ELISA (Enzyme-linked immunosorbent assay) 9th November 2021 – Tags: , ,


Enzyme-linked immunosorbent assay (ELISA) is a powerful method to detect and quantify a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method allows the analysis of protein samples immobilised in microplate wells using specific antibodies.

ELISAs are usually performed in 96- or 384-well polystyrene plates, which passively bind antibodies and proteins. This binding and immobilisation of reagents is what makes ELISAs easy to design and perform.

The immobilisation of ELISA reagents on the surface of the microplate facilitates the separation of bound and unbound material during the assay. This ability to use high-affinity antibodies and to wash out non-specifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

What is ELISA? (Enzyme-Linked Immunosorbent Assay)

The test ELISA (Enzyme-linked immunosorbent assay) is a test that detects and measures antibodies in the blood and can be used to determine whether the patient has antibodies related to certain infectious diseases.

The enzyme-linked immunosorbent assay (ELISA) is an antibody-based test method. This widely used technology is sensitive, rapid and reliable.

Antibodies are proteins that the body produces in response to harmful substances (called antigens). It is used in many laboratories to determine whether an antibody is present in a patient’s blood sample. Although the procedure is routine and simple, it involves a large number of variables, such as reagent selection, temperature, volume measurement and time, which if not adjusted correctly, can affect the successive steps and the outcome of the test.

Enzyme-linked immunosorbent assay (ELISA), also known as enzyme immunoassay (EIA), is a biochemical technique used primarily in immunology to detect the presence of an antibody or antigen in a sample. ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as for quality control in various industries, such as application in the food industry.

The technique was proposed as an alternative to radioimmunoassay, as the latter posed a health risk due to the radioactive isotopes used.

This technique differs from others based on the Ag-Ac reaction in that binding to a solid surface allows the identification of specific reactions and therefore quantitative results to be obtained.

The ELISA method is a benchmark for the quantification of pathological antigens and, in fact, many variations of this method exist. These are suited to high-throughput screening because the results are rapid, consistent and relatively easy to analyse.

The best results have been obtained with the sandwich format, using highly purified, pre-matched capture and detector antibodies. The resulting signal provides very sensitive and highly specific data.

Principles of the ELISA assay

Antibodies are proteins produced in vertebrate plasma cells as part of the adaptive immune system against structures (antigens) that the body recognises as foreign elements.

Antibodies bind to the corresponding antigens using a distinct pattern of ionic and hydrophobic interactions, hydrogen bridge bonds and Van-der-Waals forces. The interaction between the antibody and the corresponding antigen is selective and highly specific, similar to that of a lock and key.

The enzyme-linked immunosorbent assay is based on this selective and specific antibody-antigen recognition Many qualitative and quantitative ELISA assay formats have been established.

The performance of an ELISA assay requires at least one antibody specific for a particular antigen. According to the basic method, one of the immunological components is immobilised on a solid phase in the cavities of the microtitre plate. The sample analyte interacts with the antibody-antigen system. This interaction can be visualised by enzymes, bound to antigens or secondary antibodies, and indicates whether antigen-antibody binding has occurred. The bound enzyme converts an added substrate, resulting in a colour change, which can be measured by a spectrophotometer.

Types of ELISA assays

The ELISA experiment begins with the step of immobilising the antigen of a given sample in the wells of a multiwell plate. The immobilisation process can be achieved by two methods including direct adsorption to the surface of a plate or by an antibody capture probe adsorbed to the plate. The antibody probe must be highly specific towards the antigen of interest. Immobilisation is followed by the addition of the detection antibody resulting in an antigen-antibody complex. The antibody applicable for detection is usually labelled by exploiting enzymes such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

ELISAs can be classified into four main categories, namely direct, indirect, sandwich and competitive, depending on the modifications made to the basic procedures. The sandwich ELISA is the most powerful ELISA format due to its high sensitivity and robustness The choice of substrate for the assay depends on the sensitivity required, the type of instrumentation needed and its availability.

ELISA direct

ELISA plates are prepared by coating the wells with the solutions in which the antigen is suspected to be present. They are incubated with labelled antibodies. They indicate the presence of antigen in the solution tested.

