Transforming Antibody Detection

Towards the end of 2019, the National Institutes of Health (NIH) organized a Pandemic Preparedness Challenge that aimed to assess the potential response to an emerging disease outbreak, such as a new strain of influenza. In one of the resulting publications from the exercise, the role that a novel technology, Arrayed Imaging Reflectometry (AIR™), could play in quickly screening small amounts of blood for antibodies to influenza and other viruses was demonstrated.

Ardaza Biosystems has since transformed the AIR technology into a multiplex label-free biosensor platform, ZIVA, which can detect hundreds of analytes at a time from a drop of blood, plasma or serum. Technology Networks recently spoke with Bryan Witherbee, CEO, Ardaza, to learn more about ZIVA and discuss some of the applications it could benefit.

Anna MacDonald (AM): Can you tell us about AIR and give us an overview of how the technology works?

Bryan Witherbee (BW): AIR, founded in 2008, from the laboratory of Dr. Benjamin Miller at the University of Rochester, NY, is a label-free optical biosensing technique based on the ability to sensitively measure reflected light from binding events on the surface of our biosensors. The basic principle of the technology is to fine tune the surface of the biosensor that has the array of capture molecules present, such that it is antireflective (zero reflectance).  As these capture molecules specifically bind biomarkers in biological samples, they are then capable of reflecting light.  We can accurately and sensitively measure that reflected light.

Adarza is the sole exclusive owner of the technology and has developed AIR into a fully automated biomarker detection platform, ZIVA. The ZIVA platform (instrument and analysis software) enables the AIR™ technology to be completely automated and user-friendly. Using miniature silicon biosensors (approximately 5 x 5 mm), printed with customizable capture molecules (proteins, antibody, viruses) enabling 100+ targets to be interrogated. When a drop of sample is added on to the chip, biomarkers in the sample will bind to their complementary chip protein. Depending on the molecules printed on the chip, one can survey what is present or not present in the sample. Unbound microchips are antireflective. But when binding occurs, the surface thickness changes causing light to be reflected at an angle. This reflection is caught on a sensor camera and analyzed and the results correlate to the size of the molecule bound, while the printed protein map on the biosensor provides the specificity. One of the best features of ZIVA is that there is no labeling, fluorescent or otherwise required. The instrument takes about 30 mins (give or take) per run to analyze the signal. Furthermore, these biosensors can further be assembled in a smart consumable 96-well format to enable high throughput analysis of samples.

AM: What applications can AIR be used for?

BW: AIR has many applications across a variety of industries and areas. This instrument can be applicable to any field requiring large scale multiplexed protein detection from thousands of samples. While the initial launch of ZIVA is focused on both protein and antibody arrays in the areas of clinical and translational research in the academia, pharmaceutical, and government industries, there are many other areas of application.  These would include agriculture crop selection and testing, food safety monitoring, and antibody screening and production.  Our technology is extremely versatile and can be used to measure a variety of biological molecules beyond just proteins and antibodies including DNA, RNA, as well as virus and bacteria fragments/particles.

AM: Can you tell us about the Pandemic Preparedness Challenge you took part in? What were the key findings? How did this exercise impact your response to the COVID-19 pandemic?

BW: The Pandemic Preparedness Challenge we participated in was an exercise run by the national NIH flu center network to understand how we might respond to an outbreak of a new strain of influenza. Our part was to use AIR technology to understand how a newly pandemic strain of flu (in this case, dog flu) might relate to known strains, and to use this information to identify candidate vaccines and therapeutic agents. We used a 115-plex antibody array of different Influenza strains, to show that the “mock pandemic” strain was closely related to HK68 influenza, a strain known as the “mother clone” of vaccine strains. From the array, we identified several antibodies reactive with both HK68 and the “mock pandemic” strain. The exercise helped prime us for action and was definitely a blueprint for how we could leverage AIR for an outbreak of disease. We were able to quickly add SARS-CoV-2 proteins (spike, RBD, nucleocapsid) and other coronaviruses to our influenza array, which allowed us to begin assessing samples for response to the SARS-CoV-2 virus.

AM: Why is it useful to be able to analyze the human COVID-19 antibody response?

BW: Serology and serosurveillance are very important tools used to determine the prevalence of infectious disease as well as host immune response and immunity. For COVID-19, it is critical to understand how confirmed positive (via PCR) patients’ host antibody status (titer) evolves and how long immunity will exist.  Currently it is not known if antibodies generated from COVID-19 infection provide protective immunity, how much antibody is protective, and how long the duration of protection. In addition, with accurate serology testing the true prevalence of COVID-19 in the general population can be assessed.

