Immunoassays detect and quantify molecules of biological interest by relying on the selectivity and specificity of antibody reagents. If you are going to run the same immunoassay for years, a sustainable, consistent source of those reagents matters as much as the assay design. This is a short guide to how an immunoassay is developed and where the reagents come from.
Development, optimization and validation
The path from idea to routine assay usually runs like this:
- Define the critical success factors. Sensitivity, dynamic range, throughput, reagent cost and so on.
- Acquire the reagents. Antibodies, analyte, standards, sample matrices, control samples, labels and substrates.
- Test the instrument. Calibration, performance testing, linearity.
- Run proof-of-concept experiments. Establish preliminary assay parameters, reagent suitability and matrix compatibility. Look at signal-to-noise and data analysis models; this is a good point to consult an immunoassay expert.
- Optimize. Select the factors to optimize, determine matrix effects, construct precision profiles, and consult a statistician.
- Validate. Robustness, day-to-day effects, scale-up, automation, reproducibility, operator effects, percentage recovery.
- Document and implement. Write the SOP and put the method into use.
The reagents
Blocking reagents raise the signal-to-noise ratio and cut background interference, which is how you improve sensitivity. Purified gamma globulin fractions do this job well and also serve as assay loading controls and quantitation standards. Reagents such as goat gamma globulin can be supplied in large quantities as a raw material for in vitro diagnostic kits.
Reporter molecules and conjugates make the antigen-primary antibody complex visible. Protein A/G conjugates and secondary antibodies are the cost-effective options; the streptavidin-biotin system is an alternative when you need more signal amplification from a conjugated primary antibody.
Solutions, substrates and stabilizers round it out: buffers and stabilizers that promote the protein interactions the assay depends on and hold physiological conditions steady.
We supply animal serum, plasma, complement and ancillary products for immunoassay development, including human gamma globulin, goat gamma globulin, IgG/IgM/antibody-depleted complement and adult rabbit complement, all available directly from our website. Contact us to learn more, or call (800) 643-3426 or (479) 636-4361.