Enzyme-linked immunosorbent assay (ELISA) is one of the most powerful and preferred techniques for identifying antibodies for research samples.
The tests are suitable for both small-scale and large-scale needs. It is ideal for small-scale laboratory needs as well as large-scale industrial purposes. You can adopt different ELISA techniques based on the purpose and desired results.
Selecting an ideal ELISA technique depends on various factors. These factors will determine the results you get from the tests and help you choose the proper technique.
The leading ELISA techniques
There are four main ELISA techniques applicable for research and studies. Selecting the proper technique is critical for boosting the results you get from the survey and the research. Here are some of the leading ELISA techniques
Direct ELISA
In this technique, the research has to use an immobilized antigen and then use an antibody conjugated by an enzyme. This technique is much faster and only requires a few steps to achieve the results. The assay is prone to fewer errors due to the few steps and reagents required or followed.
However, this method has shortcomings, such as limited flexibility since the researchers must use a specific antibody to target each protein. There is no signal amplification which reduces the assay sensitivity.
Indirect ELISA
In the technique, the researchers use antigens immobilized on a multiwall plate detected in two steps. The first step involves unlabeled binding antibodies to specific antigens.
The second step involves conjugating the enzyme to a secondary antibody directed against the primary antibody’s host species. The Elisa Kits are ideal for laboratory and high-sensitivity research needs.
The method is susceptible since the researchers can use more than one secondary antibody to bind the primary antibody. It is economical due to the fewer antibodies needed.
It is also flexible since you can use different primary antibodies. However, this method has shortcomings due to the more extended procedure than the direct technique and the possible rise and increase in the background noise.
Sandwich ELISA
This method requires the researchers to use matched antibody pairs, including detecting and capturing antibodies. Each antibody is suitable for different and non-overlapping regions.
These antibodies can also detect different epitopes hence boosting the results. When selecting the antibody pairs, you must have both monoclonal and polyclonal antibodies.
This technique is 2 to 5 times more sensitive than other methods. It boasts high specificity due to paired antibodies, offering increased flexibility for both direct and indirect methods.
However, you need to use standardized kits and tested antibody pairs; otherwise, optimizing the antibody and boosting the results will be challenging.
Competition/Inhibition ELISA
This method is also known as blocking ELISA. It is one of the most complex ELISA techniques, but the results are more competitive and buoyant than other tests.
The method is effective when dealing with a limited amount of antibodies and antigens; hence not suitable for commercial manufacturing. The method is highly flexible and less sensitive and variable.
How to Choose the Right ELISA Technique?
The decision on which technique to consider will depend on different factors. These factors are critical for boosting the method’s results and large-scale application. Here are some vital factors
What you need to detect
The result you anticipate will determine the antigen, antibodies, and technique you select for the process. If you need to detect smaller molecules, you can use the inhibition ELISA since it is ideal for dealing with minimal antibody detection needs.
If you need to identify the presence of multiple epitopes, the sandwich ELISA is suitable due to the use of paired antibodies for capturing and detection.
When you need ELISA techniques for commercial purposes such as detecting serums and plasmas, you can use techniques with fewer steps to reduce the errors while making the process simpler and saving time to achieve the results.
You also must ensure the techniques are highly sensitive and do not react with the antigen and antibodies.
The need to measure the immunological response of the analyte
This is ideal for research and studies that require the quantification of analytes. You can select either the sandwich or inhibition ELISA. The indirect ELISA will suit your research if you need to measure and quantify the immunological response.
The type of antibody you need to use
The techniques are suitable for two types of antibodies such as monoclonal and polyclonal antibodies. If you expect to use both, the Sandwich ELISA technique is ideal. Otherwise, other techniques will be suitable for the analysis.
The species you need to use
There are different species you can use for your research. You can use animals, rats, mice, and human and animal samples for your study. Specific techniques are ideal for certain specimens and samples.
You can use many techniques for animals such as mice and rats; however, you are restricted to particular procedures and kits for certain animals such as monkeys.
The purpose of the analysis
The ELISA techniques can be ideal for both quantitative and qualitative analyses. The qualitative ones are simple and will provide results such as positive and negative.
Quantitative will help you determine the concentration of analytes. Therefore, you can select between qualitative and quantitative ELISA techniques.
Sensitivity requirements
If you use analytes with higher concentrations, you need techniques to detect a wide range of analytes. If the sample is low, you need methods with higher sensitivity.
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Conclusion
You can use different techniques such as direct, indirect, sandwich, and inhibition ELISA techniques. The decision of the technique depends on the sensitivity requirement, the purpose of your analysis, the species you need to use, and the expected goals and results.
