What is the most important feature of any plate-based immunoassay?

Reproducibility came out on top as the most important feature¹ listed by respondents in a 2018 survey compiled by Biocompare. The report summarized the responses from researchers and scientists across the immunoassay market.

Synonymous with lot-to-lot consistency, reproducibility is key for drawing conclusions from your experiments. You need to know that the immunoassay will work consistently today, tomorrow, a year, or even five years from now. You also want to have confidence that the immunoassay will return the same data in a peer’s hands and between kit lots. Reproducibility^1,2 is crucial for advancing our understanding of the natural world.

What else did they say?

Assay precision, sensitivity, low cross reactivity, and cost rounded out the top four most important features of an immunoassay¹. Improved product quality was consistently mentioned as a desired product change for a variety of immunoassays including: colorometric, chemiluminescent, fluorescent, electrochemiluminescent and bead-based assays. Other important features include ease of use and automation.

ELISAs are by far the most used immunoassay type cited by survey respondents. ELISAs are followed by immunoblotting (Western blot), immunohistochemistry, flow cytometry cell-based assays, multiplex assays, fluorescent immunoassay, and flow cytometry bead-based assays.

What drives ELISA’s popularity?  For starters, ELISA is a reliable, accurate, and sensitive technique that is well-established in most research labs. Adding to its broad appeal are advancements in ELISA technology, including instrumentation and analyzers capable of faster throughput, multi-mode detection, and sensitivity along with automation of the plate-handling steps.

Driving confidence in immunoassay results

No matter the application, consistent quality and performance are what customers seek. Meeting this demand requires tight quality control and rigorous validation measures. Since not all suppliers validate immunoassays to the same level, achieving this high level of consistency is no simple task.

At Bio-Techne, we take pride in our commitment to the science and technology underpinning our products. Tight quality control of raw materials and rigorous validation help ensure our assays deliver superior accuracy and consistency over time.

Gaining this high level of confidence hinges on the careful execution of four fundamental quality control measures that involve the use of specialized, high-quality diluents.

• Recovery. Recovery represents a measurement of immunoassay accuracy. A spike recovery analysis provides an estimate of how much of the available analyte is measurable and whether sample components might be interfering with that measurement. After spiking a concentrated recombinant protein into a complex sample matrix, the readout of the spike, (minus endogenous), should be equal to the control spike. The ideal is 100 percent equal. If your sample fails to recover above 70 to 80 percent, depending on the assay, this is an indication of something causing interference within your assay sample.

• Linearity. Linearity of dilution is another core component of immunoassay accuracy, specificity, and performance. No matter what dilution factor your sample is run at, back calculated concentration should always be the same. Without proper reagent optimization, sample values often increase as you apply higher dilutions. As you dilute your sample, the interfering elements also dilute out, increasing the concentration of the target analyte and thereby making it more accessible.

• Specificity. One of the key determining factors of immunoassay quality is making sure you are measuring the correct analyte. Immunoassays are inherently subject to potential interferences that can lead to in inaccurate values. Since not all assay manufacturers employ the same quality controls, you want to make sure your supplier verifies specificity through multiple measures.

• Consistency. Lot-to-lot consistency is essential to achieving reliable long-term research studies. Stringent quality control testing on each kit component and each complete kit help make sure standard curves, background, and sample values remain consistent. Each new standard lot is tuned against a master calibrator to ensure reliable and reproducible results between kit lots, and to help prevent drift in variability over time due to differences in protein immunoreactivity.

Keeping pace with immunoassay performance demands

Across the entire immunoassay market, the common trends and drivers reflect the need for advanced reagents and instruments for multiplexing, faster throughput, sensitivity, and ease of use. At the same time, technical advances in the characterization of new biomarkers and their detection and measurement are helping to provide researchers with greater assurance that the results they achieve are accurate and reproducible from one experiment to the next.

Learn More Immunoassay Best Practices

• How do I optimize the performance of my multiplex immunoassay?
• Which immunoassay will best meet my research needs?
• What are some other ways to improve assay performance?