An ELISA or EIA, referred to as an enzyme-linked immunosorbent assay, is one of the most proficient tests to measure and detect blood antibodies. Laboratories utilize this test to determine if a human or animal has specific antibodies related to severe infectious conditions. Furthermore, antibodies are proteins that the body generates in response to adverse substances known as antigens. Moreover, several researchers and scientific departments use ELISA as a screening tool before more in-depth tests are ordered.
How do researchers perform ELISA tests?
Performing an ELISA test is straightforward. Typically, a patient signs a consent form before the healthcare professional documents the medical necessity for the test. These remarks contain the reasoning as to why an individual has to undergo ELISA testing. When it comes to detecting antibodies concerning viruses and infections, a doctor will take a blood sample to assess the concentration of present antibodies. While it was relatively challenging to operate an ELISA in the past, modern technologies have streamlined the techniques and processes to make them more user-friendly.
Selecting the right ELISA type
There are four primary forms of ELISA: direct, indirect, sandwich, and competitive. Each method possesses distinctive suitability, advantages, and disadvantages.
Direct ELISA
In a direct ELISA, antigens are immobilized directly onto the microtiter plate’s surface and detected through a specific antibody. Following this, the antibody is conjugated to HRP or other detection molecules.
Indirect ELISA
Indirect ELISA is a method that utilizes a two-step detection process. First, primary antibodies bind to the target antigen; then, secondary labeled antibodies bind to the host species of the primary antibody. As with direct ELISA assays, the antigen is immobilized on the plate surface. This method can also detect specific antibodies within a serum sample by substituting serum for the primary antibody.
Sandwich ELISA
Several researchers worldwide utilize the sandwich immunoassay, or sandwich ELISA. This format requires two specific antibodies for different antigen epitopes. Researchers refer to these two antibodies as "matched pairs." One antibody is coated onto the plate surface and serves as the capture antibody to immobilize the antigen.
Competitive ELISA
Competitive ELISA, also known as inhibition ELISA, measures an antigen’s concentration by detecting signal interference. Each ELISA format discussed above can be adapted into a competitive format. Here, the sample antigen competes with a reference antigen to bind to a limited amount of labeled antibodies. While a reference antigen is pre-coated on the plate, the sample is pre-incubated with labeled antibodies. Once this process is complete, the mixture is added to the wells. Based on the amount of antigen available in the sample, only free antibodies will be available to bind with the reference antigens.
This implies that the more antigen present in the sample, the lower the detection of reference antigens. As a result, a higher sample concentration will produce a weaker signal.
Getting suitable results with ELISA
ELISA kits are favored for their specificity, simplicity, and cost-effectiveness, making them valuable tools in laboratories worldwide. Many commercial and pharmaceutical outlets purchase ELISA kits for a vast range of analytes. While gene synthesis also plays a significant part in these scenarios, these outlets are often aware of the limitations of certain ELISA formats and the high costs associated with them in a research setting. Such clarity enables laboratories to develop their own assays, specifically when working with stringent budgets or when numerous antibody samples need to be tested.
Even though there are several ways to use ELISA, various factors must be considered to design reliable and robust assays. Here are some tips to save time, money, and stress while achieving the best possible results.
1. Choose The Surface Wisely
Because of the diverse nature of proteins, a broad range of microplates are available with various binding capacities and properties. While developing an assay, one should assess several surfaces to determine the most suitable fit for antigen or antibody testing. Even though some plates are made of similar plastics, such as polyvinyl chloride or polystyrene, they offer different binding properties. Researchers need to identify a surface with the appropriate binding capabilities. This not only provides a competitive edge but also ensures consistency if you’re testing several samples at once. In conclusion, one must titrate the molecules used for coating to ensure an ideal concentration for the assay.
2. Checkerboard Titration
Although it may seem sensible to utilize higher antibody concentrations, especially when target molecules are present in low quantities, this can be counterintuitive. When performing a titration, ensure that the signal-to-noise ratio is optimized. The aim is to achieve the highest Optical Density (OD) with the lowest possible background for a sensitive assay. If you use an abundance of antibodies, it may lead to higher background noise due to non-specific binding. However, when concentrations are too low, the assay will lose its sensitivity. A suitable method to test this is through a checkerboard titration, conducting several serial dilutions of the coating reagents, samples, and secondary antibodies.
3. Proper Sample Dilution Is Vital
Researchers often overlook proper sample dilution while investigating assay variability. When a sample must be diluted (such as serum), precise dilution is vital to minimize pipetting errors. For instance, if the checkerboard test has identified the correct ratio, it is better to perform a 1:100 dilution rather than a single 1:10 dilution to ensure accuracy. Small errors in low-volume pipetting can lead to significant variations between experimental models. Improper dilutions can also produce misleading results when differentiating samples within the same experiment.
