Information

Serological assays measuring antibody response


Given that an appropriate immune response to a bacteria may be thwarted in an individual, including not producing all of the antibodies which are known to occur in people who have been infected, or producing insufficient amounts of antibodies specific to a bacteria --- Are all serological tests using antibodies as a basis for detection of a systemic infection inherently flawed? In this instance, I'm assuming appropriate application of a test and competent laboratory methodology. But, equally important, what criteria can routinely be used to consider the efficacy of a particular antibody test, across the board.


Antibodies used for serological tests are performed in vitro, and are not required to be effective for eliminating the pathogen. For an infected individual, antibodies may play a role in neutralization, opsonization, or activating the complement system (checkout Janeway's Immunobiology for more details). There are a number of reasons why antibodies may be present and not trigger an effective response down these pathways.

For example, E. Coli O157:H7 produces Shiga toxin and there are immunoassays to test for the presence of the toxin in feces (1). Only low amounts of the toxin is needed to cause disease. Antibodies against the toxin have been seen in individuals with the disease, suggesting the toxin is not a strong enough challenge for an immune response Discussion of (2).

You could go case-by-case for each of the immunoassays and see why their targets are difficult to turn into therapeutics.


Are all serological tests using antibodies as a basis for detection of a systemic infection inherently flawed?

No

Every diagnostic test must be interpreted in the context of the clinical features of an individual and according to the data about that particular test. Tests of serum for antibodies are no different. See Cecil Medicine Chapters 8 and 9 for a discussion of this. A key passage:

Most tests do not provide a definitive answer about diagnosis or prognosis, but instead reduce uncertainty

Except in cases of passive immunization (e.g., transfused IVIG), the presence of a specific antibody in a serum sample indicates both exposure to an antigen and an immune response. What exactly this means depends on the individual and the test. Though not generally the test of choice for diagnosing active or chronic infection, some antibody tests are excellent indicators of infection in many individuals. A good example would be antibody to HIV.

As with any diagnostic test (again, see Cecil), antibody to HIV was evaluated relative to a standard, giving us a sensitivity (how good the test is at finding disease when it is present) and specificity (how good the test is at excluding disease when it is not present). It's not perfect (I've seen one false positive in my career), and it is not useful in the first few weeks after exposure, but, in general, it is an excellent diagnostic test.

Antibody tests are used for many other things, and again, as with any diagnostic test, they are evaluated for that purpose. Sticking with the use of these tests in infectious diseases, Hepatitis B antibody tests are an interesting example, since antibody to different antigens mean different things. Surface antibody, for example, indicates immunity either from exposure and clearance or from immunization. Core antibody indicates exposure to the virus itself, since core antigen is not present in the vaccine. As with most antibody tests, the antibody class can also give you useful information. IgM antibodies typically represent the initial immune response, where IgG antibodies represent a later response, and either clearance or chronic infection.


Are all serological tests using antibodies as a basis for detection of a systemic infection inherently flawed?

You're thinking theoretically. Serological assays aren't theoretical, they're empirical.

When any assay is being characterized, it will be correlated with a particular outcome. Serological assays are no different. So long as there's a strong correlation with the targeted phenomenon, it doesn't matter if they're theoretically "flawed" (whatever that means); if they tell you what you need to know, then they're functional.

If every time a serological assay reads 40 or higher, an individual can be shown to be infected and every time it's less than 40 the individual isn't protected, then it doesn't matter what theoretical problems there might be with the assay, it can be used to predict infection. For that matter, if every time a ceramic saint wept salt tears it correlated with infection, then the saint test would be perfectly valid in the clinic, no matter how flawed the theory is.

How do you correlate your assay to the presence of infection, or whatever? Typically there are gold-standard tests that are perhaps too time-consuming, or difficult, or expensive to routinely run, and the serological assay will be cross-referenced to that for validation, and then hopefully approved by whatever regulatory body is in charge (in the US, often the FDA).

Don't confuse theoretical reasons with practical considerations.


