A Basic Understanding Of ELISA Sensitivity

ELISA, short for enzyme-linked immunosorbent assay, has long been a popular lab technique that is commonly used to determine the concentrate of antibodies or antigens within a solution. This test is set apart from others in that it separates specific and non-specific interactions, which occur with serial binding to a surface that is solid, typically a polystyrene multi-wall plate. It is also different because it can produce quantitative results.

Created in the 1970s, the ELISA test gives an end-product that is colored, which indicates how much of the antigen or antibody was in the original sample used. This test is quick and easy to complete. It is also designed to quickly be able to test large numbers of samples in parallel, making it one of the most ideal options for research and diagnostic tests. Since their creation to replace radioimmunoassays, the ELISA test is widely used and can be expanded and modified for better results, depending on what is being tested.

ELISA sensitivity depends on many different factors. A benefit of the ELISA assay is that is more sensitive than most of the currently available immunoassays. The general range of detection for ELISA is is 0.1 to 1 fmole or 0.01 ng to 0.1 ng. However, sensitivity can be changed by particular characteristics of the specific reaction between the antibody and the antigen. To enhance the results and make them even more specific, a substrate with enhanced chemiluminescent or a fluorescent signal can be used.

The direct detection method of getting ELISA results can be done with labeling the antibodies with alkaline phosphatase or horseradish peroxide. Direct detection can also happen with fluorescently labeled antibodies.

With the indirect detection method of reading results, the antibodies are added to biotin and then exposed to a streptavidin-conjugated enzyme step. If the secondary antibody used is biotinylated, an additional testing step is required for detection. This extra step is treatment with an appropriate substrate.

The indirect method of detection for ELISA has been found to give higher levels of signal, therefore, making it more sensitive. On the other hand, indirect detection can also create additional background signal, which can then reduce net specific signal levels.

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ELISA—a Precise And Highly-Reliable Diagnostic Option

The basic format differs from other assays used to locate antibodies in samples through the manner in which specific and non-specific interactions are separated. In a basic ELISA, the separation takes place through serial binding of the antibody to the surface of the well of a polystyrene plate. Another distinguishing feature is that ELISA offers quantitative results, which facilitates quicker, reliable, and very precise diagnosis.

There are three ELISA detection options—quantitative, qualitative, and semi-quantitative. Qualitative results diagnose the presence or absence of the antigen or antibody while quantitative results determine the precise concentration of the analyte in the sample. The semi-quantitative result option is used to perform comparative analysis of relative levels of the analyte. ELISA detection options are far more versatile as compared to other diagnostic options like radioimmunoassay.

ELISA result options include color-coded products with the intensity or darkness of the color correlated to the concentration of the antigen or antibody in the sample. This facilitates faster and easier analysis of the results of the assays without risk of errors or flawed analysis.

A large number of ELISAs can be done in a short period of time, which makes it a popular option for diagnosis and medical research. Parallel analysis reducing processing delays and enables the diagnostic center to offer quick diagnosis without compromising on accuracy of the results. Furthermore, the test is a remarkably simple one, especially when compared to other assays.

ELISA was introduced in the 1970’s as an improvement over radioimmunoassay. There are different formats of ELISAs in use—Basic, Dip-stick, Sandwich, and Competitive—depending on the diagnostic requirements. Modified ELISAs can be used to determine presence and concentration of more than one analyte found in the sample solution that is being tested.

The modified formats offer greater sensitivity in the readouts and higher precision in the final results. Furthermore, these versions offer the option of direct cell-based output derived from the sample.

Introducing the ELISA technique

ELISA is an acronym for the enzyme-linked immunosorbent assay. This is essentially a commonly used laboratory technique for measuring the level of concentration an analyte has in a solution. Analyte is typically used in reference to antigens or antibodies.

EIA or enzyme immunoassay is the basic form of ELISA. It can be differentiated from other types of antibody-based assays by the separation of non-specific and specific interactions. This takes place through serial binding to solid surfaces. A polystyrene multiwell plate is a solid surface on which binding commonly takes place. Quantitative results can also be attained through this method.

A colored product results from the steps of the ELISA and it correlates to the quantity of analyte found in the original sample. Carrying out ELISAs is a fast and easy process. They are intended for rapidly handling a huge numbers of samples in parallel. As such, they are a tremendously popular option for the analysis of many different diagnostic and research targets.

In the early 1970, ELISAs were developed to replace radioimmunoassays. They are still very widely used in their original format. However, modified and expanded formats have been developed. This has enabled direct cell-based output, extremely sensitive readouts and numerous analytes per well.

ELISA can use antibodies that are either monoclonal or polyclonal. Monoclonal antibodies are derived from hybridomas, which are unique antibody-producing cells with the capacity to bind to a single epitope. Polyclonal antibodies are purified from animal sera and they have the capacity to bind to multiple epitopes. Four basic ELISA formats exist, which enables some amount of flexibility. Adjustments can also be made based on required results, the antibodies available or the difficulty of the samples.

While both monoclonals and polyclonals antibodies can be used in ELISA, the latter is more commonly utilized as a secondary layer of detection in indirect ELISAs. Monoclonal antibodies are more widely used for primary detection or to capture antigen.

In its simplest formats, ELISA can provide a wealth of information. However, versions that are more complex can be used if particular reagents are unavailable. These advanced versions can also be used to produce results that are more precise and enhanced signals.