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dried sampling feasibility for Group A strep serology, vaccine research

by James Rudge, PhD, Technical Director, Neoteryx | 5 min read

An article published by Alana L. Whitcombe et al at The University of Auckland, New Zealand in the January 2022 issue of Journal of Immunological Methods, reported on development of an eight-plex immunoassay from both serum and dried blood samples. The paper is entitled “An eight-plex immunoassay for Group A streptococcus serology and vaccine development.” It describes the development and validation of a high sensitivity multiplex ligand binding assay for serological assays and vaccine development of Group A streptococcus (GAS) infection. The method deployment was highly successful, and the authors commented that this 8-plex assay '"will be a powerful tool for simultaneously measuring the prevalence and dynamics of GAS antibodies in populations affected by GAS disease to inform GAS vaccine development.”

Background on Group A streptococcus (GAS) Infections

Group A streptococcus (GAS) infections cause a wide range of problems ranging from strep throat to scarlet fever and the skin condition impetigo. Antibiotics are used to effectively treat these bacterial infections, but it is estimated that there are 1.78 million new GSA cases worldwide each year, with more than 500,000 individuals succumbing to complications from severe GAS diseases. One such complication is rheumatic heart disease (RHD), an inflammatory autoimmune disorder resulting from rheumatic fever that permanently damages the heart valves, impairing heart function. Indeed, it is estimated that out of the 500,000 deaths linked to GAS, 320,000 are as a result of RDH.

Vaccine Research, iStock-1027752634

Vaccine Development Research for GAS

The Covid-19 pandemic has demonstrated the incredible efficacy of vaccines for helping to prevent severe SARS-CoV-2 infections, which can result in serious complications. Just as vaccines have been a major step forward in the battle against Covid-19, they could have a similar impact in preventing GAS infections and the related disease complications, such as rheumatic fever and RHD. There are several GAS vaccines in development, some of which target the M-protein. Yet, because there are at least 200 variants of M-protein, targeting it for vaccines is challenging. There are, however, a few conserved proteins too, which are more appealing as vaccine targets. It is hoped that combined vaccines will be able to cover > 98% of all GAS agents.

There are several ways to diagnose GAS, such as via bacterial swab & culturing. Serology is another popular way to test for GAS and targets such as Streptolysin O (SLO), which is a cytolytic toxin released by GAS. Another popular target for such assays is DNaseB, an extracellular virulent protein with DNA-degrading activity.

The same research group at the University of Auckland had previously developed a 3-plex assay to look at these SLO and DNaseB targets as well as a novel target (SpnA). The group’s goal for this current project was to add to their 3-plex array with 5 antigens obtained from vaccine development They also wanted to look at a subset of paired venous blood to serum samples to confirm that this assay would also be compatible for dried blood samples collected remotely with microsampling devices, such as DBS or VAMS. With this more comprehensive list of targets tested on serum and dried blood, they hoped that the assay would provide “the possibility for serosurveillance and vaccine antigen response studies to be more readily conducted in remote settings and high-risk populations.”

Strep Study Methods & Findings

The research group in New Zealand manufactured and purified recombinant proteins for a number of the of the antigens, however SpyCEP, SpyAD and the GAC were supplied by the GSK Vaccines Institute for Global Health.  

  • The antigens were coupled to magnetic beads to be used on a Luminex platform (multiplex immunoassay). The beads were stored appropriately in wash buffer.

  • For standards development, they purified all 8 IgG antibodies classes from pooled IVIG. These were confirmed by ELISA and quantified using Nanodrop2000 spectrometry. They then created working standards from these.

  • They optimized the assay protocol using the 8-plex bead solution containing the antigen, which was added to serum or plasma.

  • The method was tested for specificity, sensitivity, and reproducibility.

  • As part of the validation, samples were taken from multiple sources: Sera from patients with GAS (n=7), 16 sera samples from patients with ARF, 13 samples from healthy children, and 26 healthy adults. From some of the patients, paired blood for dried blood and serum samples were also collected.

  • Dried blood samples were either collected on Whatman 903 cards (DBS) or 20 µL Mitra® devices (VAMS®). These were dried overnight and for up to 21 days at RT. Both dried sample types were shaken overnight at 4°C in PBS, BSA and Tween.

  • Serum equivalents where then calculated. To do this they approximated that 50% of the blood volume was hematocrit. As a side note, this approach to obtain serum equivalent results had been previously employed by other groups. Please consult a review of serological (and related) assays used during the Covid-19 pandemic on Mitra devices for details.

Study Authors’ Discussion & Conclusions

  • The assay showed a wide dynamic range with excellent precision, showing intra-assay CVs of up to 3.5% and inter-assay of between 6.9-10.5%.

  • When comparing single-plex and multiplex assays, no interference or cross-reactivity was observed between the antigens. This allowed for a combined multiplex assay that could be used for serological and concerned vaccine antigens.

  • The bead-based nature of the multiplex assay required far less antigen than traditional ELISA or CLIA.

  • Researchers were able to collect 512 datapoints from one plate (in only a few hours), making this assay one of the highest throughput multiplex immunoassays currently available for serology and related assays.

  • The approach could be modified to look at subtypes of antigens allowing a systems approach to study antibody interactions and dynamics. Indeed, the same group used this approach to understand the serological landscape of Covid-19 antibodies (including Ab neutralization).

  • Both dried blood sample types (DBS (N= 26) and VAMS (N = 8)) were compared with matched serum. Both showed a very high degree of correlation (R2992 for VAMS and R2 = 0.977) compared to serum.

  • The authors commented that by combining both antigens from vaccines and also natural infection, that this approach could be used to distinguish between natural and vaccine induced raised antibodies. Furthermore, they commented that this approach would have utility for future clinical trials in this disease area.

  • As there is currently no point of care test to detect GAS, the group commented that dried blood samples would be an ideal solution for remote and resource limiting settings. Indeed, they stated that dried finger-prick blood sampling “offers a non-invasive way to collect samples in these areas, while benefiting from the precision of a laboratory-based assay”.

  • The group did concede that the study was limited by the number of clinical samples, but they now plan to conduct larger studies to further cement this approach.

Final Thoughts

Coupling finger-prick sampling with new high-sensitivity multiplex immunoassays, is enabling a new frontier of serological and vaccine science. Indeed, this approach was used with great success in the Covid-19 pandemic, providing a valuable insight into the serological landscape of the disease as well as vaccine efficacy. Interestingly, such an approach was described in the 2019 paper by J Wang et al in a related serology study measuring > 30 strains of influenza using remotely collected VAMS microsamples. It is good to see that in only three years, remote approaches are becoming successful for a progressively wider range of antibody assays.

This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this review, visit the original article published in the Journal of Immunological Methods.

Image Credit: Vaccine Research, iStock

You can access this microsampling study and others in our Technical Resource Library.

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Originally published Apr 18, 2022 9:00:00 AM, updated on May 6, 2022

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