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Assay tests are analytical approaches for detecting and analyzing the qualitative and quantitative properties of a target molecule or entity. Such tests have multiple applications across different industries applying molecular biology. 

Although multiple screening assay tests exist, ELISA tests remain the most popular assay in biotechnology. ELISA (Enzyme-linked Immunosorbent Assay kits) primarily detects gene analytes from complex or crude biological specimens. The kits are popular for their high sensitivity, specificity, and reliability. 

However, commercial ELISA tests are expensive, and one study shows that they vary in quality. Therefore, click ELISA kits to learn the test’s basics or read the guide below. 

How Does An ELISA Kit Work: The Principle Of Elisa 

The ELISA test is an immunological or bioanalytical assay that leverages the antigen-antibody reaction. Antigens are disease-causing pathogens like bacteria, viruses, and fungi, while antibodies are the body’s defense mechanism that neutralizes antibodies.  

When an antigen enters the body, the immune system’s B and T cells recognize it, prompting an immune response that releases complementary antibodies. The relationship between antigens and antibodies is complementary because an antibody recognizes an antigen via specific sites called epitopes.  

Moreover, the antibody features a site called a paratope via which it binds to the antigen’s epitope, neutralizing it and preventing it from attaching to and damaging healthy cells. The complementary relationship between paratopes and epitopes means that the two binding sites have a remarkably high affinity. 

Therefore, ELISA kits use conjugated enzyme-conjugated antibodies immobilized on a microplate surface to detect the target antigen. According to one literature review, the conjugation of enzymes to antibodies allows the antibody to form covalent bonds with the antigen without losing its function. Moreover, conjugation is also a detection strategy, helping identify the target antibody. 

Each antibody conjugation enzyme has different physical characteristics like color change (chromogenic) or emitting fluorescent signals when the conjugated enzyme forms covalent bonds. ELISA kits detect monoclonal and polyclonal antibodies. Below are the ELISA formats or commercial kit types available on the market. 

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ELISA Protocol/Workflow 

The ELISA protocol refers to steps included in conducting an ELISA test, and although different test types are available, the primary protocols cut across each step format. Therefore, Below is a highlight of each ELISA protocol step.  


The First step entails coating an ELISA plate with a suitably diluted biorecognition agent (antigen or antibody). Diluting the biorecognition agent in a coating buffer immobilizes them to the walls of the ELISA plate.  

Coating entails incubating the antigens or antibodies until they become fully absorbed into the plate. Moreover, the coating buffer maximizes adsorption, increasing the test’s sensitivity. Carbonates and bicarbonate compounds are the most popular coating buffers for ELISA tests. 


Besides coating buffers, blocking buffers are also essential in the ELISA protocol. A blocking buffer is an irrelevant protein mixture incubated into the ELISA plate after complete adsorption of the coating buffer. Its sole function prevents non-specific binding to plate surfaces, although it also preserves the biorecognition agent’s bioactivity. 


Detection in ELISA kits entails adding a substrate that undergoes physical changes when the immobilized antigen or antibody binds to a target analyte. 

Final Read/Signal Measurement 

ELISA kit test results interpretation can take a qualitative, quantitative, or mixed approach. Quantitative results are ideal for straightforward “yes or no” tests, while qualitative results may require mapping a graph from the ELISA plate. 

Note that a washing step using a washing buffer is necessary between subsequent steps. Moreover, it may be required to use a washing buffer at least once within each stage, depending on the test’s specifics. 

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3. ELISA Formats 

Although commercial ELISA kits are available, you can design your kit. Nonetheless, below are the ELISA formats/types for commercial and custom kits. 

Direct ELISA 

Direct ELISA kits use a single or primary conjugated antibody that reacts to the antigen in a heterologous sample to yield results. Its advantages include that it involves fewer steps and inhibits cross-reactivity. However, it poses challenges, including minimal flexibility, and leaves zero room for signal amplification. 

Indirect ELISA 

Indirect ELISA tests feature a primary non-conjugated antibody and a secondary conjugated one. It counters a direct ELISA’s flexibility and signal amplification challenge but presents the risk of secondary antibody cross-reactivity. 

Sandwich ELISA 

A sandwich ELISA also features two antibodies. However, rather than a primary and a secondary antibody, it features a capture antibody and a detection antibody working in tandem to “sandwich” the antigen. It is ultra-sensitive and ideal for detecting complex specimens, but cross-reactivity is possible. 

Competitive ELISA 

Competitive ELISA tests entail using two antibodies or two antigens to determine antigen or antibody concentrations in an unknown sample. The two antigens or antibodies compete to form covalent bonds with the polar opposite in the specimen. Its advantages include high specificity and fewer purification steps. 

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4. ELISA Substrates 

Substrates in ELISA tests facilitate the signal generation, leading to test results and interpretation. Moreover, the substrate used also influences the test’s specificity and sensitivity. 

The standard ELISA substrate types are PPNP, ABTS, OPD, TMB, and ONGP. However, each substrate has advantages and disadvantages from ease of use, cost, detection limits, and compatibility with different protocols. Therefore, consider such factors to choose compatible substrates. 

Detection Strategies 

Elisa Kits utilize detection strategies like chromogenic, radioactivity, fluorescence, and chemiluminescence for signal detection. Different kits have unique detection strategies, so find a detection strategy that works for your project. 


The guide above is an overview of ELISA kits, their components, and their functionality. However, it is a viable basis for further research to help you navigate the complex world of biorecognition and make informed consumer choices. 

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