Although there are many advantages to using ELISA, it must be appropriately set and optimized to ensure reliable results. This guide will provide an overview of different types of ELISA and emphasize the techniques to ensure successful ELISA experiments.

ELISA can be used as a diagnostic, monitoring, and quality control tool in the medical and food industries. As shown in Table 1, there are several types of ELISA available for selection based on the research objectives. The detection target of enzyme-linked immunosorbent assay can be an antigen (e.g., if intended to detect the presence of a virus) or an antibody (e.g., if intended to detect an immune response to an antigen).

For simplicity, the following examples illustrate antigen detection, however, the same principle also applies to antibody detection, and this section will provide an overview of each type.

Table 1 | Advantages and disadvantages of different types of ELISA

Plan ahead

Advance planning can help the workflow run effectively and reduce the possibility of errors and retesting; Therefore, it is necessary to thoroughly plan all components of the workflow before it begins. Flowcharts and detection design are important planning considerations. The factors that need to be determined include: the number of samples to be tested, the number of retests, and the required number of reagent plates/kits.

The retesting of ELISA samples is important as it will highlight abnormal results. To improve reliability, it is best to use three samples, but duplicate samples can also be used if necessary. Quality control also needs to be considered in the plan, as it will indicate whether the detection is successful, whether it remains within the dynamic range, and whether comparisons and corrections can be made between detections. Using a flowchart to plan your ELISA test will also help reduce any errors in sample placement.

When using different reagents on multiple plates or multiple tests, all plates need to have clear labels and isolation of reagents to avoid confusion. In addition, all reagents must be thoroughly thawed (if necessary), mixed and dated before use, and any photosensitive reagents must be checked for spoilage.

Another factor to consider, especially for sandwich methods, is the species from which the primary and secondary antibodies come. When using antibodies, they must be tested to find the most ideal signal and minimize any cross reactions.

Many test kits on the market provide antibodies, however, if a project involves a new target, it is important to test a series of antibodies against known and unknown samples. When optimizing detection, try different antibody dilutions to improve detection sensitivity while minimizing cross reactions and background noise as much as possible. Trying a series of sample dilutions can also help with this process.

Closure is another key part of the workflow, which can reduce background signals and minimize false positive results. A good blocking buffer will reduce non-specific binding while not reacting (or only reacting with the smallest) antigens, antibodies, or detection reagents used.

Blocking buffer agents are usually protein or non-ionic detergents, and the type used depends on several factors, including the antibodies, antigens, and detergent reagents used, as well as the surface chemical properties of the plate.

The most common type of blocking agent, bovine serum albumin (BSA), is a protein. If your background signal is high and you suspect insufficient sealing, using a higher concentration of sealing agent or increasing the sealing time may be helpful. On the contrary, if you have a persistent background issue, it may be worth investing time in optimizing the type of sealant you are using.

Sample processing and dilution

Improper handling or storage of reagents and samples can affect downstream analysis, as different buffers and reagents have different storage requirements. For example, some must be stored in a refrigerator, some at room temperature, and some with a photosensitive cover (such as aluminum foil).

If there are plans to use the same samples in future experiments, they should be placed in aliquots and taken out as needed. This will avoid multiple freezing/thawing cycles and maintain accurate results. When the sample is ready for use, it should be thawed on ice (or according to the protocol).

One way to obtain consistent and accurate results is to ensure that the experimental platform is properly set up so that all components and instruments are within reach. The use of calibrated precision pipettes will also limit any inconsistencies during the pipetting process.

The precision within operation refers to the error between each hole. As mentioned earlier, adding duplicate samples is important for identifying errors and abnormal situations. The coefficient of variation (CV) is a ratio that describes the variability within a population, independent of the absolute value of observations [7]. When interpreting ELISA data,% CV is useful for identifying any inconsistencies in sample replication. This is reflected in the variation between the measured optical density (OD) values. In short, the lower the% CV, the more accurate the results.

The accuracy of the testing interval refers to the repeatability between boards or between testing intervals, such as testing completed on different days. Although commercial ELISA kits have inter assay quality control, scientists must keep a detailed notebook to record any changes in the protocol at any time, so that other researchers can perform the same experiments in the same way and obtain the same results. This also enables detection to be monitored over time to identify any systemic issues or degradation in detection performance.

Pollution can cause many problems in downstream analysis of ELISA data, such as high differences between wells, high background values, and weak or low signal intensity. There are several methods to prevent pollution:

1. Work in a clean environment;

2. Disinfect all water by deionization or distillation;

3. Use new and clean reagents for each transfer;

4. Use a new pipette tip between two samples;

5. Be careful when preparing and applying samples to avoid splashing or cross contamination;

6. If automation is used, ensure that the system is cleaned and the reagent supply is regularly updated;

7. Only remove the required portion of the reagent and avoid pouring it back into the inventory bottle, as this may introduce contaminants.

The correct washing and rinsing plan is particularly important. Insufficient, inconsistent, and/or excessive cleaning during downstream analysis can cause problems. There are many methods for washing boards, including using automatic washing machines, manual splitters, or bottle washing. When manually sucking out the liquid from the hole, be careful not to touch the bottom of the hole - the tip of the straw should be placed on one side of the hole. In order to reduce background signals, the cleaning amount needs to be large enough to remove unbound samples and reagents from the wells. However, excessive washing can reduce the target signal.

Ensure that all holes are cleaned evenly to avoid introducing signal variations throughout the entire detection board. To achieve this, if it is manual cleaning, please check if the pipette tip is securely fixed. If using an automatic washing machine, all dispensing and suction nozzles must be unobstructed. You can test them on an empty board to check if they work the same.

Accurate data analysis is a crucial step in the ELISA workflow, as it addresses the issue of inter assay variability, which may be influenced by many factors such as temperature and development. To limit the impact of these factors on the detection results, normalization should be performed by comparing the standard samples on each plate between experiments.

It is crucial to keep the test sample within the dynamic range of the plate reader, otherwise the results will not be representative. The dynamic range of the instrument can be found in the machine manual. When developing detection methods and determining sample dilution, both the highest and lowest positive samples should fall within the dynamic range of the instrument. Standard samples are usually provided together with ELISA kits; However, these can also be purchased independently.

Overall, ELISA is an important component of many laboratories. Following the above tips will help anyone produce accurate and reliable results.