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Posted on Thu, Sep 19, 2019

ELISA steps, principle, and how it works

The ELISA (Enzyme Linked ImmunoSorbent Assay) is the gold star immunoassay, which means that it is the standard procedure that all new assay technology is compared to during research and development. The ELISA is also fundamental to most clinical tests for diagnosis of disease because it is currently the most characterized and standardized method. The ELISA is an immunoassay, the principle of which relies on the specific recognition between an antibody and antigen. This specificity comes from the unique three dimensional structure of the antibody paratope and the antigen epitope. These two regions fit like a lock and key via non-covalent, charge-based, and/or hydrophobic interactions. The clinical purpose of the ELISA is to detect either antibody or antigen from a biological fluid such as blood (serum), urine, or saliva. When the ELISA is used to antibody, the assay is being used to assess whether or not the patient has been exposed to a certain antigen at some point. It is difficult to assess current infection with this method because the body retains antibodies forever after the first introduction. However, elevated amounts of antibody can be indicative of active immune response to the pathogen. One major benefit of the ELISA is that it is quantitative, meaning that an actually number of protein can be evaluated. When the ELISA is used to detect antigen it provides a better understanding of current infection since the antigen would be cleared if it was no longer active in the body. 

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Sandwich ELISA steps

The ELISA is composed of multiple steps. Many of these steps are blocking and washing steps in order to prevent non-specific binding. Non-specific binding occurs when proteins adsorb to each other or to the plastic surface of the ELISA plate via off-target interactions. These interactions are also mediated by charge or hydrophobicity, so they can be mitigated to some extent by blocking and washing. The blocking steps involve the introduction of proteins the well to adsorb to the open sites on the plastic. This prevents the target protein from accessing these sites later on in the assay. The washing steps include detergents to reduce off-target hydrophobic interactions between proteins and to remove any unbound proteins from the system.  

Step 1: Immobilization of the capture protein

The ELISA assay is performed in a 96-well polystyrene plate. The wells are chemically treated to make them “sticky” to increase the ability for proteins to adsorb to the surface. The first ELISA step is to immobilize the capture protein to the wells of the plate. There are a handful of different ELISA assay techniques, but here we will focus on the sandwich ELISA. In a sandwich ELISA the capture protein is an antibody and the target is the antigen. Therefore, the first step is to immobilize the capture antibody onto the wells of the 96-well plate. Many ELISA assay kits are available with the capture protein pre-immobilized. 

Step 2: Wash off any unadsorbed capture protein from the well surface

Step 3: Block any unbound sites on the 96-well plate

Proteins such as Bovine Serum Albumin (BSA), Casein, or aprotinin are commonly used to block the ELISA assay. These proteins will adsorb to the plate surface. This will prevent the target protein from adsorbing non-specifically to the plate surface during later steps in the assay, which results in lower background noise

Step 4: Wash away any unadsorbed blocking proteins from the well

Step 5: Incubate with the sample (serum, urine, saliva, or spiked research solution)

This is the step where the specific recognition between antibody and antigen takes place. In this sandwich format the antibodies are adsorbed the well and are recognizing antigen in the sample fluid. This step requires some incubation time in order to allow the binding kinetics to reach equilibrium. 

Step 6: Wash away the incubation fluid 

This is a crucial washing step that often requires multiple washes to ensure that any unbound antigen is washed away. Ideally, the only antigens that remain in the well are attached to capture antibodies. 

Step 7: Incubate with Detection Antibody

The detection antibody in an ELISA always has some type of conjugated label such as a flurophore for fluorimetry, or an enzyme for colorimetric detection or chemiluminiscence. This antibody recognizes a different epitope on the target antigen than the capture antibody did. As a result, the ELISA assay has a sandwich of capture antibody-antigen-detection antibody. 

Step 8: Wash away unbound detection antibody

Step 9: Apply substrate for chemical colorimetric or chemiluminiscent reactions or apply incident light for fluorescent reactions, and quantify the signal

The amount of fluorescence, luminescence, or intensity of the color indicates how much target antigen was captured from the sample. This is where a high signal and low background/noise is important. A good assay has a high signal to noise ratio and produces a clean result without much background. The better the signal to noise ratio the better the sensitivity of the assay, and the lower the limit of detection. 

A low limit of detection is the goal of any immunoassay or sensing device. A significant amount of research is directed towards creating state-of-the art devices that can detect ever smaller concentrations of target protein from complex biological samples. These ELISA steps and principles such as blocking, washing, and reducing non-specific binding are crucial to the success of any protein assay.


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