From Multiplex Serology to Serolomics-A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86-100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

Keywords: SARS-CoV-2 multiplex serology.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Schematic study diagram. COVID-19 cases…

Schematic study diagram. COVID-19 cases were recruited at Heidelberg University Clinics between March…

Antibody responses to SARS-CoV-2 proteins…

Antibody responses to SARS-CoV-2 proteins in n = 85 pre-pandemic controls and n…

Antibody responses to SARS-CoV-2 proteins…

Antibody responses to SARS-CoV-2 proteins N, S1, S2, and their respective sub-fragments (N-EP3,…


Measuring the Antibody Response to SARS-CoV-2

As SARS-CoV-2 vaccines are rolled out around the globe, it is increasingly important for clinicians to be able to measure the body’s immune response over time, to understand how effective protection is and how long it lasts. Antibody tests have a vital role to play in the fight against COVID-19, helping to assert and measure the antibody response after infection or vaccination.

To learn more about the ways that antibody tests are being used in the pandemic, Technology Networks spoke to Heather-Read Harper, European Senior Manager for Immunoassay and Clinical Chemistry at Beckman Coulter. Heather also highlighted the advantages of quantitative antibody tests and discussed why detecting both IgG and IgM can be valuable.

Anna MacDonald (AM): Can you explain some of the roles that antibody tests are playing in the fight against COVID-19?

Heather Read-Harper (HRH):
Antibody tests, also known as serology tests, tell us the phases of immune status of an individual and look for the presence of antibodies. There are two types of antibodies typically measured in blood: IgM antibodies, in general, are the body’s first line of defence against a virus, while IgG antibodies offer a more sustained immune response to a virus.

Antibody tests play a critical role in understanding the level of immunity an individual has developed against the SARS-CoV-2 virus. This type of understanding can help identify those more at risk of infection and those who have been previously infected and could safely return to work or university, for example.

Wider scale testing can also provide an overview of the immunity status of the population and potentially help guide the future management of this virus.

With vaccine programs well underway, these assays have the potential to play a vital role in understanding an individual’s immune response to the vaccine over time and how long immunity might be maintained.

AM: What are the advantages of detecting each type of antibody?

HRH:
In the first few days following infection only the virus can be detected. This is when it multiplies and is readily transmitted to others. At this point, molecular diagnostic PCR-based tests and antigen tests can be used to detect the virus. The individual’s immune system then starts to respond to the viral load as part of the acute phase of infection. This is when IgM antibodies are produced and increase to fight the virus and can be used to identify patients with a more recent infection. After about a week, the convalescent phase of the infection is reached and IgG is produced and increases, while IgM starts to decline. The time it takes, however, does vary from one individual to another individual’s response.

Following these principles of infection, we can utilize both IgM and IgG assays to understand the immunity status and stage of infection in an individual, although IgG is thought to provide the necessary immunity to reduce the impact of future infection.

AM: Beckman Coulter recently launched the Access SARS-CoV-2 IgG II antibody assay. Can you provide an overview of how the test works?

HRH:
The Access SARS-CoV-2 IgG II assay measures IgG antibodies directed to the receptor-binding domain of the spike protein of the coronavirus in response to a previous infection. It uses immobilized virus antigens on magnetic particles to capture IgG antibodies from patient samples and reveals them using labeled anti-IgG antibodies.

The assay then provides a numerical result ranging from 2.00-450 AU/mL as well as a qualitative result for SARS-CoV-2 IgG antibodies. The test has a confirmed 99.9% specificity and 98.9% sensitivity 15-60 days post symptom onset. The Access SARS-CoV-2 IgG II assay can be used in Random Access Mode (RAM) and seamlessly integrated into existing workflows without batch processing. The assay can also be used with a variety of Beckman Coulter analyzers, including the high-throughput DxI 800 designed for large labs, the DxI 600 for mid-sized labs and the Access 2 analyzers for smaller labs and healthcare clinics.

AM: Why were IgG antibodies directed to the receptor-binding domain of the spike protein chosen as the target?

