Additional Information on Competitive ELISA

The technique was developed in 1976 Yorde and his coworkers (1). In the competition ELISA for antibody detection, the test sample (containing free antibody) and enzyme-labelled antibody conjugate are simultaneously added to the antigen coated wells. They compete with each other for the antigen. If free antibody is present in the test sample it binds to the antigen and subsequent addition of substrate, after washing, does not elicit colour development. Hence, colour development in this assay is inversely proportional to the quantity of specific antibody in the test sample. In simple terms when the amount of the antigen or the antibody analyzed in the serum is low, high absorbance is obtained, whereas low absorbance is observed with greater quantities. Sera with preservatives like sodium azide are unsuitable for competition ELISA format, since the preservative may inactivate the enzyme. It is also known as a blocking/inhibition ELISA and perhaps the most complex of all the ELISA techniques. Different ELISA formats like direct, indirect, sandwitch all can be adapted to a competitive format. The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Competitive ELISA is useful for the measurement of low molecular weight targets. Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.

In a competitive/blocking ELISA, immunoglobulins from positive serum samples compete to inhibit the mAb from binding to its specific binding site on antigens and subsequently prevent color development whereas non-reactive sera produce a strong colored reaction. Competitive/blocking ELISAs have been confirmed to be significantly sensitivite and specific for detecting antibody to Bluetongsue Virus (2), West Nile virus (3), and Francisella tularensis (4), and even a higher sensitivity than i-ELISA for detection to Mycoplasma meleagridis (5) and PRRSV (6).

Competitive ELISA has also been developed and used for detection of antibody to Bluetongue virus (BTV) in other countries (7). Authors have described the development and evaluation of C-ELISA for the detection of BTV antibody using anti-core antibody which eliminate the requirement of different species-specific secondary antibody conjugate, unlike an indirect ELISA. The present assay may be used for sero-surveillance of bluetongue in small and large ruminants. The competitive ELISA detects effectively compositional differences in complex antigen mixtures, even when the specific detecting antibody is present in relatively small amounts.

A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases (8). Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, with a lowest detectable amount of 0.03 pmol of enzymatic protein. The ELISA format was sensitive, convenient for clinical laboratories and also has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. The competitive ELISA detects effectively compositional differences in complex antigen mixtures, even when the specific detecting antibody is present in relatively small amounts.

In a recent study scientists utilized competitive ELISA for the quantitative determination of CIGB-210 using an anti-CIGB-210 hyperimmune serum (9). Vimentin is one of the cellular proteins involved in HIV replication. It is a constitutive cytoplasmic protein and belongs to the family of intermediate filaments (IF) of the cytoskeleton. Vimentin knockdown MT4 cells are refractory to HIV replication. CIGB-210 is a synthetic peptide derived from human B keratin and its pretreatment with MT4 cells triggers the redistribution of vimentin IF. Also it has been shown to inhibit HIV replication at the nanomolar range.[7] The synthetic peptide CIGB-210 is a promising anti-HIV drug candidate and they showed development of a competitive ELISA to measure the levels of CIGB-210 in plasma. This report demonstrated the utility and feasibility of this analytical method for pharmacological experiments in animals as humans. The competitive ELISA developed by the authors is a valuable instrument for pharmacokinetic studies of CIGB-210 in animals and humans.

Patients with inflammatory diseases are monitored on a personalized level by monitoring of TNF-alpha inhibitors. In a recent study authors developed innovative competitive ELISAs (cELISAs) for the detection of neutralizing antibodies against infliximab and adalimumab and compare their results to reporter gene assay (RGA). It has been clinically observed that many patients receiving TNF-alpha inhibitors, with negative drug and anti-drug antibody results from bridging ELISA (bELISA), lose their drug response over time, despite dose optimization. In the study clinical data on patients showed the usefulness of cELISA which demonstrated greater clinical utility as well as represented cost, time, and labor-effective assay (10).

The competitive enzyme-linked immunosorbent assay (ELISA) can be developed in direct or indirect ELISA format as well. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody’s binding site is not occupied by either the antigen standard or antigen in the test samples. Whereas in the indirect competitive ELISA (indirect cELISA) pits plate-immobilized antigen against antigens in solution for binding to antigen-specific antibody. The antigens in solution are in the test sample and are first incubated with antigen-specific antibody. These antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. In both the formats further wells are washed to remove unbound antigen-antibody complexes and free antigen followed by addition of a reporter-labeled secondary antibody and substrate. The indirect cELISA is often used for competitive detection and quantification of antibodies against viral diseases in biological samples.


  1. Yorde DE, Sasse EA, Wang TY, Hussa RO, Garancis JC. Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin. Clin Chem 1976;22:1372–7.
  2. Reddington J, Reddington G, MacLachlan N. A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus. J Vet Diagn Investig. 1991;3(2):144–147. doi: 10.1177/104063879100300207.
  3. Hirota J, Shimizu S. A new competitive ELISA detects West Nile virus infection using monoclonal antibodies against the precursor-membrane protein of West Nile virus. J Virol Methods. 2013;188(1-2):132–138. doi: 10.1016/j.jviromet.2012.12.002.
  4. Sharma N, Hotta A, Yamamoto Y, Fujita O, Uda A, Morikawa S, Yamada A, Tanabayashi K. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay. Clin Vaccine Immunol. 2013;20(1):9–16. doi: 10.1128/CVI.00516-12.
  5. Dufour-Gesbert F, Kempf I, Kobisch M. Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis. Vet Microbiol. 2001;78(3):275–284. doi: 10.1016/S0378-1135(00)00280-7.
  6. Ferrin NH, Fang Y, Johnson CR, Murtaugh MP, Polson DD, Torremorell M, Gramer ML, Nelson EA. Validation of a blocking enzyme-linked immunosorbent assay for detection of antibodies against porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol. 2004;11(3):503–514.
  7. Afshar A, Eaton BT, Wright PF, Pearson JE, Anderson J, Jeggo M, et al. Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen. J Vet Diagn Investig 1992;4(3):231e7.
  8. Battelli MGAbbondanza AMusiani SBuonamici LStrocchi PTazzari PLGramantieri LStirpe F. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA). Clin Chim Acta. 1999 Mar;281(1-2):147-58.
  9. Duarte CA, Chávez S, Masforrol Y, Puertas S, Paneque T, Ramirez AC, Casillas D, Puente P, Garay H, Fernández-Ortega C. A competitive ELISA for the quantitative determination of the novel anti-HIV drug candidate CIGB-210 in biological fluids. J Immunoassay Immunochem. 2018 Nov 29:1-21. doi: 10.1080/15321819.2018.1547975. [Epub ahead of print]
  10. Ogrič M, Žigon P, Lakota K, Praprotnik S, Drobne D, Štabuc B Sodin-Semrl S, Čučnik S. Clinically important neutralizing anti-drug antibodies detected with an in-house competitive ELISA. Clin Rheumatol. 2018 Jul 16. doi: 10.1007/s10067-018-4213-0. [Epub ahead of print]