# Competitive ELISA

Competitive ELISA is an immunoassay in which antigen in a sample competes with a fixed amount of labeled antigen for binding to a limited amount of antibody, so the measured signal falls as the analyte concentration rises. It is widely used for small molecules and haptens (small molecules that can bind antibodies without a carrier, but must be linked to a protein carrier to elicit an immune response), such as steroids, eicosanoids, and mycotoxins, which carry only a single epitope (The specific part of an antigen an antibody binds) and therefore cannot be caught between two antibodies in a sandwich assay.<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup><sup> • </sup><sup>[2](https://www.ncbi.nlm.nih.gov/books/NBK555922/)</sup><sup> • </sup><sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup><sup> • </sup><sup>[24](https://www.ncbi.nlm.nih.gov/books/NBK10759/)</sup> The format is also called inhibition ELISA.<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup>

| Key fact | Detail |
|---|---|
| What it measures | Low-molecular-weight antigens and haptens with a single antibody-binding epitope<sup>[2](https://www.ncbi.nlm.nih.gov/books/NBK555922/)</sup><sup> • </sup><sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup> |
| Signal direction | Inverse: more sample antigen means less bound labeled antigen and lower absorbance<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup> |
| Curve model | 4-parameter logistic fit by nonlinear regression; \( \mathrm{IC}_{50} \) read at 50% inhibition<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup><sup> • </sup><sup>[4](https://www.creative-proteomics.com/resource/protocol-for-competitive-elisa.htm)</sup> |
| Reliable range | The 20% to 80% \( B/B_{0} \) portion of the standard curve; only 40% to 60% \( B/B_{0} \) if CVs below 25% are required<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup> |
| Typical sensitivity | Roughly pg/mL for steroids (6.37 to 26.4 pg/mL in commercial kits) to ng/mL for larger analytes<sup>[5](https://www.assaygenie.com/content/Technical%20Manuals/ELISA/QSES/QSES006.pdf)</sup><sup> • </sup><sup>[6](https://www.thermofisher.com/elisa/product/Estrone-3-Sulfate-E1S-Competitive-ELISA-Kit/EIA17E3S)</sup><sup> • </sup><sup>[7](https://stjohnslabs.com/content/complete%5Fpdf/STJE0010039%5Fdatasheet.pdf)</sup> |
| Precision | Routine intraassay, interassay, and day-to-day variation usually 5% to 20%<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup> |
| Origin | Enzyme-labeled-antigen immunoassays reported simultaneously in 1971 by two independent groups, building on radioimmunoassay<sup>[8](https://oap-journals.com/proteomics-and-genomics-research/article/the-evolution-of-the-enzyme-immunoassay-enzyme-linked-immunosorbent-assay-1681)</sup> |

## How it works

Competitive immunoassays are reagent-limited: the antibody is supplied in restricted amount, and binding distributions follow the Law of Mass Action, unlike reagent-excess immunometric (sandwich) assays.<sup>[9](https://booksite.elsevier.com/9780080970370/content/Texts_chapter_pages/Texts_chapter%20pages/a2_1_Principles_of_competitive.html)</sup> A fixed concentration of labeled hapten or antigen competes with the analyte for the limited antibody.<sup>[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC9798763/)</sup> When the sample contains no analyte, the labeled antigen binds freely and signal is maximal (\( B_{0} \)). When analyte is present, it occupies antibody binding sites, less labeled antigen binds, and signal drops in proportion to analyte concentration.<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup>

The dose-response curve is therefore an inverted "S": high signal at low concentration, falling to a plateau at high concentration, with a narrow near-linear central interval.<sup>[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC9798763/)</sup> Results are quantified by fitting signal (or percent inhibition) against log concentration with a 4-parameter logistic model, the regression equation of choice, and reading the \( \mathrm{IC}_{50} \), the concentration that inhibits 50% of labeled-antigen binding.<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup><sup> • </sup><sup>[4](https://www.creative-proteomics.com/resource/protocol-for-competitive-elisa.htm)</sup> Percent inhibition is calculated as:

\[ \%\ \mathrm{inhibition} = \left( 1 - \frac{A_{\mathrm{sample}}}{A_{\mathrm{control}}} \right) \times 100 \]

where \( A_{\mathrm{sample}} \) and \( A_{\mathrm{control}} \) are average absorbance values.<sup>[4](https://www.creative-proteomics.com/resource/protocol-for-competitive-elisa.htm)</sup>

