ELISA TECHNIQUE: PRINCIPLE AND TYPES

Introduction

Enzyme-linked immunosorbent assay (ELISA) technique is one of the most familiar names for the production of in vitro diagnostic products, use in medical laboratories, regulatory bodies, and extrinsic trait analysis as well as competence evaluation organizations.

Engvall and Perlmann issued their first paper on ELISA in 1971 and introduced the name. Meanwhile, van Weemen and Schuurs described similar procedures in the same year reporting the possibility of quantifying human chorionic gonadotropin concentration in urine.

It is a biochemical assay that discovers if there exist any of either antigen (proteins, peptides, hormones, etc.) or antibody in a given sample by the use of antibodies and an enzyme-mediated color. It is an easy, delicate, swift, trustworthy, and flexible assay system for the quantitative determination of antigens and antibodies. Due to the rapid development of the ELISA, it has also become a substantial upgrade to the existing serological tools.

ELISA is also referred to as “heterogeneous” enzyme immunoassays instead of the “homogeneous” enzyme immunoassays because there is no partition between free and bound reactants during the procedure. Based on specific uses of the assay, ELISAs can also develop in various numbers of configurations. The ELISA system’s configuration depends mainly on the specific nature and purity of accessible antibodies or antigens.

General Principle of ELISA Technique

The general principle of an ELISA is to use an enzyme to detect the binding of antigen and antibody for the identification of small amounts of antigens such as proteins, peptides, hormones, or antibodies in a fluid sample. 96-well microtiter plates are used to immobilize the antigen in fluid and allow the antigen to bind to a specific antibody that is thereafter detected by using a peripheral, enzyme-coupled antibody. The enzymes used are mainly alkaline phosphate (EC 3.1.3.1) and glucose oxidase (E.C. 1.1.3.4) which transforms a pale substrate (chromogen) to a visible colored product that indicates the presence of antigen. Finally, various qualitative and quantitative measures can be used that depend on the colorimetric reading for sensitivity and concentration levels of antigens in the sample.

Source: Journal of Investigative Dermatology (2013)

Types of ELISA techniques

Direct ELISA

It is the simplest form of the ELISA in which, the antigen is added to the solid phase that passively soaks up on incubation (for one hour at 37°C or can be incubated at 4°C overnight). Any unbound antigen is washed away after incubation with the use of agents (BSA, ovalbumin, aprotinin, or other animal proteins) with only the solid phase now remaining.  Then add specific antibodies for the antigen designate with an enzyme (conjugate) and incubate the plate again. The unbound conjugate is washed away again after the enzymes bind with the antigen on the solid phase. A chromophore (color carrier) is additionally supplied which acts as a catalyst for the enzyme to produce a colored product. Finally, the reaction concludes after a brief period and then the colorimetric reading is analyzed by employing a spectrophotometer.

Indirect ELISA

 Numerous antiserums can be examined for binding to a given antigen by utilizing only a single anti-species conjugate. Mostly utilized for the diagnostic applications while exploring a large number of samples. It is identical to the direct ELISA excluding the fact of having an additional washing stage and the varieties of antibody supplied later when the buffer gets removed. It needs two antibodies: the main detection antibody that binds to the protein and a peripheral enzyme-linked antibody. First of all the main antibody is added, the plates are washed and then the peripheral enzyme-linked antibody is supplied and the plates are incubated. The steps are now similar to the steps of direct ELISA subject to the detection of a color change. It offers high sensitivity compared to direct ELISA. However, we may have a different number of imprecise bindings in particular sera.

Sandwich ELISA

It is highly sensitive to other ELISA types but relatively expensive. The antigens are in layers separating the two sections of antibodies used for capturing and detection. It is important to coat the well surface with a certain amount of bound antibody for acquiring the required antigen. After adding a particular primary (capture) antibody to the plates and incubating it overnight,  the plates are buffer washed at room temperature and the plate is washed with PBS (Phosphate buffer saline) again before adding the antigen. The required antigen of concern is added and bound the antigen with the primary antibody through incubation. After washing the plate again, the detecting antibody is added and incubated at room temperature again that is accompanied by a buffer wash, Then the enzyme-linked secondary antibodies are applied to bind with the primary antibody later washing the plate to remove the unbound antibody-enzyme conjugates. Finally, the substrate that produces a color change which can later be analyzed is added.

Competitive ELISA

In competitive ELISA, there is competition for binding with the main antibody between the sample antigen and the antigen present to the wells of the microtiter plate. The main antibody with the sample antigen is incubated and is added to the well coat with the same antigen and later washed for removing the unbound antibody. The amount of the antigen or the antibody to analyze must be low so that it can achieve high absorbance or else there will be low absorbance in case of greater quantities. Then add the secondary antibody linked to an enzyme tracked by a substrate that triggers a color development or glowing signal. The secondary antibody conjugated is added to an enzyme, followed by a substrate to elicit a chromogenic or fluorescent signal. The absence of color indicates the presence of antigen in the sample. If there is a change in color of the substrate, the test is negative and there is no presence of antibody in the sample. If there is no change in the color of the substrate, we have an antigen present in the sample.

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