What is avidity?
A: Whereas affinity measures the strength of a single binding site of an antibody to its antigen, avidity takes into account the cumulative strength of multiple bindings when multivalent antibodies bind to multivalent antigens. When an immune response is elicited, the IgG antibodies that are initially produced tend to have lower avidity. Over time, these antibodies undergo affinity maturation, and higher avidity antibodies are produced. Avidity assays measure the overall stability of the antibody-antigen complex.
How can avidity testing improve the differentiation of acute from chronic infections?
IgM and IgG tests are often used to gauge infection status. Typically, IgM-positive indicates an early-stage, acute infection, while IgG-positive only suggests a later-stage or past infection. However, relying solely on IgM and IgG can be misleading due to issues like false positives and IgM's cross-reactivity with other antigens. Moreover, IgM might persist long after an infection or might be absent in early stages or immunosuppressed patients. As such, avidity testing has become indispensable in clinical diagnostics. It differentiates between acute and chronic infections by assessing the strength of antibody binding to antigens, reflecting the immune response's maturity.
What methodologies are currently available for avidity testing?
Various commercial methods for measuring IgG avidity in clinical diagnostics have been developed, commonly employing protein dissociation agents such as urea or utilizing recombinant antigens.
Assays that use dissociation agents like urea disrupt non-covalent interactions — specifically, hydrogen bonding and hydrophobic interactions — subsequently influencing the binding between antibodies and antigens. Low avidity antibodies, which are weakly bound or recently formed, are more susceptible to this disruption, leading to the detachment of the antibody-antigen complex. In contrast, high-avidity antibodies are less affected and remain bound to their antigens.
Alternatively, other assays deploy recombinant proteins as blocking agents. In these assays, recombinant proteins initially block the antibodies, followed by incubation with tagged specific antigens to replace the recombinant proteins. Low-avidity antibodies will bind with tagged antigens, triggering a signal that determines the avidity. Notably, assays involving blocking agents, without the influence of chaotropic agents, potentially demonstrate reduced variability and enhanced accuracy.
Emerging research methods include kinetic avidity assays. For example, our laboratory has crafted a “testing-on-a-probe” panel that leverages a biosensor platform that targets the receptor binding domain (RBD) of viruses, with a notable application for SARS-CoV-2. Furthermore, we have developed a chaotrope- and label-free biolayer interferometry technique for real-time, label-free interaction measurements. These assays evaluate the avidity of antibodies specific to RBD in both COVID-19 patients and healthy vaccinated individuals. Our findings suggest that these novel avidity assays could be useful in monitoring both SARS-CoV-2-infected patients and the vaccination response in individuals.
What are the challenges of avidity testing in clinical practice, and future directions for its application?
Commercial avidity tests have inherent limitations. Notably, there's significant variability in their methodologies and interpretation of results, making cross-laboratory or cross-study comparisons challenging. While a high avidity index generally rules out an acute infection, a low index complicates the diagnosis and often necessitates additional information for confirmation, depending on the specific IgG avidity test used.
Emerging research-driven avidity assays, especially label-free versions, show promise but require further refinement for clinical use. While label-free methods offer advantages, including speed, cost-effectiveness, and simplicity, their sensitivity and specificity currently lag behind, which is why they haven't yet been commercialized for clinical diagnostics. However, recent studies aiming to enhance the sensitivity and specificity of these label-free avidity tests are encouraging, suggesting potential for their routine clinical application in the foreseeable future.
Yaxin Li, PhD, is a postdoctoral research associate in the department of pathology and laboratory medicine at Weill Cornell Medicine in New York City. +Email: [email protected]