## How it is done

A representative antibody-down protocol runs as follows.<sup>[11](https://www.abcam.com/en-us/technical-resources/protocols/competitive-elisa-protocol)</sup><sup> • </sup><sup>[4](https://www.creative-proteomics.com/resource/protocol-for-competitive-elisa.htm)</sup>

1. **Coat.** Add capture antibody or fixed antigen at 1 to 10 µg/mL, about 50 µL per well; incubate 2 h at room temperature or overnight at 4 °C.
2. **Block.** Add 200 µL blocking buffer for 1 to 2 h at room temperature to cover unoccupied plastic.
3. **Compete.** Preincubate standards and samples with the labeled competitor antigen (or antibody-HRP conjugate, in the antigen-down orientation) for at least 1 h; preincubation can increase sensitivity. Add the mixture to the well and incubate about 2 h.
4. **Wash and develop.** Wash, add substrate such as TMB, and read absorbance at 450 nm (TMB) or 405 nm (ABTS).
5. **Fit and check.** Fit a 4- or 5-parameter logistic curve; accept fits with \( R^{2} > 0.99 \), spike recovery of 80% to 120%, intra-assay CV below 10%, and inter-assay CV below 15%, where \( \mathrm{CV} = \sigma/\mu \).<sup>[11](https://www.abcam.com/en-us/technical-resources/protocols/competitive-elisa-protocol)</sup>

Because competitive curves are steep and narrow, pilot studies should test at least three sample dilutions and require agreement between at least two.<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup>

## Origin

The competitive principle comes from radioimmunoassay. Rosalyn S. Yalow and [Solomon A. Berson](https://www.edgepedia.org/solomon-a-berson) published "Immunoassay of endogenous plasma insulin in man" in the Journal of Clinical Investigation in 1960, detecting endogenous plasma insulin with a radiolabeled antigen.<sup>[12](https://doi.org/10.1172/jci104130)</sup><sup> • </sup><sup>[8](https://oap-journals.com/proteomics-and-genomics-research/article/the-evolution-of-the-enzyme-immunoassay-enzyme-linked-immunosorbent-assay-1681)</sup>

ELISA replaced the radioactive iodine-125 label with enzyme-conjugated antigens or antibodies.<sup>[2](https://www.ncbi.nlm.nih.gov/books/NBK555922/)</sup> In 1971, two papers from independent groups appeared simultaneously: Eva Engvall and [Peter Perlmann](https://www.edgepedia.org/peter-perlmann), "Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G" in Immunochemistry, and B.K. Van Weemen and A.H.W.M. Schuurs, "Immunoassay using antigen, enzyme conjugates" in FEBS Letters.<sup>[13](https://doi.org/10.1016/0019-2791%2871%2990454-x)</sup><sup> • </sup><sup>[14](https://doi.org/10.1016/0014-5793%2871%2980319-8)</sup><sup> • </sup><sup>[8](https://oap-journals.com/proteomics-and-genomics-research/article/the-evolution-of-the-enzyme-immunoassay-enzyme-linked-immunosorbent-assay-1681)</sup> Engvall and Perlmann attached alkaline phosphatase as a nonradioactive tracer to rabbit IgG antigen, so the antigen in the unknown competed with labeled antigen on a cellulose immunosorbent; this is the arrangement now known as competitive ELISA, in a form seldom used today because plastic microtiter plates have replaced the centrifugation-dependent cellulose support.<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup> In 1985, Bertrand Friguet, Alain F. Chaffotte, Lisa Djavadi-Ohaniance, and Michel E. Goldberg published a homogeneous competitive ELISA in the Journal of Immunological Methods for measuring the true affinity constant of antigen-antibody complexes in solution.<sup>[15](https://doi.org/10.1016/0022-1759%2885%2990044-4)</sup>

## Variants

**Orientation.** Competitive ELISAs run in two geometries. In an antigen-down assay, the fixed antigen is coated on the plate and the sample competes with it for an antibody conjugated to a detection enzyme such as HRP. In an antibody-down assay, the antibody is coated and an enzyme-labeled fixed antigen is added; in both, more sample antigen yields lower signal.<sup>[11](https://www.abcam.com/en-us/technical-resources/protocols/competitive-elisa-protocol)</sup>

**Direct versus indirect.** Direct configurations incorporate the labeled antigen or antibody itself; indirect configurations use a reporter-labeled secondary antibody.<sup>[1](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)</sup>