HRH:
The coronavirus has four types of protein: membrane, envelope, nucleocapsid and spike. It is the spike protein that is the major surface protein of the coronavirus and mediates entry into host cells. The receptor binding domain (RBD) of the spike protein binds strongly to a receptor on respiratory cells called angiotensin-converting enzyme 2, or ACE2. ACE2 is the entry receptor of the human cell and once the viral cell membrane fuses with human cell membrane, the genome of the virus enters the human cells and begin infection.

It is believed that the IgG antibodies that bind to the RBD of the spike protein may be key to understanding the immune response to the virus and in vitro studies have shown that the existence of these antibodies may indicate neutralization of SARS-CoV-2 infection and render the virus incapable of infecting cells.

The Beckman Coulter SARS CoV-2 IgM and IgG II assays are both directed at the receptor-binding domain of the spike protein, in order to detect antibodies that are potentially protective.

AM: What are the advantages of a quantitative antibody test?

HRH:
Many of the serology assays available at the start of this pandemic were qualitative in nature. This meant only a positive or negative result was generated. The results, therefore, only provided information to clinicians on whether the patient had been exposed to the virus. The result did not give a clear picture on the immune response over time.

Quantitative assays deliver a numerical value that can quantify the level of antibodies in blood. Assays such as Beckman Coulter’s SARS CoV-2 IgG II allow clinicians to monitor trends in a patient’s antibody levels by establishing a quantitative baseline and thus assess a relative change of an individual’s immune response to the virus over time.

Heather-Read Harper was speaking to Anna MacDonald, Science Writer for Technology Networks.


Methods

Data and sampling

To understand the dynamics of malaria infection and the impact of annual mass drug administration (MDA), a prospective cohort study was conducted from 2013 to 2015 in 12 villages across five administrative regions—West Coast (WCR), North Bank (NBR), Lower River (LRR), Central River (CRR), and Upper River (URR) Regions- as described by Mwesigwa et al. [13]. Plasmodium falciparum (Pf) prevalence measured by polymerase chain reaction (PCR) ranged from 2.27 to 19.60% in the Central River and Upper River Regions respectively (Fig. 1). Residents above 6 months of age were enrolled in the study, and monthly surveys were conducted during malaria transmission season from June to December each year, during the dry season in April 2014, and prior to the implementation of MDA in May and June 2014 and 2015 (Fig. 2). Individual finger prick blood samples were collected for haemoglobin estimation and on filter paper (Whatman 3 Corporation, Florham Park, NJ, USA) for molecular and serological analysis. Clinical malaria cases included individuals presenting with symptoms at health facilities (e.g. passive case detection) or individuals identified in villages by study nurses with history of fever in the previous 24 h or axillary temperature ≥ 37.5 °C and a positive rapid diagnostic test (RDT) result (Paracheck Pf, Orchid Biomedical System, India).

Map of Malaria Transmission Dynamics Study with regions and study villages by PCR prevalence

Study timelines. Malaria Transmission Dynamics Study timeline shown in black and green. Serological study timeline shown in blue for West Coast and Upper River Regions (low and moderate transmission settings, respectively). Serological analysis was conducted on samples from whole-village monthly surveys in N’demban and Besse in the West Coast Region (a), Njaiyal and Madina Samako in the Upper River Region (b), and longitudinal samples from individuals with a positive rapid diagnostic test (RDT) or polymerase chain reaction (PCR) test result during the Malaria Transmission Dynamics Study. Samples for serological analysis were processed on the Luminex MAGPIX samples from monthly surveys were analysed using microscopy, rapid diagnostic tests (RDTs), and polymerase chain reaction (PCR)

