The ELISA technique is a powerful and effective immunoassay. The key to an effective ELISA assay, however, is development and optimization. The development of a large immune complex can be difficult, with the success of the test being determined by the failure or incorrect optimization of any component. ELISA signal development can be influenced by a variety of circumstances.
Before proceeding with several samples of unknown composition, it is recommended that you first build and improve a standard curve for your analyte of interest. You can process the samples if your standard curve displays range, sensitivity, and linearity.
Plate Type and Adsorption
The absorbance of the colorimetric compounds used in ELISA is measured using a laser that shines up the base of each well; hence a flat bottomed plate with a transparent base is required. If fluorescent detection is implemented, the plate must be opaque white or black, and fluorometric plate readers can read the plate from above or below. The use of black or white opaque plates with a transparent bottom is required for chemiluminescent detection. Chemiluminescence is best accomplished on white plates since they enhance the signal. Black plates are suitable for fluorescence detection because they release less background.
Polystrene, polypropylene, or polycarbonate plates can be used for an ELISA. Many of the plates are gamma irradiated to create a positive charge that helps with coating.
Passive adsorption is the most usual process of securing proteins to plates. In addition to some electrostatic forces, this approach relies on hydrophobic interactions. Carbonate-bicarbonate (0.2M sodium/bicarbonate pH 9.4) is a widely used coating buffer. The high pH of this solution enhances the solubility of many proteins and peptides, as well as ensuring that proteins are unprotonated and have a net negative charge, which aids in attaching to a positively charged plate. Tris-buffered saline (TBS) or phosphate buffered saline (PBS) at physiological pH are other buffer alternatives. Depending on the requirements of your assay, pre-coated plates are also an option.
Antibodies
Not all antibodies are appropriate for ELISA and will need to be tested before using experimental samples. The antigen’s three-dimensional structure may be changed during the adsorption process, causing it to lose its ability to attach its target epitope. Furthermore, if an antibody is created against a peptide sequence that is part of the internal sequence of the antigen of interest, the antibody may not bind the entire antigen when immobilized on a plate.
In addition for an antibody to work in an ELISA experiment, it must bind selectively to the antigen and not cross react with blocking buffer components. When two antibodies are needed, such as in a sandwich ELISA, the selected antibodies must react with different antigen epitopes. The detection antibody must be capable of binding with its own epitope without steric hindrance from the first antibody on the plate if the antigen is immobilized by a capture antibody. Various species require different levels of capture and detection.
The concentration at which the antibodies will be utilized is another crucial thing to consider when preparing an ELISA examination. This will involve optimization and may be influenced by the substrate.
Blocking Buffer
A blocking buffer is a solution of irrelevant protein, protein mixtures, or other substance that links to all of the plate’s remaining binding surfaces passively. When a blocking buffer increases an assay’s sensitivity by minimizing background interference and increasing the signal-to-noise ratio, it’s effective.
Target Antigen
The target antigen should be in a matrix or a buffer to provide interaction with a pre-coated capture antibody, or it should be directly attached to the plate. In rare cases, the antigen’s 3D structure may be changed during adsorption to the plate, and it will no longer bind the target epitope. A plate pre-coated with a binding protein (capture antibody) is indicated in this scenario.
A standard curve of a protein of known concentration is required for quantitative analysis. A standard curve displaying concentration versus absorption is created when a serial dilution of the known protein is completed and assessed. The concentration of unknown-concentration samples is extrapolated from the standard curve.
The amount of enzyme that binds will directly influence the amount of signal obtained, hence the concentration of enzyme conjugate used is a crucial part of ELISA optimization. A low signal with a low signal-to-noise ratio can result from too little enzyme. In addition to creating background noise, too much enzyme may impact the signal-to-noise ratio.
Washing the Plate
TBS (Tris-buffered saline) and PBS (Phosphate-buffered saline) with 0.05 % TWEEN are two commonly used ELISA washing buffers. To wash the plate, aspiration or plate inversion should be used to repeatedly fill and empty the wells. 3 x 5-minute washes are generally recommended after incubation with the coating and detecting antibody. 6 x 5-minute washes are recommended after incubation with the enzyme conjugate.
Substrates and signal detection
The choice of substrate is determined by the assay’s sensitivity and the equipment available. Antigen detection in the sub-picogram per well range is possible with chemiluminescent substrates, which are the most sensitive. Colorimetric and chemifluorescent substrates can detect antigen levels in the mid to low picogram range per well.
A standard plate reader with the suitable filters is used to detect colorimetric signals. Chemifluorescence is detected with a fluorometer, whereas chemiluminescence is measured with a luminometer; nevertheless, some plate readers can detect chemiluminescence.
ELISA Tips and Tricks to Optimize Results
Tip1: Kit Compatibility
Assure your kit is suitable with your target and the nature of your sample before buying it. The kit you select should, ideally, have been characterized in the same matrix as the sample you’re testing, such as plasma, serum, urine, tissue culture, saliva, and so on. Check that the kit’s detection ranges and sensitivity are appropriate, and that it can detect the target in the species you’re working with.
Tip 2: Before you begin testing, make sure you have a thorough understanding of the assay.
Each kit is specialized to identify certain targets and has unique capabilities; it is not a “one-size-fits-all” solution. Knowing your ELISA kit’s sensitivity and specificity, as well as how to add precision to each step will result in an accurate and dependable standard curve that displays quantification of your target. Each kit will include reagents and buffers that are specific to the target. Each set of parameters, such as the type of antibody, incubation periods, temperatures, and reporting system, must be known and understood before proceed. Preparing for this ahead of time will save you a lot of time and frustration.
Tip 3: Sample Preparation
Establish the correct dilution range by using a small sample prior to beginning your specialized tasks. Your samples must be suitable for use in a microtiter plate assay. The number of biological markers analyzed will change. Use the kit’s instructions as a guideline; you are searching for data that fits inside your sample standard curve. Remember that samples containing interfering factors include bilirubin will yield erroneous findings.
Tip 4: Maximize Your Samples
To get the most out of your kit, Biorbyt recommends running a few test assays with control samples at various dilutions to generate standard curves. Maintain your most valuable samples safe until you have figured out what dilutions to use. You can plan the optimum layout of your plate once you know the samples and dilutions to use. Use all of the wells according to the kit’s instructions. If necessary, add more detection reagent based on the information supplied.
Tip 5: Washing
To decrease background signal caused by unbound, conjugated antibody, thorough washing is required. As a result, the signal-to-noise ratio of the assay will improve. Washing at each phase aids in the preservation of the unique binding events. Ascertain that the wash volume is sufficient to eliminate all traces of antigen or antibody from the wells, hence reducing undesired background signal. Maintain the proper distance between the well’s bottom and the wash tips to prevent residual antibody/antigen-containing fluid from affecting your signal.