**Blocking (serology) format.** For antibody detection, an enzyme-conjugated antibody and the test serum's antibodies compete for antigen coated on the plate. A color change indicates a negative result, because the conjugate bound the antigen; absence of color indicates a positive result, because serum antibodies blocked the conjugate. The color logic is inverted relative to antigen quantification.<sup>[2](https://www.ncbi.nlm.nih.gov/books/NBK555922/)</sup>

## Applications

**Hormones.** [Steroid](https://www.edgepedia.org/steroid) quantification is a core use, including estradiol, testosterone, estrone sulfate, and ethynylestradiol.<sup>[16](https://food.r-biopharm.com/wp-content/uploads/2021/02/manual-ethynylestradiol-elisa-5081estr501.21.pdf)</sup>

**Mycotoxins.** A validated direct competitive ELISA quantified five mycotoxins in human serum, aflatoxin B1, deoxynivalenol, fumonisin, ochratoxin A, and zearalenone, with LLOQs of 0.61 ng/mL (AFB1), 19.53 ppb (DON), 4.88 ppb (FUM), 19.53 ppb (OTA), and 0.15 ppb (ZEA), and recoveries of 73% to 106%.<sup>[17](https://www.mdpi.com/2072-6651/14/11/727)</sup>

**Signaling molecules and affinity.** Competitive kits serve small signaling molecules such as \( \mathrm{IP}_{3} \) and CTRP15.<sup>[7](https://stjohnslabs.com/content/complete%5Fpdf/STJE0010039%5Fdatasheet.pdf)</sup><sup> • </sup><sup>[18](https://aviscerabioscience.com/Product_Systems/CTRP15/SK00393-10.pdf)</sup> Competition ELISA also determines \( \mathrm{IC}_{50} \) values for ligand-receptor blocking, fitting background-corrected 450 nm absorbance against \( \log_{10} \) peptide concentration over 10 nM to 100 µM.<sup>[19](https://pmc.ncbi.nlm.nih.gov/articles/PMC4828978/)</sup>

## Limitations and alternatives

**Sensitivity and range.** A competitive immunoassay is not as sensitive as a sandwich ELISA and is more sensitive to matrix effects, especially serum matrix.<sup>[20](https://www.ncbi.nlm.nih.gov/books/NBK92434/)</sup> Its useful dynamic range is smaller, and the narrow standard curve is more susceptible to the routine 5% to 20% assay variation than sandwich curves are.<sup>[3](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)</sup>

**Robustness and specificity.** Incubation timing is less robust than in sandwich assays: the \( \mathrm{IC}_{50} \) of the standard curve shifts with minor changes in incubation of the steps.<sup>[20](https://www.ncbi.nlm.nih.gov/books/NBK92434/)</sup> [Covalent labeling](https://www.edgepedia.org/covalent-labeling) of the hapten can change its affinity for the antibody, so the label's effect must be tested.<sup>[20](https://www.ncbi.nlm.nih.gov/books/NBK92434/)</sup> Cross-reactivity is a practical limit: manufacturers determine cross-reactivities in buffer, values may differ in samples due to matrix effects, and the test cannot discriminate between the target analyte and cross-reactive substances.<sup>[16](https://food.r-biopharm.com/wp-content/uploads/2021/02/manual-ethynylestradiol-elisa-5081estr501.21.pdf)</sup>

**Hook effect.** The hook effect (prozone phenomenon) produces unexpectedly low signal or inaccurately low results at high analyte concentration; sample dilution and two-step protocols mitigate it.<sup>[21](https://pmc.ncbi.nlm.nih.gov/articles/PMC11808753/)</sup>

**Affinity ceiling.** Competitive assay sensitivity depends on antibody affinity, and preparing antibodies exceeding \( 10^{-12} \) M affinity is described as nearly impossible with existing technology.<sup>[22](https://www.sciencedirect.com/science/article/abs/pii/S0308814621002740)</sup> The Friguet homogeneous competitive ELISA, though considered an excellent affinity method, does not account for antibody bivalence and underestimates \( K_{\mathrm{d}} \) by a minimum factor of two.<sup>[23](https://www.sciencedirect.com/science/article/abs/pii/S0022175921000879)</sup> No published head-to-head comparison of competitive ELISA with LC-MS/MS or HTRF for small-molecule quantification is available.