AM: In what ways can AIR benefit vaccine or therapeutic antibody development?

BW: Vaccine development starts with finding the right “agent” to generate a desired immune response. AIR can be used in the screening process to find the right vaccine that results in the desired immune response by interrogating many “agents” in an arrayed format, leveraging our custom array services. Because AIR is label-free, an immune profile is easily generated with no need for a secondary labeled reagent.  Conversely, the AIR technology can also be used to help vaccine development to quantify the “agent” in the vaccine (whether a toxin, protein, or microbe) and is especially beneficial for multi-valent characterization.  For example, AIR can be used in influenza vaccine development by enabling the use of arrays of monoclonal antibodies to different hemagglutinins and quantify them without the need to develop a cumbersome, tedious, traditional sandwich ELISA. Last, AIR technology can quickly assess the efficacy of the host immune response by looking at the serological “signature” in response to the vaccine and monitor vaccine efficacy in the general population.

Therapeutic antibodies are monoclonal antibodies raised against a particular antigen and widely used in the treatment of autoimmunity, cancer and inflammatory diseases. ZIVA’s array is capable of detecting and differentiating the antibody panel of a patient generated in response to a disease (e.g. COVID-19), classify them based on their abundance and sort them as potential therapeutic antibodies to treat the disease. Therefore, ZIVA is applicable to both the preventative and the therapeutic workflow.

AM: How does AIR compare to other methods of detecting human antibodies?

BW: AIR, the technology driving the ZIVA platform, is different from other methods because of its label-free approach and multiplexing capabilities. Other technologies need a label to see any immune signature from the host.  AIR enables the immediate detection of immune response without the need for an additional step, measuring total immunoglobulin (Ig).  The ZIVA workflow can be further designed to measure classes of Ig (IgG, IgM, etc.) if desired, by a simple additional step.  The traditional approach only allows measurement of one class of Ig at a time and is difficult to get a total Ig measurement.  Because the human response to viruses and other infectious agents is complex and a multitude of antibodies are produced to fight infections, ZIVA can provide a very cost-effective, customizable, and simple approach to obtaining a serological “signature”.

Another point that we want to convey is that with ZIVA we have the ability to scale from low to high multiplexing. ZIVA not only can detect multiple molecules simultaneously, it can also differentiate accurately the entire biomarker continuum. We can affordably and effectively provide low multiplex/single plex formats as well. At this moment, ZIVA has features that allows for multiplexed testing, high or low, in an accurate and efficient fashion requiring a small amount of sample.

AM: What are the benefits of multiplex testing?

BW: Multiplex testing by definition is testing for the presence of a variety of molecules simultaneously from a single sample. The benefits of multiplex testing are in the high definition of detail that can be provided to assess immune response. It also provides a more sensitive and specific profile compared to measuring a single host antibody response by providing a high definition view of a person’s immunity status. In our current product (Acute Respiratory Viral Serology Array) we provide researchers the ability to understand past exposures to influenza and other coronaviruses.  This is important as exposure to past coronaviruses can lead to false positives in tests that are looking only at COVID-19 infection.  Leveraging our multiplexing capabilities, we have designed an orthogonal analysis by having multiple versions of the COVID-19 proteins and other coronaviruses – thus if there is a positive response it can be verified and can identify potential false positives due to past exposure to other coronaviruses.  Last, while the single point tests are generally accurate in confirmed PCR positive patients, they do not provide the robustness needed to assess prevalence of disease in the general population and can lead to false positive results in as high as 30% of those screened.  The multiplex, orthogonal approach is a much more robust prevalence tool for epidemiology.   

AM: Can you tell us what next steps you have planned?

BW: For the serology market, we plan on expanding the respiratory panel to include more influenza “pandemic” content to aid in surveillance and monitoring. ZIVA assays are very customizable so the addition of content to interrogate other viruses or even new pandemic viruses, such as swine flu, can be done quickly.

For the protein detection market, we will be launching our human and mouse cytokine screening (pg/ml level of sensitivity) and ultra-sensitive arrays (fg/ml level of sensitivity) in Q4 of 2020. We continue to expand in this market with more content in 2021 to address biomarker detection needs in metabolism, cardiology, oncology, neurology, and toxicity.

Bryan Witherbee was speaking to Anna MacDonald, Science Writer, Technology Networks.