Commentary: How can multiplex immunoassays support vaccine development?

Well-established assay formats that can be used throughout the development continuum are  a great benefit to researchers

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Commentary: How can multiplex immunoassays support vaccine development?

The drive to develop a safe and effective vaccine for SARS-CoV-2 has meant that vaccines have been in the spotlight more than ever over the past year. The concerted and coordinated efforts of scientists worldwide resulted in the structure and function of SARS-CoV-2 being deciphered rapidly, thereby enabling the use of diverse vaccine design strategies, with three main types of COVID-19 vaccine (mRNA, protein subunit, and vector) either already or soon to be in Phase 3 clinical trials in the United States. [1]

Developers and manufacturers are also under increasing pressure to utilize tools and techniques that will aid and speed the development of these complex biologics while building in ‘quality by design’. The application of well-established assay formats, which can be used throughout the development continuum, are consequently a great benefit to researchers in terms of both saving time and ensuring presentation of high-quality data to the FDA.

Multiplex immunoassays

A prime example of vaccine developers taking advantage of proven technologies to help fast-track potential vaccines through pipelines is multiplex immunoassays. Offering the flexibility to create competitive, sandwich, direct and indirect assays, multiplex immunoassays have been used for more than 15 years to evaluate the efficacy of an immune system response. Requiring only very small amounts of sample, multiplex immunoassays can be applied at all stages of vaccine development, including at the preclinical stage, and therefore stand out as a proven tool to monitor critical safety and efficacy aspects of the process.

Multiplex immunoassays are essentially bead-based derivatives of ELISAs, such as xMAP® (multi-analyte profiling) technology [2]. However, in comparison with traditional methods such as ELISA, where you can look at only one marker at a time, or flow cytometry, where you may have 10 to 12 markers, the bead format allows for up to 500 markers per assay, empowering consideration of complex antibody interactions within the multiplex assay. For cytokine profiling during upfront development, for example, this gives researchers a much more comprehensive and accurate picture of what is happening.

High throughput is enabled in bead-based multiplex immunoassays through their ability to detect multiple analytes, using varying ratios of different fluorescent dyes in each bead. The specified target analyte is detected based on the fluorescence intensity of the xMAP® reporter molecule, phycoerythrin. These assays are run on 96-well plates and read on Bio-Plex or Luminex instruments, which can distinguish each bead region associated with an analyte and measure fluorescence intensity, enabling the development of both quantitative and qualitative assays.

From early discovery to clinical trials

Serological studies can be used through the entire process of vaccine research and development, spanning animal models in research, the preclinical phase, clinical trials, and even post-market surveillance, allowing continuity throughout. Several papers have been published on Gardasil, a vaccine for human papillomavirus (HPV), demonstrating the utility of the indirect multiplex immunoassay format for serological assays to evaluate immunogenicity and monitor efficacy of the vaccine.  In this format, multiple antigens are bound to different beads, allowing antibodies specific to the bound pathogen proteins to be measured in serum and plasma samples.

In one of these studies, viral subunits used in the vaccine were coated on beads and used to quantify the immune response with and without adjuvant formulation in non-human primate models, showing that the inclusion of an adjuvant resulted in higher antibody titers [3]. A competitive assay was also developed, using beads coated with known neutralizing epitopes to detect levels of neutralizing antibodies in samples after vaccination. Used for human serum in clinical trials, the competitive assay format demonstrated that Gardasil successfully induced seroconversion and protective immunity. In a separate study, the multiplex competitive assay format also enabled the detection of neutralizing antibodies to Gardasil’s multivalent vaccine, thereby increasing testing throughput. [4]

The value of the competitive assay format also extends to post-market surveillance. For Gardasil, the efficacy, immunogenicity, and safety of the vaccine was assessed ten years post-vaccination. The results from this assay showed that seroconversion to all four quadrivalent HPVs was achieved, with peak titers after the third dose. Of the thousands of patients tested, all had high levels of antibodies against the four virus-like particles, implying a sustainable and strong immunological response, and all were confirmed to be negative for HPV infections.