The serological study presented here is a subset of the Malaria Transmission Dynamics Study and included all available samples (n = 4599) from a selection of monthly surveys in four villages, totalling 1795 individuals (Fig. 2). In the West Coast Region (Besse and N’Demban), samples processed for serological analysis were from surveys conducted at the start of the transmission season in July 2013 (N = 534) and at the end of the season in December 2013 (N = 524). In the Upper River Region (URR), serological analysis included all samples collected in Njaiyal and Madina Samako in July 2013 (N = 778), December 2013 (N = 628), April (dry season) 2014 (N = 799), and December 2014 (N = 737) (Table 1). These regions represent extremes of two transmission intensities, with months selected at the start and end of the transmission season. Samples from clinical PCD cases were linked by study participant identification code to samples from the same individuals collected during routine monthly surveys. To further estimate the association between individual-level antibody responses and concurrent clinical or Pf infection, whole-village monthly survey samples in the West Coast Region and Upper River Region as described above were combined with an additional subset of 1244 longitudinal samples from 316 individuals who experienced a positive RDT or PCR test result or presented with clinical symptoms at any point during the Malaria Transmission Dynamics Study (Fig. 2). For these individuals, all available samples from the study were processed to longitudinally capture their serological responses before and after a positive RDT or PCR test result.

This study was approved by The Gambia Government/MRC Joint Ethics Committee (SCC1318). Verbal consent was first obtained at village sensitisation meetings, followed by individual written informed consent for all participants. Parents/guardians provided written consent for children less than 17 years, and assent was obtained from children between 12 and 17 years.

Antigen selection and design

Antigens were selected from an initial screen of 856 candidates on an in vitro transcription and translation (IVTT) protein microarray based on correlation with malaria infection in children [11]. Antigens were generated and expressed in Escherichia coli (E. coli) as glutathione S-transferase (GST)-tagged fusion proteins [20,21,22], with the exception of PfAMA1 expressed in Pichia pastoris as a histidine-tagged protein [15]. Protein purification was conducted by affinity chromatography (Glutathione Sepharose 4B, GE Healthcare Life Sciences) or HisPur Ni-NTA (Invitrogen) for GST- and His-tagged proteins, respectively, and concentration, quality, and purity of antigen yield assessed using a Bradford assay and SDS-PAGE. Bacterial lysate from culture of untransformed E. coli was used in assay buffers to eliminate background reactivity to E. coli proteins that were not specific to malaria target proteins.

Additionally, to account for potential non-malaria reactivity against GST-tagged fusion proteins, GST-coupled beads were included to quantify GST-specific immunoglobulin (IgG) responses and correct for non-specific binding. After laboratory processing, there were 71 participant samples with GST antibody responses above 1000 median fluorescence intensity (MFI), which was defined as the threshold to indicate potential non-malaria-specific binding, and were excluded from further analyses. Tetanus toxoid (TT, Massachusetts Biologic Laboratories) was also included as an internal positive control, assuming that vaccinated Gambians would show antibody responses to this protein target. A summary of antigen constructs and coupling conditions are detailed in Table 2.

Laboratory procedures

Antigen-specific antibody responses were quantified using the Luminex MAGPIX protocol described in Wu et al. 2019 [23]. Plasma was eluted from 6 mm dried blood spots (DBS) (4 μl whole blood equivalent) and shaken overnight at room temperature in 200 μl of protein elution buffer containing phosphate buffered saline (PBS) (pH 7.2), 0.05% sodium azide, and 0.05% Tween-20, yielding an initial 1:50 sample dilution. One day prior to assay processing, samples were diluted to a final 1:500 dilution using 10 μl of the 1:50 pre-dilution sample and 90 μl of blocking Buffer B to prevent non-specific binding (1xPBS, 0.05% Tween, 0.5% bovine serum albumin (BSA), 0.02% sodium azide, 0.1% casein, 0.5% polyvinyl alcohol (PVA), 0.5% polyvinyl pyrrolidone (PVP), and 1500 μg/ml E. coli extract). Negative and positive controls were also incubated 1 day prior in Buffer B, with negative controls prepared at a 1:500 dilution and Gambian pooled positive controls in a 6-point 5-fold serial dilution (1:10–1:31,250). The positive control was based on a pool of 22 serum samples from malaria hyper-immune individuals in The Gambia, and ten individual plasma samples from European malaria-naive adults were used as negative controls.

