Achieving a high signal-to-noise ratio and solid standard curve are goals of any ELISA development project, yet several complex issues may arise, presenting challenges to assay optimization. Many issues stem from differences between the sample and calibrator matrices and nonspecific binding of immunoassay components.
Most of the time invested in developing a new ELISA will involve selecting the proper calibrator matrix that matches most closely to the often very complex biological sample matrix. Assay Diluents are additive components used in an ELISA to equalize any differences between the sample matrices (serum, plasma, urine, cell culture fluid) and the calibrator diluent used to generate the standard curve. The Assay Diluent Optimization Pack provides 100 mL of each of our four Assay Diluent formulations to create an economical method for addressing complex matrix effects and other assay concerns in ELISA development projects.
To quickly determine which Assay Diluent formulation is best for the assay, all four Assay Diluents can be run in parallel on the same IgG-coated ELISA plate.
1. Coat antibody or antigen onto the ELISA plate using ICT’s Antibody Coating Buffer or Antigen Coating Buffer.
2. Incubate 8–24 hours at room temperature (RT).
3. Aspirate the coating solution.
4. Wash each well twice with ICT’s ELISA Wash Buffer.
5. Block the uncoated regions of the ELISA plate by pipetting 300–400 µL of block buffer into all wells and incubate 8–24 hours at RT.
6. Designate wells for the evaluation of each Assay Diluent formulation, and then load 50-100 µL of each Assay Diluent into the appropriate wells. Note that all wells should receive Assay Diluent.
7. Prepare standards and samples spiked with known amounts of target analyte and then load 50-200 µL into appropriate wells.
8. Proceed with the rest of the ELISA protocol.
9. Analyze results to determine which Assay Diluent formulation gave the lowest background signal, highest signal-to-noise ratio, and most accurate recovery of any samples spiked with known amounts of analyte.
• pH: 7.4 at 1X
• Contains: General Assay Diluent – Mammalian proteins
IgM-Reducing Assay Diluent – Mammalian proteins
Neptune Assay Diluent – Non-mammalian proteins
Antigen-Down Assay Diluent – Mammalian proteins
• Supplied At: 1X
• Storage: 2-8°C