It is necessary to include negative controls, which are samples of the same type as those analysed (blood, urine, etc.), but in which the absence of the antigen sought is certain. Positive controls are also included (solutions in which the antigen sought is found).

  • Fast and with minimal procedural steps.
  • Minimal precursor requirement makes it less error prone.
  • Antigen immobilisation is not specific, so background-related problems are observed..
  • It offers less flexibility in terms of the primary antibody.
  • The absence of signal amplification reduces sensitivity.
ELISA indirect

ELISA plates are prepared in the same way as above. The positive and negative controls are the same. The detection system uses two antibodies: a primary antibody against the antigen and a secondary antibody labelled against the primary antigen.

The detection is more sensitive because of the signal amplification due to the binding of two or more secondary antibodies to each primary. It is the most popular assay, as is indirect immunofluorescence, because the same secondary labelled and enzyme system allows the quantification of a wide variety of antigens; it is therefore a more versatile and cheaper method, although it loses some precision by having an extra link compared to the direct method.

The dilution of the solution containing the primary antibody – e.g. blood serum – is a very important factor to take into account to avoid false negatives, because if the sample is too dilute, it will not be positive if the antibody titration is too low (i.e. although antibodies are present, the test is not positive because the concentration of specific antibodies against the antigen that is stuck to the bottom of the well is not sufficient to give a detectable signal).

The ELISA indirect is the method of choice for detecting the presence of serum antibodies to the human immunodeficiency virus. (VIH), the causative agent of acquired immunodeficiency syndrome (AIDS).

According to this technique, recombinant HIV envelope and core proteins are absorbed as solid-phase antigens into the wells. HIV-infected individuals produce serum antibodies against epitopes on these viral proteins.

In general, the indirect ELISA can detect serum antibodies to HIV from as early as six weeks after infection.

  • It offers high sensitivity and flexibility as the number of secondary antibodies can bind to a primary antibody and one type of secondary antibody can tag different primary antibodies.
  • It is cheaper, as fewer labelled antibodies are needed.
  • Higher signal-to-noise ratio.
  • Time-consuming and labour-intensive.
ELISA «sándwich»

(Antigen capture and immunocomplex detection assay).

This is a widely used assay in which the well is coated with a first anti-antigen antibody. After washing away the excess antibody, the test sample containing the antigen, which will be retained in the well upon recognition by the first antibody, is applied.

After a second wash that removes the non-retained material, a solution with a second labelled anti-antigen antibody is applied.

Thus, each antigen molecule will be bound to an antibody at the base that retains it and at least one second antibody that labels it.0 This assay has a high specificity and sensitivity due to the signal amplification allowed by the second antibody.

  • Offers high sensitivity compared to direct or indirect ELISA
  • It introduces a highly specific reaction due to the participation of two antibodies for the detection of the antigen.
  • Both direct and indirect techniques are used for detection.
  • Optimisation in terms of antibodies is problematic due to cross-reactivity issues.
  • For epitope-specific recognition, only monoclonal antibodies can be applied as matched pairs.
  • Obtaining monoclonal antibodies is a tedious process in the case of matched pairs and is more expensive than polyclonal antibodies.
ELISA inhibitor

This type of ELISA is the most complex. It is used to detect or quantify antigens present in low quantities. It is so called because a reference antigen is used which will compete with the antigen in the sample for binding to the antibody. The simplified procedure would be as follows:

  1. The reference antigen is immobilised on the plate.
  2. On the other hand, an excess of unlabelled primary antibody is incubated with the sample containing the antigen of interest, resulting in the formation of antigen-antibody complexes.
  3. The antigen-antibody mixture is added to the plate, where the reference antigen will compete with the antigen in the sample for binding to the antibody.
  4. The plate is washed to remove soluble antigen-antibody complexes.
  5. An enzyme-labelled secondary antibody is added to the plate which will bind to the primary antibody anchored to the reference antigen.
  6. The substrate is added, which upon reaction with the enzyme will provide a visible signal.
  • Negligible sample processing is required and can be applied to raw samples.
  • Less sensitive to experimental errors.
  • Good reproducibility and flexibility.

ELISA (enzyme-linked immunosorbent assay) is a widely used application for detecting and quantifying proteins and antigens from a variety of samples. Target-specific ELISA kits are available from a variety of manufacturers and can help streamline your immunodetection experiments.

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