Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-Linked Immunosorbent Assay (ELISA) is an antibody application extensively used in many settings, from basic research to diagnostics. This assay is the preferred method to determine the titer of antisera and purified antibodies and can also be successfully employed for the quantitative assessment of an antigen in a sample, often devised in convenient easy to use kit formats. These are some of the key characteristics that make ELISA an ideal method of testing: 


Versatile: Many systems using different combinations of reagents can be used. Allows passive immobilization of reagents to a solid phase and easy separation of bound/unbound reactants using washing steps. 


Simple: Multiple samples can be tested in a variety of formats using microtiter plates and compatible equipment that provides high capacity, rapid and inexpensive assays feasible to kit development. 


Sensitive: Readouts come from enzyme catalysts or fluorescent tags that amplify signal efficiently and provide range of detection optimal for critical assays including diagnostics. 


Quantitative: Signal is detected by multichannel spectrophotometers and therefore the collected data can be stored and analyzed statistically.  


Although all the different systems operate within the common parameters of immobilization to a solid phase, separation of bound and free reagents by washing steps and readout from colorimetric, fluorescent or luminescent signals, each configuration differ in the nature and number of stages needed as well as their possibilities for specialized applications like detection and quantification of antibodiesproteinspeptides and even small molecules.  

Types of ELISA 


The following are the core systems that constitute the basis to the many possible configurations: 


1. Direct ELISA: It is the simplest configuration in which the antigen is bound by passive adsorption to the solid phase, washed to remove any unbound molecules and then directly incubated with a conjugated antibody. Following the incubation period and additional washing, substrate is added to produce signal that is allowed to develop. After certain time, the substrate reaction is stopped and the resulting signal quantified. It is commonly used for tittering conjugated secondary antibodies and very useful to estimate antigen cross-reactivity.  





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2. Indirect ELISA: In this system, initial antigen binding and washing steps are the same as the direct method. The main difference in this case is the use of unconjugated antibody to bind the immobilized antigen upon incubation at optimal temperature (usually 37⁰C). Following a washing step to remove unbound antibodies, the remaining antigen-bound antibodies are targeted by a conjugated secondary antibody that will generate the readout signal as described for direct ELISA. This system has been widely applied in diagnostics because it allows large number of samples to be screened with a single conjugated secondary antibody.  




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3. Sandwich ELISA: This assay requires a compatible antibody pair that recognize different antigenic targets (epitopes) on the same antigen. The first antibody, called capturing antibody, is coated on the plate and used to immobilize the antigen upon binding during incubation with the sample. Free antigen is removed by a washing step and then a detecting antibody is added to bind the captured antigen and enable subsequent detection. Sandwich ELISA is divided into two main systems: 


  • Direct Sandwich: This assay uses a conjugated detecting antibody to an enzyme or fluorescence tag. Following incubation with the antibody-antigen complex immobilized on the plate well, signal detection is performed upon successive addition of substrate and stopping solution or as described before or appropriate excitation/emission of the fluorescent tag.  

  • Indirect Sandwich: This assay uses either an unconjugated or biotinylated detecting antibody that once is bound to the antibody-antigen complex on the well is subsequently detected by either an anti-species antibody or streptavidin  conjugated to an enzyme or fluorescent tag. Detection is then performed as above.  




Please find more details on the sandwich method and convenient, easy-to-use ELISA kits here

Competitive and Inhibition ELISA 


Each of the core systems described above can be further modified to measure molecules based on their ability to interfere with a well-known pre-titrated assay. Such assays can be used to study either antigens or antibodies and they can be competitive or inhibitory based on the specific conditions of each assay. In both types of assays, a pre-titrated system is challenged with the presence of the testing sample whose binding activity is then determined from the degree of the resulting interference in the established system. 



Competitive Fluorescence ELISA

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For the most part, inhibition assays give the testing sample the advantage of binding (i.e. incubated in advance) before the other system’s components are added whereas in competitive assays the testing sample is mix incubated and mixed with the other components of the pre-titrated system.


Overview of Steps in Different ELISA Systems


Below is the description of the main steps in the most common type of ELISA assays and the recommended reagents to use throughout the whole assay. Details on recommended protocols and  technical tips are also provided.


Overview of Steps in Different ELISA Systems




tittering elisa immunoassay

Recommended Reagents



1. The choice of blocking buffer is critical in obtaining genuine intensity signals and reduced backgrounds


For this purpose and to account for unspecific binding between conjugated secondary antibody and the antigen, it is also necessary to include controls of antigen coated wells incubated with the conjugate only. Efficient blocking buffers include BSA and gelatin.

2. Optimize the amount of conjugated secondary antibody to be used


Different conjugates will provide different ranges of signal depending on how much antibody they will bind. Because the goal is to bind the entire antigen-antibody complex present in each well, you want to make sure you will use the right amount of conjugate in your assay by performing a serial dilution assay in advance (see tip #2). Always use high quality antibody conjugates. For alkaline phosphatase conjugates replace use TBS over PBS.

3. Adjust Incubation times to improve the performance of your assay


In general, antigen-antibody complexes will form within two hours incubation at room temperature but in some cases significant stronger specific signal might be obtained with longer incubation times. Following the addition of the substrate, it is important to read the plate within the recommended time for the specific reporter system being used. Chromogenic and chemiluminescent substrates, particularly those used by peroxidase-conjugated antibodies can saturate the signal intensity and reduce the dynamic range of the assay.

4. Once your assay is optimized, always keep the conditions unmodified throughout all steps


Because Enzyme-Linked Immunosorbent Assays are very sensitive, even minor changes in buffer composition, volumes, washing times, antigen/antibody concentrations, temperature or incubation times can have a significant impact in the performance of the assay. It is highly advisable to run all assays in duplicate and include controls that help to account for plate to plate variability.   


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