Immunoassay in a Kit

Using an immune-related molecule, usually an antibody, to measure an analyte in a sample makes up the basics of an immunoassay. The best-known version is the enzyme-linked immunosorbent assay (ELISA). With an ELISA, says Ricardo Brandwijk, R&D scientist at Hycult Biotech, measuring “biomarkers in the fluid phase for quantificational purposes is the most applied application.” Part of the wide use of immunoassays in general comes from the ability to use many kinds of samples, from blood and synovial fluid to lysed cells and tissues. Consequently, scientists use immunoassays for basic and clinical work. As Brandwijk says, “In most cases, results between healthy/control and diseased/affected are compared.”

Other analytical methods, especially ones that require culturing samples, slow down progress. “Rapid diagnosis is hampered due to the time required for the cultivation and identification of infecting microorganisms,” says Sabrina Dominici, senior R&D researcher at Diatheva. “Therefore, a great deal of interest has been shown in the development of new practical techniques for the rapid diagnosis of infections, and most of these techniques involve immunoassays.”

A great deal of interest has been shown in the development of new practical techniques for the rapid diagnosis of infections.

These tools get turned on an increasing number of biological and medical problems. “A number of recent studies have demonstrated the utility of blood-based protein biomarkers for early detection of cancer, neurodegenerative disease progression, and monitoring response and resistance to immunotherapy,” says Jeremy Lambert, director of marketing at Quanterix. “Traditionally, protein biomarkers found in ultra-low levels, particularly neurological ones, have only been detectable in cerebral spinal fluid, but being able to detect such critical biomarkers in blood and serum has the potential to transform how disease and brain injuries are diagnosed.” Immunoassays can provide that level of detection.

To make it all as easy as possible, though, scientists want the technology in a kit. Such ready-to-use products, says Dominici, “are highly adaptable and can be applied to many formats depending on the needs of the end user.”

Keys to the right kit

Even with kits available, scientists must know what matters. Typically, that includes affordability, consistency, sensitivity, specificity, and more. As James Murray, immunoassay product development scientist at Abcam, says, “An immunoassay kit should be fit-for-purpose so that it helps the user to reliably achieve accurate and consistent results.”

Just what fit-for-purpose means depends on the task at hand. “Requirements for sensitivity and dynamic range can vary widely,” Lambert explains. “For example, researchers measuring proteins that are expressed at high concentrations in cell culture or tissue samples may not require low limits of detection from immunoassays, but researchers measuring inflammatory biomarkers in blood or neurological biomarkers in cerebral spinal fluid samples will benefit from assays with very low limits of detection and/or low sample volume requirements.”

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The number of molecules that will be measured should also be considered. Some kits measure more than one protein in one sample, which can increase throughput—by not needing multiple tests—and reduce the needed sample volume—by looking for more than one thing in the same sample.

To ensure that a kit does what a supplier says, data must back up the claims. “Thorough validation, including primary sample-specific quantitative data, should be provided with each immunoassay,” Murray notes.

Despite all of the amazing advances in immunoassay kits, nothing is perfect. To get the best results from one of these tools, scientists must know the shortcomings that can arise. For one thing, says Dominici, “immunoassays may be subject to interference by a variety of molecules that can chemically react with assay reagents or biological molecules, confounding the assay readout by producing apparent biological activity.” In general, suppliers do what they can to remove the possibility of interference. Still, challenges exist. “Some classes of compounds display non-specific reactivity rather than target binding,” Dominici notes. “Fortunately, there are many experimental tools available to researchers to mitigate this potential problem.”

Interesting apps

How scientists use tools reflect their real value. When asked to describe an interesting recent use of an immunoassay kit, Brandwijk mentions a paper by Daniel Ricklin, a molecular pharmacologist at the University of Basel (Switzerland), and his colleagues.¹ “The paper emphasizes that the reliability of immunoassays is best challenged with patient cohorts where effects of treatments are studied in the actual environment,” he says. “The assay should be modified to that purpose, and a standard curve is not full proof that an ELISA works with biomarkers in their natural matrix.”

Scientists explore many biomarkers with immunoassays. As an example, Lambert pointed out that researchers from the Mayo Clinic and Lilly Research Laboratories used Quanterix’ immunoassay technology “to measure the protein biomarker, tau, as a prognostic marker for cognitive decline and dementia.”² In addition, scientists from the Karolinska Institute and the University of Gothenberg demonstrated that measuring serum neurofilament light chain protein serves as a biomarker of the response to multiple sclerosis therapy.³ “In both examples, the protein biomarkers are only detectable using advanced ultra-sensitive immunoassay methods,” Lambert explains.

Changes in one area of research can also drive advances in others. For example, as Murray says, “In an era where genomic data is readily available, it’s important to understand how this information is translated into the proteome and how it’s utilized within cells via transcriptional regulation—at the RNA level—and between cells and tissues through signaling pathways—the protein level.” He adds, “Only in the context of the proteome does genomics make sense.” So, Abcam created a multiplex assay platform that can measure miRNA or proteins. “This technology allows scientists to more easily study the relationship between miRNA-regulated transcriptional expression and cytokine-regulated protein pathways in organisms and disease states,” Murray says.

In some cases, assays must aim at very specific states of a disease. As an example, Dominici points out listeriosis. “During pregnancy, this infection can cause a variety of diseases, ranging from a mild chill to a severe illness,” she says. “Unfortunately, listeriosis could be asymptomatic and/or a pregnant woman could not be aware of being exposed to listeria.” Her company developed an immunoassay that detects antibodies against listeriosis, which can reveal the infection even without any symptoms.

The simpler and more specific offerings in immunoassays make them more valuable than ever. Moreover, ongoing refinements give these kits a wider range of application, even with less training.

Cells stained for beta Tubulin using Anti-beta Tubulin antibody (ab6046) detected by Goat Anti-Rabbit IgG Fc (Alexa Fluor 647, ab150091), with DAPI nuclear counter-stain. (Image courtesy of Abcam.)

References

1. Ricklin D, et al. “Complement in clinical medicine: Clinical trials, case reports and therapy monitoring,” Mol. Immunol. 2017 [epub ahead of print, PMID: 28576323].

2. Mielke MM, et al. “Association of plasma total tau level with cognitive decline and risk of mild cognitive impairment or dementia in the Mayo Clinic study on aging,” JAMA Neurol. 2017 [28692710].

3. Piehl F, et al. “Plasma neurofilament light chain levels in patients with MS switching from injectable therapies to fingolimod,” Multiple Sclerosis Journal 2017 [28627962 ].