Written and maintained by CASRAI Editorial Board
Last updated
A sandwich ELISA measures an analyte by trapping it between two antibodies: a capture antibody fixed to a plate and a detection antibody that binds a different site on the same molecule. The signal from the detection step is read against a standard curve to estimate concentration. This page explains the principle, what the format can and cannot detect, how detection formats differ, and what to confirm on a kit datasheet. It is general laboratory information, not a protocol.
Short answer
In a sandwich ELISA, a capture antibody is immobilized on a plate, the sample is added, and the target antigen is held by the capture antibody. A second, detection antibody then binds the captured antigen at a different epitope, and an enzyme-linked signal is read and compared with a standard curve (Thermo Fisher; Addgene). It is widely used because it is sensitive and specific and works with complex samples such as serum and lysates.
What does a sandwich ELISA detect?
It detects an analyte that carries at least two distinct binding sites, because the analyte is bound between two primary antibodies that each recognize a different epitope (Thermo Fisher). The two antibodies must recognize different, non-overlapping epitopes, and the detection epitope must not be hidden once the antigen is bound by the capture antibody. Addgene makes the same point: the format needs distinct, non-overlapping epitopes.
A decision checklist: does the sandwich format fit?
- Can two antibodies bind the analyte at once? Small molecules cannot bind two antibodies at the same time, so Addgene notes that competitive ELISA suits them better.
- Is there a validated pair? Thermo Fisher describes the two antibodies as a matched pair and notes that suppliers often list validated pairs. Using the same antibody for capture and detection can limit dynamic range and sensitivity.
- Is the sample complex? Thermo Fisher lists suitability for complex samples such as serum and lysates as an advantage of the format.
- Is development time acceptable? Thermo Fisher says the sandwich format requires more optimization (finding pairs, checking cross-reactivity), and Addgene describes assay development as challenging, with a longer protocol than other types.
For how capture and detection antibodies are chosen and paired, see ELISA antibodies: capture, detection and matched pairs.
How does a sandwich ELISA work?
Described in prose, the workflow has four stages. A plate is coated with the capture antibody. The sample is added and the antigen is held by that antibody, followed by a wash. The detection antibody is added and binds the captured antigen. A signal is generated and read, and the reading is converted to concentration using a standard curve (Addgene). For curve fitting, see four-parameter logistic ELISA curve fitting.
What makes an antibody a secondary antibody?
The antibody that binds the antigen is a primary antibody. A secondary antibody is one that binds the primary antibody rather than the antigen, and it carries the label. In a sandwich ELISA the detection antibody may be enzyme-conjugated directly, or it may be unconjugated and revealed by a conjugated secondary antibody (Addgene). Thermo Fisher notes that sandwich assays typically use indirect detection and that it is critical the secondary does not react with the capture antibody. Its suggested safeguards are different host species for capture and detection (for example mouse capture and rabbit detection) and cross-adsorbed secondaries.
Detection formats compared
| Format | What carries the label | Sourced characteristics |
|---|---|---|
| Direct detection | The detection (primary) antibody itself | Fewer steps and no secondary cross-reactivity; labeling may affect antibody activity; minimal amplification (Thermo Fisher) |
| Indirect detection | A labeled secondary antibody | Signal amplification; an extra incubation step; possible nonspecific secondary signal (Thermo Fisher) |
| Streptavidin-HRP (one vendor example) | Streptavidin-HRP with a TMB and peroxide substrate | Absorbance read at 450 nm, with wavelength correction at 540 or 570 nm if available (R&D Systems) |
Thermo Fisher also notes that the same capture antibody can be used with different detection methods.
What is the protocol for an ELISA kit?
Incubation volumes, times, concentrations and the stop solution are kit-specific, so the kit insert is the protocol to follow. R&D Systems states, for example, that the stop solution depends on the kit datasheet. A general overview is in ELISA protocol basics.
Where it matters for labs and supply decisions
Checklist: what to confirm on a kit or matched-pair datasheet
- Whether the capture and detection antibodies are listed as a validated pair (Thermo Fisher).
- Whether the detection antibody is enzyme-conjugated or needs a separate conjugated secondary (Addgene), and whether the host species differ (Thermo Fisher).
- The read wavelength and any correction wavelength, which determines whether your reader can be used (R&D Systems; see what a plate reader is).
- The stop solution named on the datasheet (R&D Systems).
- That the analyte has two binding sites; otherwise consider a competitive format (Addgene).
Related ELISA formats
| Format | Note from sources |
|---|---|
| Direct ELISA | See direct ELISA assay |
| Competitive ELISA | Better suited to small molecules that cannot bind two antibodies (Addgene) |
| ELISpot | A related but different assay; see ELISpot assay |
Addgene describes the sandwich format as the most common type of ELISA.
Common misunderstandings
- Any antibody pair will work. The pair must bind non-overlapping epitopes, and the detection epitope must stay accessible after capture (Thermo Fisher).
- A secondary antibody is always needed. Direct detection uses a labeled primary and skips it (Thermo Fisher).
- Sandwich assays suit every analyte. Analytes that cannot bind two antibodies are better served by competitive ELISA (Addgene).
FAQ
How do ELISA assays work?
An antigen is captured on a plate by an immobilized antibody, bound by a detection antibody, and revealed by an enzyme-linked signal that is read and compared with a standard curve (Addgene).
What do we detect in sandwich ELISA?
An antigen with two distinct, non-overlapping epitopes, in samples such as serum or lysates (Thermo Fisher).
What is the protocol for an ELISA kit?
The one in the kit insert; times, concentrations and stop solution are kit-specific.
Sources
- Thermo Fisher Scientific, Overview of ELISA, read 2026-11-07.
- Addgene blog, Antibodies 101: The Four ELISAs and When to Use Them, read 2026-11-07.
- R&D Systems, sandwich ELISA ancillary reagent protocol (PDF), read 2026-11-07.
