Immunoassays - Critical Success Factors

Immunoassays are performed to obtain quantitative and qualitative measurements of minute quantities of an analyte - a specific substance - in a blood or body fluid sample using an immunological reaction. The high specificity and sensitivity of immunoassays result from the use of antibodies and purified antigens as reagents. In fact, immunoassay reagents and controls are the crux of an immunoassay system and the level of sensitivity and specificity of the immunoassay depends on the proper selection of reagents.

Preserving long-term stability of analytical procedures is an important goal for clinical laboratories. Experts point out that laboratories need to consider several factors when using reagents and controls for immunoassays:

• Type of equipment: ELISA could be an enzyme-, chemiluminescence-, or even fluorescence-based assay. Laboratories can use the type of system they are most comfortable with.
• Compatibility: It is necessary to obtain the right type of reagents. Antibody reagents are generally optimized and validated for the type of sample that is being assayed. There are kits specifically for plasma, serum only, or plasma with citrate or EDTA and it is important to ensure that the kit purchased is suitable for the sample in question. Leading manufacturers of immunoassay systems offer reagents and controls for the systems they supply.
• Sensitivity: This depends on a number of factors, especially the antibodies used. In sandwich ELISA (Enzyme Linked Immunosorbant Assay), which quantifies antigens between two layers of antibodies, the plate is incubated with a blocking solution after it is coated with analyte-specific antibodies, to lessen non-specific binding.
• Lot-to-lot consistency: When a new reagent lot is received by the laboratory, it is necessary to ensure that there is no significant difference from the previous lot of the same reagent. Many factors could affect the performance of a new lot of reagent lot such as a change in raw materials versus an antibody, a change in the stability of the reagent, or faulty calibration. By verifying each new reagent lot that is used, labs can ensure that the lot maintains consistent results for patient specimens. This will help to preserve the long-term stability of analytical procedures. In addition, lot-to-lot verification is also a compliance requirement of bodies such as CLIA and Joint Commission.

There are thousands of immunoreagents and reagents available to detect diverse molecules, including those that can detect minute quantities of the target molecule. However, misinterpretation is a risk that can be avoided only with carefully conceived staining study design.