ELISA Diluent: Overview, Types, & Applications

ELISA Diluent: Overview, Types, & Applications

ELISA Diluent: Overview

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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How Does ELISA Diluent Work?

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.

Types of ELISA Diluent

Two types of ELISA Diluent frequently used in assays include:

Sample Diluent

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: 

  • Minimize non-specific binding
  • Reduce background noise
  • Reduce sample variation

The types of sample diluent available in the market include:

  • Plasma sample diluent: A BSA-based buffer enriched with additives that can prevent clotting (thrombin inhibition) and complement activation during the incubation stage of the ELISA plate. It has applications in an antigen-down ELISA format with plasma samples and an antibody sandwich ELISA with plasma samples. 
  • General serum diluent: A buffered BSA solution having a role in maintaining a constant solute and pH environment during the assay for specific antigen-antibody interaction. They are frequently used to dilute mammalian and chicken serum samples. 
  • Neptune sample diluent: A non-mammalian protein solution. It’s used with serum and plasma samples in an antibody sandwich ELISA assay.
  • Sample diluent optimization pack: A mixture of all the three above-mentioned diluents. It’s used to determine which diluent will be suitable for your research use. 

Assay Diluent

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:

  • Resolve matrix problems in antigen-down or antibody-sandwich ELISAs
  • Reduce sample matrix interference
  • Minimize non-specific interaction
  • Reduce background noise

The types of assay diluent used in lab applications include:

  • General assay diluent: Inhibits clotting and complement activity and is extensively used with plasma or serum samples in sandwich ELISA assays. 
  • Neptune assay diluent: Inhibits thrombin and complement activity. It’s used with serum and plasma samples in an antigen-down ELISA. 
  • IgM-reducing assay diluent: Functions by inhibiting complement and thrombin activity while also minimizing IgM-mediated conjugate bridging interference. It’s used with plasma and serum samples in sandwich ELISA. 
  • Antigen down-assay diluent: Used to prevent the clotting of samples in the ELISA microplate. It has application in antigen-down ELISA with plasma and serum samples. 
  • Assay diluent optimization pack: Contains all the four above-mentioned diluents to help you determine which diluent suits best your research applications.  

What is ELISA Diluent Used For?

ELISA diluent is used to dilute samples and equalizes the matrix. This is to obtain accurate and reliable results in the in vitro assay. 

Sample Dilution

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:

  • Reduce background signal
  • Minimize non-specific binding
  • Equalize the matrix and reduce variations

What Industries Use ELISA Diluent?

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: 

  • Blocking antibody-coated ELISA plate microwells
  • Diluting ELISA standards and samples
  • Diluting biotinylated detection antibodies or tertiary reagents (such as Avidin-HRP or Streptavidin-HRP).


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.

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