Samples were prepared for diagnostic PCR as described by Mwesigwa et al. [13]. Briefly, DNA was extracted from three 6-mm DBS using the automated QIAxtractor robot (Qiagen). Negative and positive (3D7) controls were included to control for cross contamination and DNA extraction efficiency, respectively. The DBS were lysed by incubation in tissue digest buffer at 60 °C for 1 h and digested eluates were applied onto capture plates, washed, and the DNA eluted into 80 μl. The extracted DNA (4 μl) was used in a nested PCR, amplifying the multi-copy Plasmodium ribosomal RNA gene sequences using genus and species specific primers [24]. All PCR products were run using the QIAxcel capillary electrophoresis system (Qiagen), using the screening cartridge and 15–1000 bp-alignment marker. Results were exported and double scored using both the QIAxcel binary scoring function and manually by visualisation of the gel images and discrepancies were scored by a third independent reader. All readers were blinded to participant survey data.

Statistical analyses

Data analysis was based on total IgG levels to five antigens as potential markers of sero-incidence [11]—early transcribed membrane protein 5 (Etramp5.Ag1), gametocyte export protein 18 (GEXP18), heat shock protein 40 (HSP40.Ag1), erythrocyte-binding antigen 175 RIII-V (EBA175), and reticulocyte binding protein homologue 2 (Rh2.2030). Three antigens associated with long-lived antibody response—P. falciparum merozoite surface antigen 1 19-kDa carboxy-terminal region (PfMSP119), P. falciparum apical membrane antigen 1 (PfAMA1), and P. falciparum glutamate-rich protein, region 2 (PfGLURP.R2)—were included as a comparison, which have historically been used to assess sero-conversion rates over time [8, 9] and have also previously been studied in The Gambia [6, 7]. For antigens associated with long-lived antibodies (PfMSP119, PfAMA1, PfGLURP.R2), individuals residing in an endemic region previously exposed to malaria, but not recently infected, may still have residual antibody levels that are significantly higher than malaria-naïve individuals in non-endemic regions. Therefore, a two-component Gaussian mixture model was used to define distributions of negative and positive antibody levels, expressed in units of median fluorescence intensity (MFI). Sero-positivity thresholds were defined as the mean log MFI values plus two standards deviations of the negative distribution [25]. Mixture models were estimated using the ‘normalmixEM’ function in the ‘mixtools’ package v1.0.4 in R version 3.6.1. For antigens associated with shorter-lived antibodies where statistical evidence of a bimodal distribution of antibody responses in the population was not strong given the more rapid decay of antibody levels post-infection—Etramp5.Ag1, GEXP18, HSP40.Ag1, EBA175, and Rh2.2030—the sero-positivity threshold was defined by the mean log MFI plus three standard deviations of 71 malaria-naïve European blood donors used as negative controls.

Individual-level association between antibody response and the concurrent odds of clinical malaria (passively detected via the health facility or study nurses in the community) or asymptomatic P. falciparum infection (actively detected using PCR from monthly survey samples) were assessed using generalised estimating equations (GEE). Analysis was adjusted for age group (1–5 years, 6–15 years, and greater than 15 years) and use of long-lasting insecticide-treated nets (LLINs) in the last 24 h and allowed for clustering at the compound level, where compound is a collection of households centrally located around a main residence. The magnitude of the association between antibody response and odds of infection was evaluated for interaction with age group. Based on a subset of longitudinal samples, individual-level association between antibody response and recent infection in the previous 4 months (as opposed to current infection at the same time point) was also assessed using a GEE model, adjusted for age group, LLIN use, and random effects at the compound level, as above.

Using a GEE model, individual-level odds of clinical malaria or asymptomatic infection was assessed for association with residing in the same compound as a sero-positive individual. Similarly, the association between individual odds of infection and compound-level sero-prevalence (< 50% or > 50%) was assessed using a mixed-effects generalised linear model adjusted for age group and LLIN use and random effects at the compound level, which also accounts for the potential effect of geographical differences in transmission intensity. To ensure that estimates are not biased by the sero-prevalence of small compounds, analysis was weighted by the number of individuals in the compound (whose sero-status was assessed) and only included compounds with at least four individuals. Models were fit using the ‘geeM’ and ‘lme4’ packages in R version 3.14.