4. Not All Antibodies Are Equal
Even if one has multiple antibodies for a single protein, experts suggest testing each one individually. There are various considerations to keep in mind while selecting antibodies. Polyclonal antibodies offer a broader target range and are less costly; however, they may affect an assay’s specificity. On the other hand, monoclonal antibodies are relatively expensive but are more specific if the epitope is visible.
More often, the only way you can determine the best fit is by testing various antibodies from different manufacturers. Fortunately, numerous manufacturers provide small sample sizes for such experiments or can offer them upon request. Consequently, while choosing matched pairs for sandwich ELISAs, this option becomes essential as the antibodies must recognize distinct epitopes.
5. Understand Blocking Surprises
Innumerable investigators test various blocking agents during the assay’s development stages and choose the one that provides the ideal signal-to-noise ratio. Meanwhile, researchers often overlook batch-to-batch variation in certain blockers.
Common blockers act as biological agents, such as BSA (Bovine Serum Albumin), which is a practical and efficient blocker for a wide assay range. However, because of the reagent’s biological nature, it can differ between batches; some may comprise cross-reactive antibodies that hinder the experiment. As a result, if blockers like fetal calf serum or BSA are used, you must test various batches to ensure consistency in results.
This is one of the essential considerations that most individuals forget to incorporate. If you observe batch-to-batch variations, manufacturers are likely willing to hold specific lots for you. Since researchers know the lot numbers that work, they can plan how much reagent will be needed for their practice.
6. Secondary Antibodies - Batch Testing
Similar to certain blocking agents, secondary antibody batches can also fluctuate. Even though the antibody's primary activity may not change, the amount required for optimal results can vary significantly. Therefore, when purchasing new batches of secondary antibodies, one must run a side-by-side experiment with the previous batch to determine if the concentration utilized is still appropriate. Experienced professionals can quickly identify increased background noise and trace it back to the batch change. This is often a surprising yet unwelcome change in the lab.
7. Consider Normalizing
Often overlooked, normalization between experiments and plates reduces the impact of variables on the final results. A straightforward approach to normalization, especially where researchers use serum, is to calculate the ratio of every serum sample against a negative serum control. By performing this specific step, variations from external factors like development time and room temperature are minimized. By dividing the test sample’s OD by the control’s OD, one can express the results as fold changes.
Since this is not an absolute quantification, an absolute figure is not essential. Nevertheless, this allows an individual to obtain a consistent set of results and compare various samples more accurately.
8. Know Your Dynamic Range
When developing a test that utilizes standard curves to determine sample concentrations, one must remain within the instrument’s dynamic range. Simply put, you should not exceed the detection limit of the plate reader. If you are unsure of this limit, you can find it in the manufacturer's manual. Typically, many machines have an upper limit of around 2.0 to 3.0 OD. While preparing the standard curve for your experiment, the most concentrated standard must have an OD below the dynamic range's highest limit.
If you place standards above the specified range, extrapolating the results becomes risky, which adversely influences the accuracy of the standard curve. If the OD falls above the instrument's linear range, the resulting concentration might be inaccurate. While many modern plate readers can adjust and maneuver the dynamic range, if a researcher uses a model where this isn’t possible, they must consider the maximal OD during the standard curve design.
Moreover, every sample should fall within this range. This is where dilution plays a significant role. Correcting for range and dilution retrospectively can be highly frustrating. However, once the operator identifies the expected concentration in the samples, you can use a standard dilution to fit within the calibrated range and provide ideal results for further testing.
9. Follow Recommended Temperatures, Incubation Times, and Conditions.
Adhering to the incubation recommendations provided in the assay manual is essential for achieving optimal color development. Time, temperature, and shaking can considerably affect an assay's results if you do not follow them strictly. Reducing the incubation time can prevent the reagents from reaching chemical equilibrium before moving to the next step in the assay. Furthermore, higher temperatures significantly impact binding kinetics, potentially leading to higher background or false positives. If the protocol suggests shaking, it is ideal to stick to that guideline.
In some specific assays, deviating from these conditions may lead to significant changes in the OD. Consistency and precision during plate incubation will greatly enhance the performance of your assay.
10. Obtain Data Analysis and Optimal Detection
To achieve optimal detection, read the plate at the assay’s specified wavelength. You will find this information in the assay manual to ensure you correctly configure the data analysis software. Several researchers suggest using a 4-parameter logistic (4PL) curve fit to analyze data and acquire accurate results. However, if you’re not familiar with your plate reader’s software, consider reaching out to the manufacturer to address your concerns. If you already have the required software, the ELISA kit manufacturers can often guide you through the setup of your plate template.
It is essential to note that properly configuring the template streamlines the data analysis that occurs immediately after the plate is read.
The Bottom Line
Performing ELISA tests and experiments can be challenging if one does not thoroughly understand the concepts required to achieve ideal results. Since there are several ways to operate ELISA kits, one should not ignore critical factors like optimal detection, incubation periods, dynamic range, normalization, and adequate sample dilution.