ELISA Types and Applications
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Direct answer
ELISA, or enzyme-linked immunosorbent assay, detects antigen or antibody through an enzyme reaction whose colour change is proportional to the amount of target. The four classic formats are direct, indirect, sandwich and competitive, and each suits a different target. Indirect ELISA detects antibody, sandwich ELISA detects antigen with high specificity, and competitive ELISA suits small molecules such as drugs.
What you must remember
- Direct ELISA: an enzyme-labelled primary antibody detects antigen bound to the plate, which is quick but gives less signal amplification.
- Indirect ELISA: an enzyme-labelled anti-human immunoglobulin detects patient antibody, amplifying the signal, which makes it the basis of most antibody screens.
- Sandwich ELISA: a capture antibody and a detection antibody bind two different epitopes of one antigen, giving high specificity, as with HBsAg.
- Competitive ELISA: sample antigen competes with a labelled reference, so the signal falls as the sample concentration rises.
- IgM capture ELISA: anti-human IgM captures serum IgM first, used to diagnose acute infection, for example IgM anti-HAV.
- Enzymes and substrates: horseradish peroxidase and alkaline phosphatase are common enzymes, with optical density read on a plate reader.
- Cut-off value: results are read against a calibrated cut-off, and borderline values need confirmation by a second test.
- Window period: antibody ELISA can be negative early in HIV infection, which is why combined antigen-antibody assays add p24 antigen detection.
Choosing a format by target and purpose
A format is chosen by asking two questions: is the target an antigen or an antibody, and does the antigen have more than one epitope? The table below sets out the five formats most often tested.
| Format | Target detected | Key feature |
|---|---|---|
| Direct | Antigen | Single labelled antibody, fast and simple |
| Indirect | Antibody | Labelled anti-human Ig, signal amplified |
| Sandwich | Antigen | Two antibodies on two epitopes, high specificity |
| Competitive | Small antigen or antibody | Signal falls as sample concentration rises |
| IgM capture | IgM antibody | Diagnoses acute infection |
The sandwich format needs an antigen large enough to carry two epitopes, so small molecules are measured with competitive formats instead. The choice between direct, indirect and sandwich methods then depends on whether the sample is expected to contain antibody or antigen. In practice, each kit is validated against known positive and negative controls, and results are reported as a ratio to the cut-off so batches can be compared.
Where the exam tries to catch you
ELISA questions usually test the match between format and target, so the trap is choosing the format that looks familiar rather than the one that fits the analyte.
- Trap: using a sandwich ELISA to detect a small hapten such as a drug Correct: a small molecule has only one epitope, so two antibodies cannot bind, and a competitive format is the right choice.
- Trap: choosing indirect ELISA to detect antigen in serum Correct: indirect ELISA detects antibody, whereas antigen detection uses direct, sandwich or competitive formats.
- Trap: reading a negative antibody ELISA as excluding recent infection Correct: antibody can be absent early in infection, so IgM capture or antigen detection is needed for acute disease.
Frequently asked questions
Which ELISA format detects HBsAg in serum?
The sandwich ELISA format detects HBsAg in serum, because a capture antibody and a labelled detection antibody bind two different epitopes of the surface antigen. This two-site binding gives high specificity, which is why sandwich ELISA is preferred for antigen detection.
Why is indirect ELISA used for antibody screening in HIV?
Indirect ELISA is used for HIV antibody screening because the patient's antibodies bind a coated viral antigen, and an enzyme-labelled anti-human immunoglobulin then amplifies the signal. Newer combined antigen-antibody assays add p24 antigen detection, because antibodies can be undetectable during the early window period.
What is the principle of competitive ELISA?
In competitive ELISA, the sample antigen competes with a labelled reference antigen for a limited number of antibody binding sites. The signal therefore falls as the sample concentration rises, which makes competitive ELISA suitable for small molecules such as drugs and hormones with a single epitope.
Why is IgM capture ELISA used to diagnose acute infection?
IgM capture ELISA is used to diagnose acute infection because IgM appears early, before IgG, so capturing serum IgM reveals recent exposure. Anti-HAV IgM is commonly tested this way for acute hepatitis A, although IgM can persist for some months after the infection.