SARS-CoV-2 serology assays validated for DBS & serum samples
by James Rudge, PhD, Technical Director, Neoteryx | 5 min read
An article by Patrick J Bouic and Leo Maritz et al published in the May 2022 edition of Bioanalysis investigated the development and validation of SARS-CoV-2 serology assays for both serum and dried blood spot (DBS) samples. The DBS samples were collected with hemaPEN® microsampling devices. The paper is entitled “Validation of high-throughput, semiquantitative solid-phase SARS coronavirus-2 serology assays in serum and dried blood spot matrices.” The study showed that the hemaPEN dried blood samples correlated well with the traditional serum samples. Furthermore, the assays were able to measure IgG, IgA, IgM antibodies and neutralizing antibodies with high diagnostic sensitivity and specificity.
Pandemic Lingers with New Covid-19 Waves
In early October 2022, the BBC reported that cases of Covid-19 were again on the rise in the United Kingdom. According to the BBC, an estimated 1.3 million (or 1 in 50) people tested positive for the illness in October. There was a marked increase in infection rates among people over age 70 and even higher rates for those over age 85. There were no reported increases in Covid-19 safety measures other than the continued recommendation to get vaccinated against the virus and its variants – a measure that has proven to be very effective. Some health agencies recommended that people take extra care to protect against the latest “Covid-19 wave” as people spend more time indoors during the winter season.
Continued Research into Covid-19
The third year of the Coronavirus Pandemic is drawing to a close, and research on SARS-CoV-2 continues as scientists seek to understand how the virus and its variants affect us. They hope new insights will help physicians treat Covid-19 and related viral infections. For example, research institutions are continuing to develop tools to understand the serological landscape of Covid-19 antibodies. This work includes studies that detect and monitor neutralizing antibodies found in dried blood samples collected remotely by people who have been exposed to the virus. Such studies include those who have recovered from Covid-19 infections and those who have received Covid-19 vaccines.
Research centers at Stanford University and the National Institutes of Health (NIH) have used microsampling devices to collect dried blood samples for their infectious disease serology studies. Some serology studies have paired dried blood microsamples with analytical tools like the SIMOA (recently reviewed in a previous blog).
Throughout the pandemic, the use of remote microsampling solutions such as DBS and volumetric microsampling devices have proven to be effective in screening populations, while also keeping them safe at home. Remote devices that gained favor early in the pandemic as effective tools that helped people safely continue participating in studies have become increasingly popular. In March 2022, the NIH reported their researchers were working on a new study using the Mitra® device to measure rates of undiagnosed Covid-19 in households with a child who has mitochondrial disease. This important research will be covered in a future blog.
The hemaPEN: An Effective Microsampling Tool for Covid-19 Serology
Bouic and Maritz et al commented that the serological landscape of Covid-19 continues to rapidly evolve and the need to continue monitoring immunity and vaccine efficacy is paramount. The group chose to use the hemaPEN® microsampling device to develop solid phase immunoassays for measuring raised IgG, IgM and IgA antibodies, as well as neutralizing antibodies, to SARS-CoV-2. A reason they reported for choosing hemaPEN over DBS cards was due to the limitations of DBS, such as volume variability, hematocrit-based biases, and risk of cross contamination. The volumetric sampling enabled by the hemaPEN, however, overcomes the limitations of DBS while capitalizing on its benefits, such as excellent stability of antibodies in dried blood and enabling remote, decentralized sample collection.
SARS-CoV-2 Serology Study Methods & Findings
- Validated semiquantitative, high throughput, wet serum and dried blood immunoassays (using hemaPEN) for detection of IgG, IgM and IgA isotypes as well as SARS-CoV-2 neutralizing antibodies (nAbs).
- Care was taken to optimize shake time, shake speed and incubation temperatures.
- Correlative data between serum and dried blood was: R2 = 0.937, 0.839, 0.939 IgG, IgM, IgA respectively. Moreover R2 =r 0.501, for nAb. Spearman correlation matrices data was p < 0.005 for IgG, IgM, IgA and nAb (r = 0.872, r = 0.500, r = 0.781 and r = 0.814, respectively).
- In terms of diagnostic sensitivity (DSn) and specificity (DSp), IgG (or total isoform) showed the highest sensitivity, though reduced DSn was seen for individual IgM and IgA samples.
- The assays showed corelation compared to viral neutralization. This is important, as assay neutralization often requires biosafety level 3 (BSL-3) in labs for handling live viruses, which could have potential for negating the need for viral neutralization tests.
- The group commented that the assays were suitable for surveillance post-vaccine rollout.
- The inclusion of 3 antibody isotypes as well as nAbs mitigated the potential for reduced specificity.
- Data from receiver operating characteristic (ROC) curve analyses (from 58 SARS-CoV-2 naive individuals and 21 convalescent individuals) and positive control (low, medium, and high) samples for nine independent assays were shown to be high.
- IgG showed ROC = 0.9997 with assay cut point (ACP) @ % mean of high positive control (HPC) of 43.660
- IgM showed ROC = 0.9460 with assay cut point (ACP) @ % mean HPC of 45.010
- IgM showed ROC = 0.9622 with assay cut point (ACP) @ % mean HPC of 40.200
- A dynamic ACP approach used for nAbs due to low ROC curve data
- Within run positive controls (PC), values were 1.0–3.1%, 4.1–12.0% and 3.1–5.5% for IgG, IgM and IgA, respectively. For the neutralizing Ab, %CVs were 5.2–8.0%.
- Cross reactivity (normalized to the HPC mean) from an African test population of 105 subjects where incidents of HIV, hepatitis B, and autoimmune diseases were high, ranged from 85.7–92.4%.
Study Authors’ Conclusions
The researchers validated a SARS-CoV-2 serology serum assay for IgG, IgM, IgA and nAbs, which showed high DSn, DSp and low cross-reactivity with patients that had chronic inflammation. The assay correlated well with the hemaPEN samples and worked well as a “gold standard” in vitro viral neutralization assay. The utility of the assay would be suitable for Phase 4 clinical trial monitoring of vaccine efficacy (including long-term post-marketing antibody level surveillance).
As the Covid-19 Pandemic continues, it is important that we also continue to develop new tools to effectively monitor populations. Capillary blood sampling using remote microsampling devices such as the hemaPEN and Mitra devices are demonstrating that highly correlative results can be obtained compared to blood taken from standard phlebotomy. This opens new avenues for remote and hybrid clinical trials, as well as decentralized population studies.
This article 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 blog, visit the original article in Bioanalysis.
Image Credits: iStock, Neoteryx, Trajan
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