A boost for screening

Clone screening to ensure that only the best candidates are selected for further testing is a vital step at various stages in vaccine research and development but can be very time- and resource-intensive. Singleplex assays limit the number of clones that can be picked per screen, requiring a single assay per protein, which can have a significant impact on throughput when multiple proteins must be assessed. The ability to conduct multiplex assays increases not only throughput, but also the number of clones that can be picked, and in turn improves the chances of identifying a high-yield clone that will produce a high-quality protein that meets all the required clinical specifications. Multiplexing immunoassays during clone screening can therefore have a significant effect on overall development time.

For example, GlaxoSmithKline has used a single-step cloning strategy in which fluorescence-activated cell sorting deposits single cells in 96-well plates, where clones are initially screened for robust growth through imaging. This approach combines existing technologies to shorten the CHO cell cloning process to a single step. A sensitive, custom multiplex sandwich immunoassay is then used to quantify expression of the multi-subunit viral protein used in the vaccine. This process was used to narrow 4,000 clones to fewer than 80 for passaging — about 80% less than with traditional methods, which would usually result in ~300 clones. [5]

Maximizing immunogenicity through adjuvant selection

Adjuvant formulation as an element of vaccine development ensures maximum immunogenicity for specific populations — when vaccines are to be administered to very young children whose immune systems are not yet fully developed, or to older people who may be going into immune senescence, for example. In these types of studies, where multiple experimental conditions, tissues, and timepoints must be monitored across a variety of formulations and multiple models, the high throughput and small volume requirements of multiplex immunoassays mean they are an ideal solution.

Mouse cytokine panels have been used to study T cell responses, allowing researchers to determine whether the vaccine adjuvant is stimulating the innate immune system, thereby strengthening the adaptive immune response, by assessing titer levels of antibodies against the pathogen in question. In one study, Bio-Plex Pro Mouse Cytokine Assays were used to monitor the effects of adjuvants in influenza vaccines in serum and lung homogenate of mice of varying ages. The researchers found that cytokine response varied depending on age and whether an adjuvant was included. They also found that post-infection cytokine increases in vaccinated mice were localized, rather than systemic. [6]

In another study, Bio-Plex Pro Human Cytokine Assays were run on cell culture supernatant samples to evaluate cytokine profiles related to stimulation from different influenza antigens. Cytokine levels were studied in conjunction with seroconversion pre- and post-vaccination to understand whether there were predictive cytokine biomarkers that correlated with developing acquired immunity to an influenza vaccine. [7]


Multiplex immunoassays offer a well-established, flexible assay format that can make significant contribution to time and sample savings throughout the various stages of vaccine research and development, from early-stage concept all the way through to the clinic. Within the context of COVID-19 vaccines currently under development, multiplex immunoassays have proven useful for confirming efficacy and profiling the immune response. The COVID-19 vaccines development task is particularly urgent and the scale immense, meaning that tools to increase the efficiency of the end-to-end process are more vital than ever.




[3] Ruiz W et al. (2005). Kinetics and isotype profile of antibody responses in rhesus macaques induced following vaccination with HPV 6, 11, 16 and 18 L1-virus-like particles formulated with or without Merck aluminum adjuvant. J Immune Based Ther Vaccines 3, article 2.

[4] Smith JF et al. (2008). Evolution of type-specific immunoassays to evaluate the functional immune response to Gardasil, a vaccine for human papillomavirus types 16, 18, 6 and 11. Hum Vaccin 4, 134–142.

[5] Sargent B (2020). Single-Step Cloning Strategy Speeds Timeline for Novel Vaccine Candidates., accessed November 30, 2020.

[6] McDonald JU et al. (2017). Inflammatory responses to influenza vaccination at the extremes of age. Immunology 151, 451–463.

[7] Nakayama T et al. (2018). Cytokine production in whole-blood cultures following immunization with an influenza vaccine. Hum Vaccin Immunother 14, 2,990–2,998.

BIO-RAD and BIO-PLEX are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. The Bio-Plex Suspension Array System includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation. Luminex is a trademark of Luminex Corporation. All trademarks used herein are the property of their respective owner.

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