Selecting the Right ELISA and Optimization
The enzyme-linked immunosorbent assay (ELISA), first described in 1971 by Engvall and Perlmann. During that time the common assays used radioactively labeled antibodies and antigens and so ELISA was a welcome change. Initially, it was used for the detection of immunoglobulin G. An observation in the 1960s that antibodies or antigens can be adsorbed to a solid surface and can still participate in the high-affinity binding led to the development of the ELISA. Today, term ELISA refers to a wide range of immunoassays some involving enzymatic reactions as well. The antibodies play a major role in determining the sensitivity and specificity of the assay. They are widely used across several areas of biomedical research and discovery as well as for diagnostic purposes. There are different ways to do ELISA and usage of each one depends on several factors. Determining the right ELISA to address specific research question necessarily depends on the desired sensitivity and exactly what is being detected. Also before choosing an ELISA technique the different types of ELISAs, focusing on the advantages and disadvantages of each should be known to the user. The first is what format to use; direct, indirect, sandwich, Competition or Inhibition ELISA. Formats differ in how the target antigen is captured and detected.
Selecting an ELISA technique
There are many factors that determine which ELISA technique is appropriate. For example, a small molecule such as a hapten detection needs competitive ELISA whereas, a large protein with multiple epitopes, such as a cytokine needs sandwich ELISA. If only one antibody is available for an antigen of interest then a direct or competitive ELISA can be applied. In order to detect or quantitate an analyte, a sandwich or competitive ELISA can be utilized. For measuring an immunological response, a direct or indirect ELISA is most suitable.
Optimization of the ELISA technique
Optimization of the plate coating conditions for the antigen or antibody coating is the first step while developing a new ELISA. A microplate with a minimum protein-binding capacity of 400 ng/cm² and CV value (coefficient of variation) <5% is preferred. The choice of plate color depends upon the signal being detected. Plates should be inspected visually before use. Imperfections or scratches in the plastic should be avoided as it will cause aberrations when acquiring data from the developed assay. Clear polystyrene flat bottom plates are used for colorimetric signals while black or white opaque plates are used for fluorescent and chemiluminescent signals.
It is important to note that optimal coating conditions and plate binding capacity can vary with each protein and must be determined experimentally. During coating process, passive adsorption of the protein occurs to the plastic of the assay microplate. In this process, hydrophobic interactions occur between the plastic and non-polar protein residues. Different proteins may require specific conditions or pretreatment for optimal binding. Coated plates can be used immediately or dried and stored at 4° C for later use, depending on the stability of the coated protein. Generally, coating is done with more capture protein that can actually be bound to facilitate the largest working range of detection. However, competition ELISAs are an exception to this. On the other hand, antibodies are coated on the plates at a concentration lower than the maximum binding capacity. This is done to prevent nonspecific binding which might affect the sensitivity of an assay.
Passive adsorption works for proteins. Problems arising from passive adsorption are improper orientation, denaturation, poor immobilization efficiency and binding of contaminants along with the target molecule. Antibodies can be attached to a microplate through the Fc region using Protein A, G, or A/G coated plates. This binding orients them properly and preserves their antigen binding capability. Fusion proteins can be attached to a microplate in the proper orientation using glutathione, metal-chelate, or capture-antibody coated plates. Peptides and other small molecules, which typically do not bind effectively by passive adsorption, can be biotinylated and attached with high efficiency to streptavidin or NeutrAvidin protein coated plate. Biotinylated antibodies also can be immobilized on plates precoated with biotin-binding proteins. Using pre-coated plates in this manner physically separates the antigen or capture antibody from the surface of the plate as protection from its denaturing effects. Pre-coated ELISA plates are good options for the above-reported problems.
The antibody used for capture and detection can be either monoclonal or polyclonal in sandwich ELISA systems. A polyclonal is often used as the capture antibody to pull down maximum antigen, whereas a monoclonal is used as the detection antibody in the sandwich assay to provide specificity. Recombinant monoclonal antibodies are also in use for ELISA. They have an edge in terms of no lot-to-lot variation over traditional monoclonals.
To reduce the background signal and improve the signal-to-noise ratio blocking buffers are used. A blocking buffer can be a solution of irrelevant protein, a mixture of proteins, or other compounds. They passively adsorb to the remaining binding surfaces of the plate. Nonspecific binding, including various protein: protein interactions unique to the samples and antibodies involved. The ideal blocking buffer reduces background without altering the epitope for antibody binding. While optimizing new ELISA, different blockers should be tested for the highest signal: noise ratio in the assay. The signal: noise ratio is measured as the signal obtained with a sample containing the target analyte in comparison to sample without the target analyte. An appropriate concentration of blocker is important as less amount reduces signal: noise ratio and excessive concentrations of blocker may mask antibody-antigen interactions causing reduction of the signal: noise ratio.
Washing steps play a very important role in ELISA to remove nonbound reagents and decrease background. Ultimately they increase the signal: noise ratio. Washing buffers are physiologic buffers Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) with a detergent such as Tween-20. Detergent is added to remove nonspecifically bound material. Dilute solution of the blocking buffer along with some added detergent is also used. Insufficient washing leads to a high background, while excessive washing might result in decreased sensitivity
The detection step in all ELISA systems involves an enzyme substrate. The enzyme converts the substrate to a detectable product and the intensity of the signal produced is directly proportional to the amount of antigen captured in the plate and bound by the detection reagents. Enzyme-conjugated antibodies (especially those involving horseradish peroxidase, HRP) offer the most flexibility in detection and documentation methods for ELISA because of the variety of substrates available for chromogenic, chemifluorescent and chemiluminescent imaging. Chromogenic ELISA substrates allow direct visualization and enable kinetic studies. Furthermore, chromogenic ELISA substrates are detected with standard absorbance plate readers common to many laboratories. Fluorescent or chemiluminescent substrates are more sensitive but are not as common and require a fluorometer. For assays requiring many plates to be read, the signal might begin to decay before plates are read.