Village-level sero-prevalence amongst children aged 2–10 years in the West Coast Region and Upper River Region in July and December 2013 (n = 1001) was compared against all-age clinical and P. falciparum infection incidence rates from the same months, the latter of which were previously reported by Mwesigwa et al. [13]. Monthly clinical and P. falciparum infection incidence rates were defined respectively as the number of new clinical cases or P. falciparum infections (PCR-positive individuals who were PCR-negative in the previous monthly survey) divided by total person years at risk (PYAR). This age range was selected to align with other routine surveillance metrics, such as annual parasite index (API), commonly using this age group as a sentinel population. The strength of the relationship between village-level incidence rates and sero-prevalence for each antigen was assessed using Pearson’s correlation coefficient. Data from April and December 2014 were not included in the analysis because clinical incidence and Pf infection rates from these surveys have not yet been reported.


SARS-CoV-2 Multiplex Serology Assays

ProcartaPlex Serology Assays

The proteins that serve as primary antigens to stimulate an immune response producing IgA, IgM, and IgG antibodies during SARS-CoV-2 infection include the nucleocapsid (N), the spike (S) protein, and sub-regions of the spike protein such as the receptor binding domain (RBD) and the S1 regions. The Nucleocapsid (N) protein has the highest homology (90%) between SARS-CoV-1 and SARS-CoV-2 (1). Serology test kits available during the early phase of the SARS-CoV-2 pandemic were developed to detect antibodies against the Nucleocapsid, which displayed significant cross-reactivity, and thus higher false positive readings for subjects exposed to SARS-CoV-1 (1). The SARS-CoV spike (S) protein assembles into a trimerized structure to form a crown-like (hence corona) appearance and is composed of a S1 and S2 subunit. Within S1, the receptor binding domain (RBD), which is located in the C-terminal subdomain, has higher identity (74%) between SARS-CoV and SARS-CoV-2 than the N-terminal domain, consistent with the view that SARS-CoV-2 may use ACE2 as its receptor for entry into host cells like SARS-CoV (2). The RBD has been identified as one of the immunodominant sites of the SARS-CoV-2 spike protein, with antibodies against the spike protein correlating well with neutralization. In addition, it is important to test serologic cross-reactivity with endemic and seasonal coronaviruses to rule out false-positive results. The Human Coronavirus Ig Total 11-Plex ProcartaPlex Panel enables screening of four SARS-CoV-2 antibodies (Spike trimer, S1 subunit, RBD, and Nucleocapsid), six coronavirus strains (SARS-CoV-1, MERS, CoV-NL63, CoV-KHU1, CoV-229E, CoV-OC43 ), and one negative control in a single well using Luminex xMAP technology. Simultaneous detection of anti-SARS-CoV-2 antibodies and related coronavirus antibodies in one assay can save time to provide a complete, holistic data set using plasma or serum samples.

Figure 1. Screening results from 39 Covid PCR (+) and 168 healthy PCR (-) controls for SARS-CoV-2 antigens. Results show antibody levels detected for spike timer, S1, RBD, and Nucleocapsid antigens correspond to the expected results of high antibody levels for PCR (+) and low antibody levels for PCR (-) samples.


SARS-CoV-2 Assay Documents & Resources

Documents

Bio-Plex Pro Human SARS-CoV-2 Serology Assays Instruction Manual (PDF 601 KB)

Learn more about the assay with detailed instructions, workflows, kit contents, storage, and troubleshooting tips with this instruction manual.

Bio-Plex Pro Human SARS-CoV-2 Serology Assays Quick Guide (PDF 68 KB)

Find out how to prepare and run a full 96-well Bio-Plex SARS-CoV-2 Serology Assay plate with this quick guide.

Bio-Plex Pro Human SARS-CoV-2 N/RBD/S1/S2 4-Plex Panel Product Information Sheet (PDF 439 KB)

Obtain product details such as assay characteristics and cross-reactivity results with this product information sheet.

Bio-Plex Pro Human IgG SARS-CoV-2 Semi-Quantitative Assay Protocol (PDF 96 KB)

Produce results with this semiquantitative protocol for use with the Bio-Plex Human IgG SARS-CoV-2 N/RBD/S1/S2 Serology Assays.