## References

1. [Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA)](https://cshprotocols.cshlp.org/content/2017/7/pdb.prot093740)
2. [Enzyme-Linked Immunosorbent Assay (ELISA) - StatPearls - NCBI Bookshelf](https://www.ncbi.nlm.nih.gov/books/NBK555922/)
3. [How to Obtain Reproducible Quantitative ELISA Results](https://www.oxfordbiomed.com/sites/default/files/2017-02/ELISAArticle.pdf)
4. [Protocol for Competitive ELISA - Creative Proteomics](https://www.creative-proteomics.com/resource/protocol-for-competitive-elisa.htm)
5. [Canine E2 (Estradiol) Quickstep ELISA Kit technical manual (Assay Genie)](https://www.assaygenie.com/content/Technical%20Manuals/ELISA/QSES/QSES006.pdf)
6. [Estrone-3-Sulfate (E1S) Competitive ELISA Kit (EIA17E3S) - Invitrogen](https://www.thermofisher.com/elisa/product/Estrone-3-Sulfate-E1S-Competitive-ELISA-Kit/EIA17E3S)
7. [Universal IP3 (Inositol Triphosphate) Competitive ELISA Kit datasheet (St John's Labs)](https://stjohnslabs.com/content/complete%5Fpdf/STJE0010039%5Fdatasheet.pdf)
8. [The Evolution of the Enzyme Immunoassay/Enzyme-Linked Immunosorbent Assay](https://oap-journals.com/proteomics-and-genomics-research/article/the-evolution-of-the-enzyme-immunoassay-enzyme-linked-immunosorbent-assay-1681)
9. [Principles of Competitive and Immunometric Assays (The Immunoassay Handbook, Elsevier)](https://booksite.elsevier.com/9780080970370/content/Texts_chapter_pages/Texts_chapter%20pages/a2_1_Principles_of_competitive.html)
10. [Improved Sensitivity and Wide Range Detection of Small Analytes Using a Two-Antigen-Combined Competitive Immunoassay](https://pmc.ncbi.nlm.nih.gov/articles/PMC9798763/)
11. [Competitive ELISA protocol | Abcam](https://www.abcam.com/en-us/technical-resources/protocols/competitive-elisa-protocol)
12. [Rosalyn S. Yalow, Solomon A. Berson (1960). IMMUNOASSAY OF ENDOGENOUS PLASMA INSULIN IN MAN. Journal of Clinical Investigation.](https://doi.org/10.1172/jci104130)
13. [Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G (Immunochemistry, 1971)](https://doi.org/10.1016/0019-2791%2871%2990454-x)
14. [Immunoassay using antigen—enzyme conjugates (FEBS Letters, 1971)](https://doi.org/10.1016/0014-5793%2871%2980319-8)
15. [Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay (Journal of Immunological Methods, 1985)](https://doi.org/10.1016/0022-1759%2885%2990044-4)
16. [Ethynylestradiol ELISA manual (r-Biopharm)](https://food.r-biopharm.com/wp-content/uploads/2021/02/manual-ethynylestradiol-elisa-5081estr501.21.pdf)
17. [Analytical Validation of a Direct Competitive ELISA for Multiple Mycotoxin Detection in Human Serum](https://www.mdpi.com/2072-6651/14/11/727)
18. [Myonectin/CTRP15 (Mouse, Rat, Human) Competitive ELISA Kit (Aviscera Bioscience)](https://aviscerabioscience.com/Product_Systems/CTRP15/SK00393-10.pdf)
19. [An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions](https://pmc.ncbi.nlm.nih.gov/articles/PMC4828978/)
20. [Immunoassay Methods - Assay Guidance Manual (NCBI Bookshelf)](https://www.ncbi.nlm.nih.gov/books/NBK92434/)
21. [An overview of ELISA: a review and update on best laboratory practices for quantifying peptides and proteins in biological fluids](https://pmc.ncbi.nlm.nih.gov/articles/PMC11808753/)
22. [Binding affinity-guided design of a highly sensitive noncompetitive immunoassay for small molecule detection](https://www.sciencedirect.com/science/article/abs/pii/S0308814621002740)
23. [Development of a competitive inhibition kinetic ELISA to determine the inhibition constant (Ki) of monoclonal antibodies](https://www.sciencedirect.com/science/article/abs/pii/S0022175921000879)
24. [ncbi.nlm.nih.gov](https://www.ncbi.nlm.nih.gov/books/NBK10759/)

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*Topic: Encyclopedia › Life and health › Biological foundations › Immunology and immune-system biology*

*Initially written Sep 29, 2026 · Edited: Oct 15, 2026*

*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*

License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license