ELISA or enzyme-linked immunosorbent assay is a simple and cost-effective technique of detection that’s quite popular. It’s also referred to as enzyme immunoassay (EIA). This test uses enzymes, antibodies, and antigens to identify substances in liquid samples, such as plasma or serum. ELISA is a wet analytical method and is different from the dry lab analytical methods that use dry strips.
It’s a plate-based assay method involving solid surfaces such as a polystyrene multiwell plate, separating it from other antibody assays. During an ELISA, the analyte, or substance under analysis within the sample, is immobilized on the solid surface. Then, all the other compounds within the sample are removed using a detergent solution.
This allows the separation of the analyte from the sample. After it’s immobilized, the detection custom antibody, linked to the enzyme, is added to form an antibody-antigen combination. The final step involves adding an enzymatic substrate to react with the enzyme. This reaction usually generates a visual cue such as a color change that can then be measured.
Due to the popularity of ELISA kits, you will find countless of them in the market and commercials, but not all of them are built equally. So how do you get through the wide variety and pick a good one? Below are some essential tips to help you.
1. The Species Under Study
Samples are obtained from various models such as mice, rats, and humans. These three are the more common models, and finding an ELISA kit for them is pretty straightforward. On the other hand, if the models are non-classical, such as monkeys, the number of available kits goes down. For rare models, you have to select a validated kit for the species that show similar homology as the species in your sample.
2. The Analyte to Be Detected
Analytes are usually protein in nature, and you have to understand what they are before detecting them. Sandwich ELISA kits are generally known for their suitability in detecting large proteins with numerous epitopes like cytokine. On the other hand, competitive ELISA kits are great for small molecule detection, such as hapten.
Almost all commercial ELISA kits measure plasma and culture supernatants. Reading through the product instructions in detail to make sure the kit is suitable for your sample. For instance, the collection method of plasma samples can affect the type of ELISA kit you choose. Additionally, other facets such as lipids in the sample and hemolysis can disrupt the assay’s performance.
Taking these factors into consideration before selecting your ELISA kit is important. If you are unsure, you can first buy a trial size ELISA kit to conduct a pretest to verify its suitability and sensitivity to your samples.
3. Have a Clear Purpose for the Analysis
ELISA kits are tools that can be used for both quantitative and qualitative analyses. Quantitative ELISA shows the concentration of the analyte within a sample through a standard curve, while qualitative ELISA gives a simple negative or positive result for the sample tested.
You have to know whether you want to quantify the analyte within your sample or simply detect its absence or presence. Having a clear goal of the analysis will help you choose between qualitative ELISA and a quantitative one.
4. The Type of Antibody
Various ELISA kits have different antibodies. It can either be a monoclonal antibody or a polyclonal one. In sandwich ELISA, having both is sometimes a great idea — a polyclonal antibody for capture and a monoclonal one for detection.
5. The Required Level of Sensitivity
If you’re unsure about the concentration level of the analyte within your sample, some ELISA kits have a broad range of detection to give you a correct reading. However, if the concentration of the analyte within the sample is too low, the ELISA kits with high sensitivity are the way to go.
If the concentration is on the other side of the spectrum, diluting your samples to the level of detection by the ELISA kit is recommended.
6. The Quality of Antibody Pairs
Suppose you’re thinking about using a sandwich called ELISA. In that case, a pre-coated ELISA kit will utilize a matched antibody pair more effectively. This is because the antibody will bind to various epitopes of the antigen molecule that is not overlapping. Having polyclonal and monoclonal antibody pairs is better, and it prevents competition from a similar binding site on an antigen. Sandwich ELISA kits of superior quality will have extensive testing to pick the best antibody pair for analyte detection.
As a rule of thumb, only pick ELISA kits containing information about the antibody pairs that the supplier includes.
7. The System for Detection
There are various methods of detection in ELISA, such as fluorescent, colorimetric, and luminescent systems. All ELISA entails the immobilization of the analyte on the surface, enzyme labeling, and matching of the substrate.
Selecting the correct enzyme and a matching substrate is critical. Additionally, the conditions for enzyme-substrate reactions, the detection device, and the microplate should be chosen correctly.
8. The Kit Specificity
One of the major drawbacks of doing an ELISA is cross-reactivity and obtaining a false-positive result. This happens when the antibodies attach themselves to another molecule rather than the intended analyte. When you’re in the market for an ELISA kit, remember to look for any cross-reactivity data provided by the manufacturer.
9. Rely On References
ELISA kits used in other research before and achieved success can also help you in your research. If they’ve been reported in the literature, there is a big possibility that they’re trustworthy. In addition, kits and makers who have certification are also more reliable.
10. Availability of Recovery and linearity Data
Recovery and linearity experiments key in the assessment of ELISA kits performance. Recovery data helps you know whether the differences in sample matrices will disrupt the analyte detection process. Therefore, a higher recovery is usually better.
On the other hand, the linearity of dilution usually determines the extent of the dose-response of the analyte and how linear it is to a single diluent. Hence, the concentration of the samples should ideally be similar for all the dilutions. Most ELISA kits manufacturers give the linearity and recovery data and other critical parameters like dynamic range and sensitivity. However, ELISA kits from different makers have different data, and you can compare these data parameters to find the suitable ELISA kit for you.
The enzyme-linked immunosorbent assay is recognized by scientists globally for its numerous benefits. It’s convenient, simple, and quick to perform. Hopefully, these ten tips give you some insight into buying one that will serve its purpose for your research.