Sample diluents are used to dilute samples into the functional range of the assay and to create the standard curve. Due to the finite binding capacity of the plate well-coated proteins (e.g., antibodies, antigens), highly concentrated samples must be diluted in order to obtain absorbance readings within the sensitivity detection limits of the instrument. Properly formulated sample diluents will also reduce background noise associated with non-specific bridging of signal-generating conjugates to the plate well surface.
ICT’s Protein-Free Sample Diluent provides a non-protein-based buffered, neutral pH environment that is highly compatible with antibody-antigen interactions. Antimicrobial agents allow for room temperature bench-top use and extensive storage stability at 2-8°C.
Protein-Free Sample Diluent is a protein-free matrix used for the dilution of biological samples (e.g. serum, cell culture media) into the useful range of antibody-sandwich or antigen-down ELISA format assays. This unique buffer contains a heterogeneous mixture of proprietary molecules that help reduce background noise associated with non-specific bridging of signal generating conjugates to the plate well surface.
An example of the protocol for Protein-Free Sample Diluent:
1. Prepare standards in Protein-Free Sample Diluent.
2. Dilute samples in Protein-Free Sample Diluent.
• Serum samples should generally be diluted at least 1:50 in order to minimize backgrounds caused by non-specific antibody binding.
• To dilute the sample 1:100, add 1 part sample to 99 parts Protein-Free Sample Diluent. For example, add 10 µL sample to 990 µL ProteinFree Sample Diluent for a total of 1,000 µL.
• Highly concentrated samples may need to be diluted 1:1,000 or more.
3. Once diluted, run the assay according to the specific ELISA protocol.
4. Analyze the data. If the samples were diluted 1:100, the dilution factor must be considered when calculating the value. For example, if the sample generated an OD value that correlates to 500 pg/ mL based on the standard curve, multiply by the dilution factor of 100 to yield a true concentration of 50,000 pg/mL = 50 ng/mL in the sample.
Protein-Free Sample Diluent is available in 100 mL, 500 mL, and 1 L sizes.
Bulk volumes are available upon request.
ICT provides all the components you need to build a better ELISA. ICT’s coating buffers, blockers, sample and assay diluents, conjugate stabilizers, and wash buffer all work together to minimize the build up of unwanted proteins to generate a very clean signal. These products work together to address common issues during ELISA development, such as specificity, sensitivity, reproducibility, and shelf-life. ICT’s ultimate goal is to help you develop optimized ELISAs that have a high specific signal and low background noise. If you need assistance in your assay development, or are looking for assay development services, please see our available services. View our ELISA CATALOG
For more ELISA protocols and information, please visit www.immunochemistry.com.