Choosing the Best Sample and Assay Diluents for Your ELISA Assay
What are Sample and Assay Diluents?
What exactly are Sample and Assay Diluents, how do they differ from one another, and what role do they play in optimizing your ELISA?
ImmunoChemistry Technologies offers a number of Sample and Assay Diluents. Understanding the function of these reagents will assist you in selecting the best Sample Diluent and the best Assay Diluent for your specific ELISA needs.
View our webinar Benefits of Sample and Assay Diluents for ELISAs as an additional resource.
Sample Diluents
Sample Diluents are used to create the standard curve and to dilute the sample so that the concentration of the target analyte falls within the dynamic range of the ELISA - this is essential to ensure accurate quantitation. Additionally, Sample Diluents can help overcome assay interference issues commonly encountered with certain types of samples.
Which Sample Diluent should I use?
ICT offers four distinct Sample Diluents - each specially formulated to address different experimental scenarios/challenges.
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Looking for a good all-around Sample Diluent?
General Serum Diluent (also available as a 2X concentrated stock General Serum Diluent, 2X) is formulated with BSA and is designed for diluting a range of sera (goat, rabbit, mouse, bird and human), cell-culture media, ascites fluid and other aqueous-based biological samples for use in ELISA assays. It is also excellent as a generic matrix for diluting antigen standards for use in antibody-sandwich ELISAs. Additionally, it is an excellent serum dilution medium for antigen-down ELISAs designed to assess humoral immune responses. It minimizes non-specific binding of sample components to coated and blocked plates.
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Assaying plasma samples that can't be highly diluted - issues with thrombin and complement interference?
Plasma Sample Diluent is designed for dilution of neat plasma and serum samples. A key feature of this sample diluent is that it is specially formulated to prevent clotting events in serum and plasma samples - a particular issue if the samples need to be assayed without being highly diluted due to assay sensitivity limitations. Additionally, the mammalian protein-based formulation reduces non-specific binding of IgG onto coated and blocked plates. Read our blog Highlighting our Plasma Sample Diluent
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Assaying plasma or serum from mouse, pig, cow or rabbit in antigen-down ELISA format?
Sample Diluent-Non Mammalian-based (formerly Neptune Sample Diluent) contains non-mammalian proteins and is highly recommended for diluting serum or plasma samples from mouse, pig, cow or rabbit in antigen-down ELISAs. It reduces non-specific binding of IgG in the sample - it can be particularly useful with pig serum IgG which is heavily glycosated and "sticky." It is also suitable for samples such as serum or cell culture media in both antibody-sandwich and antigen-down ELISAs.
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Need a protein-free Sample Diluent?
Protein-Free Sample Diluent contains a heterogenous mixture of proprietary non-protein blocking molecules that reduce non-specific binding of signal-generating conjugates to the surface of the plate. It can be used for diluting a rnage of biological samples (including serum and cell culture media) for use in both antigen-down and antibody-sandwich ELISA formats. Learn about our Protein-Free Sample Diluent
If you want to efficiently and economically screen for the best Sample Diluent for your particular experimental circumstances, ICT offers a Sample Diluent Optimization Pack containing 100 mL of each of our Sample Diluents (Sample Diluent - Non-Mammalian-based, Protein-Free Sample Diluent, Plasma Sample Diluent and General Serum Diluent).
Assay Diluents
Assay Diluents are added to all wells (sample, standards and controls) of the ELISA to address the so called "matrix effect." This is the sum effect that all of the non-target components in the sample have on the final ELISA readout. Several elements can contribute to the matrix effect depending on the nature of the sample.
The matrix effect can be particularly problematic if the sensitivity of the assay is such that the sample cannot be highly diluted.
- The high viscosity of complex sample matrices can inhibit the binding kinetics of the
target analyte in the sample, compared to the standards, leading to a substantial underestimate of sample analyte concentrations (see Sample Matrix Viscosity, Another Common Contributor to Signal Inhibition in Enzyme Immunoassays).
- Complement binding to the Fc region of immunoglobulins may interfere with the assay readout by blocking analyte-specific binding sites via steric hindrance.
- Other immune system components can also interfere with antibody-sandwich antibodies leading to false negative or false positive readings (see Endogenous Heterophile and Human anti-animal Antibodies Mode of Action with Respect to ELISA Interference).
ICT's Assay Diluents are formulated to address the various elements contributing to the matrix effect. All contain a complex matrix that can markedly reduce the complexity differential between the sample and the standards. All are formulated to inhibit clotting in plasma and serum samples.
Which Assay Diluent should I use?
ICT offers four Assay Diluents, each specially formulated to address different experimental scenarios/challenges.
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Looking for a good all around Assay Diluent?
General Assay Diluent (also available as a 2X concentrated stock General Assay Diluent, 2X) is a mammalian protein-based diluent formulated for use with serum, plasma, urine and cell culture samples in antibody-sandwich ELISAs. As well as minimizing the difference in matrix complexity between the sample and the standards, it also reduces non-specific binding of sample proteins to the assay plate and so reduces background noise. Additionally, it inhibits thrombin and complement activity in serum and plasma samples. Read our blog post ICT Answers Your Questions: Can General Assay Diluent be used as sample/standard diluent?
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Problematic serum and plasma samples - IgM interfering with your assay readout?
IgM-Reducing Assay Diluent is formulated to reduce IgM-mediated interference due to conjugate bridging in serum and plasma samples assayed in antibody-sandwich ELISAs - it reduces binding of "sticky"IgM to the plate surface and capture antibody and helps prevent false positives. It also reduces interference due to thrombin and complement activity.
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Need an animal-free Assay Diluent?
Neptune Assay Diluent is a non-mammalian protein-based reagent formulated to equalize matrix complexity differentials between sample and standards that are often encountered when evaluating serum and plasma samples. In addition to equalizing matrix complexity and non-specific binding, it also inhibits thrombin and complement activity.
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Issues with clotting in plasma and serum samples in antigen-down ELISAs?
Antigen-Down Assay Diluent can be particularly useful for antigen-down ELISA assays where clotting of plasma or serum samples is an issue. Calcium chelating agents and proprietary additives eliminate clotting and minimize interference due to thrombin and complement activity. The mammalian protein and detergent formulation can be especially helpful in reducing non-specific binding when assaying serum samples from human, pig and cow - samples that can be unusually "sticky."
If you want to efficiently and economically screen for the best Assay Diluent for your particular experimental requirements, ICT offers an Assay Diluent Opimization Pack containing 100 mL of each of our Assay Diluents (General Assay Diluent, IgM-Reducing Assay Diluent, Neptune Assay Diluent, and Antigen-Down Assay Diluent).
Additional Resources
ELISA & Assay Reagents Catalog
ELISA & Assay Reagents Advantages Catalog
Guide to Developing Optimized ELISAs