What is an ELISA Kit
In the first publication describing the Enzyme Linked Immunosorbent Assay (ELISA), published in 1971
1, the assay was used to determine the concentration of antigen specific antibodies in a sample of rabbit antisera.
Since that time it has established itself as a cornerstone technique for detecting and quantifying proteins or peptides.
Now the market offers a wide range of ELISA kits to an ever growing number of targets, each claiming specificity, sensitivity, and reliability.
Here we will delve into the key factors that should be considered when selecting an ELISA kit, ensuring that your choice aligns with the goals and requirements of your research.
What Type of ELISA Should You Choose
The diagram below briefly summarizes the components and steps for performing the four most common ELISA types.
In the most straightforward version, the 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.
In the competitive ELISA, unlabeled primary antibodies are first complexed with sample antigens.
These are added to wells coated with antigens.
After washing, only free, unbound antibodies will bind to the coated antigens.
After the addition of conjugated secondary antibodies and substrate, a signal is produced that is inversely proportional to the amount of antigens in the sample; the greater the amount of antigen in the sample, the lesser the signal.
Another variant of this ELISA uses primary antibodies to coat the well.
Labeled antigens will then compete with sample antigens for binding.
The resulting detection signal remains inversely proportional.
Which of these ELISA types to use depends on your research goals and the potential abundance of your antigen.
For instance, if the analyte of interest is in low abundance in a complex sample, then a sandwich ELISA may be the best choice.
This will allow for multiple washes to lower the background noise and still allow for signal amplification from the secondary antibody to ensure overall sensitivity.
If you are interested in detecting specific immunoglobulins, then the indirect ELISA is the appropriate choice.
If assay speed and simplicity are the most important factors than a direct ELISA could be your best bet.
Identifying what the overall goals of your assay are will help you figure out which ELISA type will be best suited to your needs.
For more information see the ELISA Crash Course: The Major Types.
Target Antigen Specificity
Before delving into the technical details of an ELISA kit, it is paramount to establish the specificity of the target antigen.
Consider the nature of your experiment and the protein of interest.
Does the kit recognize the full-length protein or a specific epitope?
Assessing the compatibility of the kit with your target ensures the accuracy and relevance of your results.
Cross reactivity can also be an issue with antibodies that have been generated to peptides so ensuring that the proper controls are in place is important.
ELISA Sensitivity and Dynamic Range
The sensitivity of an ELISA kit is a pivotal parameter, especially when dealing with low-abundance proteins.
Evaluate the lower limit of detection (LOD) and the dynamic range of the kit, as these factors determine the kit's ability to accurately quantify proteins across a range of concentrations.
A high-sensitivity kit with a broad dynamic range is often preferable for versatile applications.
One major contributor to the sensitivity of the ELISA is the choice of the substrate that is being detected.
In general a chemiluminescent substrate will be 10-20 fold more sensitive than a colorimetric one.
3
If even greater sensitivity is required you can even consider platforms that use electrochemiluminescent detection, immune-PCR or even single molecule assays, although all of these solutions, while very sensitive, will require the purchase of specialized equipment and reagents.
3
To learn more, read
Five Ways to Improve Immunoassay Sensitivity.
Assay Duration and Workflow Complexity
Consider the practical aspects of the ELISA kit, such as the overall assay duration and workflow complexity.
Some experiments may demand a quick turnaround, while others may allow for longer incubation periods.
Assess the protocol's steps, including sample preparation, incubation times, and detection methods, to ensure compatibility with your lab's schedule and resources.
One potentially overlooked part of the ELISA process is how the assay results will be read.
Will you need just a simple microplate reader that measures single analyte absorbance? Or do you need a multimode reader?
Read up on what to look for
when choosing an ELISA Plate Reader.
Preparing Samples for ELISA
Different ELISA kits are optimized for specific sample types, ranging from serum and plasma to cell lysates and tissue homogenates.
Verify that the chosen kit is suitable for the sample type you intend to analyze.
Some kits may require additional sample preparation steps, and overlooking this aspect can lead to compromised results.
There are a few general rules surrounding sample prep for ELISAs; minimize freeze-thaw cycles, add protease inhibitors to sample buffers, be sure to evaluate potential matrix effects and be sure to optimize sample dilution.
To get more detail about these tips and more you can read
Optimizing ELISA Sample Prep.
Cross-reactivity and Interference in ELISAs
Examine the potential for cross-reactivity with related proteins or interference from common substances present in your samples.
Assessing the kit's specificity and its ability to discriminate against closely related molecules is crucial to obtaining accurate and reliable data.
It is important to remember that manufacturers may not test their ELISA kits against all samples so there is always the potential for cross-reactivity and/or interference.
Running the appropriate positive and negative controls is critical to ensure that your results are valid.
ELISA Kit Manufacturers
Reliable ELISA kits undergo rigorous validation processes to ensure consistent performance.
Look for kits that have been validated for specificity, sensitivity, and reproducibility.
Quality control measures, such as lot-to-lot consistency, are indicative of a manufacturer's commitment to delivering reliable results.
If your lab has worked with a particular manufacturer in the past (and had good results) then that is an obvious place to start.
However, if a kit for your particular antigen of interest is not available, then reading about other people’s experiences with other ELISA vendors can be helpful.
You can read through
reviews of different ELISA kits
on Biocompare to help you narrow your search for the right supplier.
ELISA Data Analysis
Evaluate the data analysis tools and reporting features provided by the ELISA kit manufacturer.
User-friendly software with robust data analysis capabilities simplifies result interpretation and enhances the overall efficiency of your experiment.
If you are performing a quantitative ELISA, and even if you’re not, be sure to pay attention to your standard curve.
Figure out if you need to plot your data simply using a linear regression model or if you should use a 4-parameter logistic (4PL) model.
You can read more about this here:
How to Perfect an ELISA.
Customer Support and Documentation
A reputable manufacturer provides comprehensive documentation, including detailed protocols and troubleshooting guides.
Additionally, reliable customer support can be invaluable when encountering challenges during the experimental process.
Prioritize suppliers with a strong reputation for customer service.
For more information regarding troubleshooting tips, you can check out our
ELISA Troubleshooting Guide.
Conclusion
Selecting the right ELISA kit is a pivotal decision that significantly influences the outcome of your experiments.
By carefully considering factors such as target specificity, sensitivity, workflow, and compatibility, you can make an informed choice that aligns with the goals of your research.
Remember that investing time in the selection process is an essential component of ensuring the success and reliability of your ELISA-based assays.
References
1. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol. 1972;109(1):129-135.
2. Dalton K. Elisa Crash course: The major types. Biocompare. August 20, 2019. Accessed March 2024.
3. Easthope E. Five Ways to Improve Immunoassay Sensitivity. Biocompare. October 08, 2019. Accessed March 2024.
4. Easthope E. Choosing an ELISA Plate Reader. Biocompare. May 11, 2023. Accessed March 2024.
5. Easthope E. Optimizing ELISA Sample Prep. Biocompare. June 13, 2023. Accessed March 2024.
6. Easthope E. How to Perfect an ELISA. Biocompare. June 30, 2020. Accessed March 2024.
7. Easthope E. ELISA Troubleshooting Guide. Biocompare. November 9, 2020. Accessed March 2024.
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