Enzyme-linked immunosorbent assay (ELISA) is a regularly performed biochemical plate-based assay. It’s used in immunology and biochemistry labs to analyze the presence and amount of several biochemicals such as peptides, proteins, antibodies, and hormones.
The assay requires many reagents including buffers, conjugate stabilizers, blockers, wash buffers, and sample and assay diluents to perform better. They work in an orchestrated manner to avoid unwanted protein build-up and generate clean signals.
The two primary diluents used in the assay include assay diluent and sample diluent.
Sample diluent is a solution used to dilute ELISA test samples, whereas the assay diluent is used to equalize the matrices of any given sample, such as serum or plasma samples. Both types of diluent help to reduce background noise and contribute to obtaining accurate and reliable results.
How do the diluents work in the assay and interact with other components to provide the results? What workflows involve the use of diluent? What industries frequently use them? You’ll find the answers to all these questions in this article.
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Many types of solutions are used to perform the ELISA assay, such as blocking buffer, coating buffer, conjugate stabilizers, assay diluents, substrates, and stop solution. Each contributes to the accuracy of immunoassay results in a specific way.
Further, the ELISA assay involves two types of solutions to run: standards solution and test samples. Standards solutions are the highly purified forms of the target analyte that are used to plot a standard graph, whereas the test sample contains the analyte of interest in a complex mixture.
The analytes in the samples are often present in complex matrices, such as serum or blood. So, the difference in the sample matrix and the standard curve matrix leads to an inaccurate determination of the amount of analyte of interest.
Such complex assay issues are addressed by using assay diluent.
The addition of the assay diluent to all wells of an ELISA plate containing samples and standard calibrators creates a different composition or environment for the calibrators/standards compared to the samples. It’s necessary for accurate calibration and measurement in the ELISA assay.
Two types of ELISA Diluent frequently used in assays include:
The sample diluent is a specific buffer solution used in the first step of indirect ELISA assay to evaluate antibodies and proteins. In immunoassays, such as ELISA, and Western Blotting assay, the diluent helps to:
The types of sample diluent available in the market include:
Assay diluent helps in “equalizing the problematic sample matrix”. In simple words, it balances each component present in any given sample, such as plasma, serum, or cell culture samples, that could affect the accuracy and reliability of the assay results. It ensures the assay results are consistent and precise.
The uses of assay diluent are to:
The types of assay diluent used in lab applications include:
ELISA diluent is used to dilute samples and equalizes the matrix. This is to obtain accurate and reliable results in the in vitro assay.
ELISA diluents are used to dilute ELISA test samples, such as serum and plasma samples, to bring them into the appropriate functional range of the assay.
High-titer samples can overwhelm ELISA plates, leading to inconsistent results and decreased signal. The right sample diluent reduces false positives, minimizes background noise, dilutes interfering proteins, and improves sensitivity, ensuring accurate and reliable assay outcomes.
Here’re some roles played by the diluent in the ELISA assay:
ELISA assay diluent is a buffer solution (often a phosphate-buffered saline (PBS) containing bovine serum solution) used either to dilute ELISA samples or equalize the assay matrix.
This buffer is designed and validated for ELISA applications, including:
ELISA diluents are used to dilute various biological samples, such as blood, serum, and plasma. This ensures optimal concentrations for effective binding and detection of the target analyte, therefore providing accurate and reliable assay results.
Biotechnologists frequently use ELISA assays for various research applications, such as analyzing proteins and antibodies. This makes it essential to obtain accurate results without the need of performing them over and over again.
This is contributed by involving an ELISA diluent that dilutes the samples, reduces background noise, and minimizes unwanted interaction in the ELISA reaction.
The efficacy of the commercially available ELISA diluent is confirmed through validation using serum/plasma obtained from healthy human blood donors. They have been specifically designed to enhance the performance of ELISA kits and antibodies sourced from the companies.
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ELISA diluent is a crucial buffer solution used in ELISA assays to dilute samples. Its primary purpose is to minimize non-specific interactions, reduce background noise, and mitigate sample matrix interference. There are two types of diluents utilized in immunoassays: sample diluent and assay diluent. Although they serve distinct functions, both aim to deliver improved accuracy, reliability, and quality of results.
When selecting the appropriate diluent for your assay, it is essential to evaluate the available options in the market carefully. Thoroughly examine the provided citations, product information sheets, safety data sheets (SDS), certificate of analysis, and any other relevant documents accessible on the provider's website.
Additionally, refer to the frequently asked questions (FAQs) section for more specific product-related information.
Maintaining high equipment standards is just as crucial as using quality reagents. The combination of top-notch reagents and equipment is instrumental in achieving publishable and dependable results. Therefore, it is imperative not to compromise on either aspect.
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