Indirect Sandwich ELISADrug discovery, Diagnostics, Food testing
ELISA, a Gold standard for diagnosis, detection, and validation
Enzyme-Linked Immunosorbent Assay, typically known as ELISA, is a qualitative and quantitative immunoassay that uses antibodies as probes to specifically detect and quantify molecules of interest, such as proteins, peptides, antibodies, and hormones. It relies both on the ability of immunoglobulins to bind specifically to a target antigen and a detection system whose measurable signal directly reports on the presence and quantity of the antigen. Besides being a powerful research tool for antibody development and screening, the ELISA technique is broadly used in clinical diagnostics as a biomarker detection assay in various areas including cancer, diabetes, cardiovascular disease, viral infection, and inflammation. It is also useful for toxicology testing and detecting allergens in the food industry.
Although several types of ELISA have been developed, they all comprise four steps: coating, blocking, detection, and signal measurement. The experimental setup of ELISA will then vary depending on the biological question, the antigen or analyte to be investigated, and the detection strategy. A typical ELISA test begins with immobilizing the target antigen on a solid surface, generally the polystyrene surface of a multi-well microplate, either by passive adsorption in alkaline conditions (direct coating) or indirectly (indirect coating) using antigen-specific antibodies adsorbed onto the plate surface. Following incubation with the antigen sample, all unsaturated surface-binding sites are covered with a blocking agent to prevent non-specific binding of proteins to the plate, thereby maximizing the signal-to-noise ratio. The detection of the bound antigen involves two different methods: direct or indirect. The direct detection uses enzyme or fluorophore-conjugated antibody that directly recognizes the antigen; while the indirect method consists of probing the antigen-specific primary antibody with a labeled secondary antibody. A common adaptation of indirect detection involves the use of a streptavidin/avidin-enzyme conjugate to bind the biotinylated detection antibody, resulting in signal amplification. Washing between steps is crucial to ensure that non-specific (low affinity) binding events are removed, thus eliminating the background noise. Finally, when using an enzyme, the appropriate substrate is added to trigger the enzymatic reaction and consequently produce a measurable signal, which is either a change of color, fluorescence or luminescence. The intensity of the signal is proportional to the amount of antigen bound to the antibodies and can, therefore, be used as a readout to determine the antigen concentration in the sample by comparing to a standard curve for the equivalent antigen (generally a purified recombinant protein). The most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). The choice of the substrate will depend on the instrumentation available for signal measurement but also on the degree of sensitivity required for the assay. Using chemiluminescence, ELISA is potentially able to detect antigens in the sub-picogram per well range, while colorimetric and chemi-fluorescence detection are able to measure low-to-mid picogram levels of antigen per well. When using a fluorophore, no enzyme or substrate is required. The fluorescent moiety is covalently attached to the secondary antibody or in some cases to the detection antibody or the streptavidin/avidin molecule. The signal is measured using a fluorometer.
Sandwich or Capture ELISA
In a Sandwich ELISA, the target antigen is recognized by two specific antibodies, thus forming a sandwich-like complex. In the simplest form of sandwich ELISA, the first antibody, named capture or coating antibody, is adsorbed onto the well surface and recognizes specifically the target antigen (Figure 1). the second antibody binds to the captured antigen and is called the detection antibody. These two antibodies form a matched pair that essentially undergoes development and validation in order to ensure their ability to work in combination and bind to two distinct, non-overlapping epitopes on the antigen molecule. When developing a matched pair, it is crucial to use two antibodies that have been raised in different host species to prevent cross-reactivity between the coating and detection antibodies. Moreover, a set of monoclonal and polyclonal antibodies can be used, where the monoclonal acts as a coating antibody (for high-specificity binding) and the polyclonal for detecting the antigen. The detection antibody can be either labeled with an enzyme for direct detection (Figure 1) or probed by an enzyme-conjugated secondary antibody specifically directed against the detection antibody (indirect detection). Alternatively, a biotinylated detection antibody can be introduced, which then enables the use of streptavidin-enzyme conjugate (Figure 1). Sandwich ELISA is a highly specific assay because only the target antigen/analyte is captured and detected with the matched antibody pair. This makes it suitable to analyze complex protein samples since the antigen/analyte does not require purification prior to measurement. Sandwich ELISA often requires more optimization than traditional (direct and indirect) ELISAs but delivers higher sensitivity and signal-to-noise ratio (minimal background signal).
Figure 1: A schematic illustration of Sandwich ELISA highlighting two different detection systems; Direct using enzyme-conjugated detection antibody and indirect using the Streptavidin-enzyme conjugate to probe the biotinylated detection antibody.
General Sandwich ELISA Protocol
This protocol represents a general sandwich ELISA involving a biotinylated detection antibody and a streptavidin-HRP conjugate for indirect colorimetric detection using the TMB (tetramethyl benzene) substrate. The protocol can be easily modified and adapted depending on your samples and the working concentrations of your matched antibody pair and enzyme conjugate.
The protocol comprises 4 steps:
- Protein Standard Preparation,
- Plate coating with capture antibody and plate blocking prior to the addition of standards and samples,
- Incubation with biotinylated detection antibody and enzyme conjugate,
- Addition of substrate and colorimetric detection.
The “Protein Standard Preparation” step consists in preparing a serial dilution of the antigen/analyte of interest in order to generate a standard curve. The Concentration of the antigen/analyte in samples is then extrapolated from the standard curve within the appropriate dynamic range. To obtain accurate concentration measurement, the standard curve should display low variation between replicates and a good linearity.
Recommendations and Good Practices
To achieve high accuracy and performance in ELISA, it is crucial to:
- Ensure sample quality and integrity to obtain consistent results,
- Adhere to incubation parameters (time, temperature, light exposure) for optimal antigen binding, detection, and signal intensity,
- Perform proper and thorough blocking and washing to thwart any non-specific interference and eliminate background noise,
- Use suitable multi-well plates, such as polystyrene (PS) or polyvinyl chloride (PVC) 96-well flat-bottom microplates,
- Do not allow the plate to dry out during the assay.