The variety of existing assay types reflects the great diversity of biological analytes and the different ways they can be measured. A popular method is the immunoassay, which uses specific antibody-binding to detect or quantify target antigens, such as proteins, pharmaceuticals and other metabolites. This broad application can come in many formats and procedures—perhaps none as popular or common as ELISA.1
In the enzyme-linked immunosorbent assay (ELISA), a target analyte is immobilized in the wells of a microtiter plate by chemical coating or by capture antibodies. Detection antibodies, along with chemiluminescent substrate, are used to generate a signal proportional to the amount of analytes in the sample. Commercial ELISA kits provide a rapid and reliable means of specific antigen detection for a wide range of biological samples, including lysates, homogenates, supernatants and biofluids.1
There are four ELISA techniques: direct ELISA, indirect ELISA, sandwich ELISA and competition or inhibition ELISA. Choosing which one is appropriate depends on the antibodies available, the results required and the complexity of your samples. With direct ELISA, conjugated primary antibodies specifically bind to antigens coated in microplate wells. Indirect ELISA follows a similar principle, but uses unlabeled primary antibodies paired with a conjugated secondary antibody. The sandwich ELISA coats wells with primary antibodies, which in turn bind the sample antigens. A new set of antibodies will be added, forming a sandwich on the antigens.2 The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Essentially, sample antigen or antibody competes with a reference for binding to a limited amount of labeled antibody or antigen, respectively. The higher the sample antigen concentration, the weaker the output signal, indicating that the signal output inversely correlates with the amount of antigen in the sample.3
Species
Commercial ELISA kits for commonly used species—such as human, mouse, and/or rat—are readily available. For more unique species like monkey, canine or porcine, there will limited numbers available. In this case, choose the kit that has been validated for species that show homology with your sample species. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.4
For example, the biomarker ELISA kit for NT-proANP was developed for human use but due to the high sequence homology between species, the kit is successfully used to measure NT-proANP as a cardiac safety biomarker in various animal models (rat, mouse, rabbit, monkey).5
Always check to see if your sample type has been validated by the manufacturer. ELISA kits can be validated for a wide range of samples, including cell culture supernates, serum, plasma (heparin, EDTA), cell lysate, and tissue.4
Specificity
One of the most frustrating issues after performing an ELISA is when you realize that the strong signal obtained is actually a false positive result. In this case, cross-reactivity has occurred. The antibodies have attached themselves to some other molecule instead of the target analyte. On the other hand, interference happens when components in the sample matrix change antibody-antigen interaction, inhibiting the antibodies to find the target analyte. Thus, it is critical that antibodies used in your ELISA kit do not cross-react with non-target proteins.4
Sensitivity
The sensitivity is the lowest detection level of the marker that the antibody pair used in the ELISA kit can detect. Assays offering high sensitivity offer a different dynamic range than assays with a lower sensitivity. The dynamic range of an assay indicates the range of concentrations over which an assay can accurately quantify the analyte.
For example, if you are unsure about the concentration of the target analyte in your sample, consider an ELISA kit with a broad detection range. However, if you know your samples contain extremely low levels of the target analyte, you need an ELISA kit with high sensitivity for detection.
Additionally, look into the validation data of the kit, often documented in the ELISA protocol booklet or in the validation data files. Reference values and pathological values in serum and/or plasma of the biomarker of interest are sometimes documented, as well. This data can be helpful in selecting an appropriate assay.4
Linearity
While parameters like include sensitivity and precision are important considerations for ELISA kits, other parameters are more predictive of ELISA performance for typical sample types. Percent recovery and linearity of dilution measure the target protein in real samples, such as plasma, serum or cell culture media.
Percent (%) recovery is determined by spiking a known amount of purified target protein into a biological sample type. The spiked sample is measured in the ELISA and the concentration is calculated from the standard curve. This calculated concentration is compared with the known concentration of the protein and is expressed as a percentage. For example, 100% recovery means that the observed concentration was the same as the actual concentration of spiked protein in the sample. It suggests that the proteins and other molecules in the sample type did not interfere with the quantification of the protein. If % recovery measures less than 80% for a specific sample type, then a different ELISA kit should be selected for quantification.5
The linearity of dilution is a good companion to % recovery because it measures native rather than spiked protein in biological samples. The linearity of dilution is determined by measuring multiple dilutions of known positive samples by ELISA. The concentration of the target protein is determined by multiplying the dilution factor by the calculated concentration. For best results, the concentration of the samples should be similar for all dilutions. If a difference of more than 20% from the undiluted sample is observed, then a different ELISA kit should be chosen for accurate quantification.5
Overall, these tests confirm an ELISA is within the desired range of measuring the analyte and has a robust immune response.
Last thought
Choosing the appropriate ELISA kit is critical to the success of your experiments. Factors such as target specificity, sensitivity, workflow requirements and overall compatibility with your study design should guide your decision. Taking the time to evaluate these elements carefully helps ensure your assays produce reliable, reproducible results that support your research objectives.