Bio-Plex Multiplex Systems Brochure (PDF 15.9 MB)

Learn about how how you can reduce time to results and samples while discovering more with Bio-Plex multiplex immunoassays, instruments, and software.

Quantitative Bio-Plex Pro Human IgG SARS-CoV-2 Serology Assays Using a Standard Curve Application Note (PDF 420 KB)

Learn how the VIROTROL SARS-CoV-2 Serological Control is validated for use as a reference material to quantitate levels of SARS-CoV-2&ndashspecific IgG antibody using the Bio-Plex Pro Human IgG SARS-CoV-2 Serology Assays in this application note.

Bio-Plex Analyte Guide (PDF 2.2 MB)

Explore the different multiplex immunoassays and panels available on the Bio-Plex platform. Evaluate assay performance characteristics and sample data to choose the panel that fits your application.

Webinars

Role of Aberrant Cytokine Activity in Host Immune Response to COVID-19

In this webinar, we discuss early research and nascent hypotheses regarding the pathophysiology of SARS-CoV-2 induced COVID-19 disease by evaluating cytokine and chemokine profiles, the role of chronic inflammation in comorbidities, and the arc of immune resolution of historical virulent pathogens, such as SARS and MERS.

Multiplex Immunoassays in Vaccine Research and Development

Get the most data out of your sample, without spending time on individual ELISAs. Learn how multiplex assays are being used on the front lines to understand patient immune response during infection, and discuss the most commonly asked questions around targets, panels, and multi-plate data analysis.

Vaccine Development and Manufacturing

Tips for overcoming developmental hurdles, technologies that can shorten timelines, and novel tools for manufacturing and quality control.

Immunological Response Factors to SARS‑CoV‑2 in Acquired Immunity

In this webinar, we will be discussing the immune response to SARS‑CoV2, both in the context of natural exposure to the virus as well as induced immunity from vaccination. We will also investigate the relationship between symptom severity and the longevity of acquired immunity.


SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients

FIG 1 ACE2 receptor expression and affinity. (A) Overview of soluble ACE2 receptor design (ACE2-IgHu). (B) Affinity of SARS CoV-2 receptor binding domain for immobilized ACE2-IgHu assessed by SPR (27 nM) curves are concentrations of RBD X, Y, and Z. (C) Affinity of ACE2-IgHu for immobilized SARS CoV-2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgHu for competition assays (∼EC90, red arrow) requires high signal without excess receptor present. FIG 2 ACE2 receptor competition assay development. (A) Competition ELISA schematic displaying immobilized anti-His pAb (red) capturing His6×-tagged SARS-CoV-2 spike protein (rainbow). Premixed ACE2-IgHu (green, blue) at a constant concentration with a dilution series of competitors (green, red) is added, and anti-human HRP (green) determines the amount of ACE2-IgHu remaining in the presence of competitors through a colorimetric readout. (B) Four constant concentrations of ACE2-IgHu were tested with various concentrations of the ACE2-IgMu competitor to establish an optimal ACE2-IgHu concentration which displays a full blocking curve (red, 0.10 μg/ml) from the competitor dilution series while retaining a wide range in signal. (C) Pseudovirus neutralization curves for a control antibody (non-SARS-CoV-2) in red and for ACE2-IgHu in blue.

Animal IgG and serological competition.

FIG 3 Animal IgG and serological competition. (A) IgG and serological competition schematic. Anti-His pAb captures SARS-CoV-2 spike protein. Immunized sera or IgG from small animals are used as competitors to block ACE2-IgHu receptor binding when premixed. ACE2-IgHu remaining is determined from an anti-human-HRP colorimetric readout. (B) IgGs present in a vaccinated BALB/c mouse block ACE2-IgHu binding with greater effect when the full-length SARS-CoV-2 S1-S2 spike protein is immobilized versus the S1 subunit by itself. (C) Area under the concentration-time curve (AUC) schematic displaying the larger area for uninhibited ACE2 binding versus the area from curves showing competition with ACE2. (D) AUC of IgGs purified from immunized rabbit sera (IgGr low dose, blue IgGr high dose, red) versus naive IgGr or day 0 IgGr. (E) AUC of sera from immunized rabbits (low dose rabbit sera, blue high dose rabbit sera, red) versus naive rabbit sera or day 0 rabbit sera. (F) AUC of sera from immunized guinea pigs at week 2 (dark blue) and individual animals (blue), naive sera (gray), and pooled day 0 sera from all animals (black). The pooled immunized curve displayed a comparable AUC to the average AUC from all individual immunized animals.

