELISA a capture antibody is coated on the plate. The target in your sample binds it, then a detection antibody against a different epitope binds the captured target. The detection antibody is usually biotinylated and revealed with streptavidin-HRP, which adds another layer of amplification.
Because the target must be recognised by two antibodies at once, sandwich assays are highly specific and work well in complex samples such as serum and tissue homogenates. They need a matched antibody pair, and the target must be large enough to bind two antibodies, so they suit proteins rather than small molecules. Most commercial quantitative ELISA kits for cytokines, growth factors and tissue proteins use this format.
Small molecules such as cyclic GMP, cortisol, steroid hormones and short peptides have only one site an antibody can bind, so they cannot be sandwiched. In a competitive ELISA the target in your sample competes with a labelled or plate-coated version of itself for a limited amount of antibody.
The more target in the sample, the less labelled target binds, so the signal falls as concentration rises. The standard curve slopes downward, which surprises people reading their first competitive plate.
| Format | Coated on plate | Signal vs target | Strengths | Typical use |
|---|---|---|---|---|
| Direct | Antigen | Rises | Fast, few steps | Antibody screening |
| Indirect | Antigen | Rises | Amplified, flexible | Serum antibody titres |
| Sandwich | Capture antibody | Rises | Specific and sensitive | Proteins in serum, plasma, lysates |
| Competitive | Antigen or antibody | Falls | Measures single-epitope targets | Hormones, cGMP, peptides, drugs |
If you are building an assay rather than buying a kit, the monoclonal vs polyclonal guide explains why sandwich pairs often combine a monoclonal capture antibody with a polyclonal or second monoclonal detection antibody.
Every quantitative ELISA plate carries a dilution series of a known standard. You plot absorbance against concentration and read your samples off the curve.
Samples outside the curve need a different dilution, which is why the sample preparation guide suggests running a dilution test on a few samples before the full set.
| Problem | Likely cause | Fix |
|---|---|---|
| High background in every well | Incomplete washing or blocking; substrate exposed to light | Wash thoroughly and tap plates dry; keep TMB in the dark |
| Weak or no signal | Expired or wrongly stored reagents; a missed step | Check storage, order of addition and incubation times |
| Poor duplicates | Pipetting error, bubbles, uneven washing | Use calibrated pipettes and reverse pipetting for viscous samples |
| Edge wells read differently | Uneven temperature across the plate | Seal plates and pre-warm reagents to room temperature |
| Samples above the top standard | Concentration too high | Dilute further and rerun |
For proteins, a sandwich ELISA with a biotin-streptavidin amplification step is usually the most sensitive and specific format. Sensitivity also depends heavily on the antibody pair, so compare the lowest detectable dose on kit datasheets rather than the format alone.
That is how competitive ELISA works. The target in the sample competes with labelled target for antibody binding, so more target in the sample means less labelled target bound and a lower signal. The standard curve slopes downward.
In indirect ELISA the antigen is coated on the plate and detected with a primary antibody plus a labelled secondary. In sandwich ELISA a capture antibody is coated, the antigen from the sample binds it, and a second detection antibody binds the antigen. Sandwich measures antigens in samples; indirect usually measures antibodies.
Only over the small central part of the curve. ELISA standard curves are sigmoidal, so a four-parameter logistic fit is the standard and gives accurate values across the whole range.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
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