Primate serological competition.

FIG 4 Primate serological competition. (A) Competition ELISA schematic displaying immobilized His6×-tagged SARS-CoV-2 spike protein (rainbow). Preblocking of the spike protein with primate sera (blue) at various concentrations was added followed by ACE2-IgMu (green, blue) at a constant concentration. Anti-mouse HRP (green) determines the amount of ACE2-IgMu remaining in the presence of competitors through a colorimetric readout. (B) Affinity of ACE2-IgMu for immobilized SARS-CoV-2 S1+S2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgMu for competition assays (red arrow, 0.4 μg/ml) requires high signal without excess receptor present. (C) Optimal ACE2-IgMu concentration which displays a full blocking curve (0.40 μg/ml) from the competitor dilution series (ACE2-IgHu) while retaining a wide range in signal. (D) NHP sera pooled from five vaccinated animals were used as competitors in the primate competition assay. The AUC from vaccinated NHP sera (blue) versus day 0 NHP sera (black). (E) Human sera from nine SARS-CoV-2-positive COVID-19 patients were tested in the primate competition assay and compared with 16 naive human sera collected prepandemic. The AUC of the COVID-19 patient serum (purple) is significantly decreased compared to the prepandemic human serum (gray). The median is shown as a solid black line, and quartiles are shown as dashed black lines. (F) Human sera were analyzed by a pseudovirus neutralization assay. The samples and the coloring are the same as in (E). Statistics include a two-tailed t test with P values indicated. FIG 5 ACE2 receptor blocking correlates with pseudovirus neuralization. A symbol represents each of the individual datapoints where we had a paired AUC blocking and pseudovirus ID50 values. The human samples are in triangles, the mice in circles, individual Guinea pigs in squares, Guinea pig pools in diamonds, and rabbit pools in hexagons. SARS-CoV-2 spike-experienced samples are shown in color. Naïve samples and healthy donors are shown in gray. Least-squares fit line is shown with P value and R squared from Prism.

SPR-based assay for ACE2 receptor blocking.

FIG 6 ACE2 receptor competition assay development. (A) Overview of SPR experiment depicting SARS-CoV-2 RBD capture by streptavidin-biotin interaction, sera injected as analyte, and ACE2 injected as second analyte. (B) Sensorgram for ACE2 blocking SPR assay with ACE2-IgHu injected as sample (ACE2sample) as indicated and ACE-IgHu injected as receptor (ACE2receptor) as indicated. Sample responses were referenced to blank injections. Each curve corresponds to a 3-fold dilution of ACE2sample starting at 1,500 nM as indicated on the right, and the ACE2receptor was injected at a constant concentration of 100 nM to all curves. (C) Response in RUs measured at the end of sample (ACE2sample) injection (blue) and receptor (ACE2receptor) injection (red) at each concentration of sample. (D) ACE2 inhibition curve derived from RUs at each concentration.

Acknowledgments

We thank all the participants involved in the study Bernie McCudden and Jenny Mitchell for support with the cohort and Jill Garlick, Janine Roney, Anne Paterson and the research nurses at the Alfred Hospital. This work was supported by the Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grant to KK (#1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of the Hong Kong Special Administrative Region, China (#T11-712/19-Ncdf) to KK, the Jack Ma Foundation to KK, KS and AWC, the Medical Research Future Fund (#2005544) to KK, SJK, AKW and AWC, the a2 Milk Foundation to KS, MRFF Award (#1202445) to KK, NHMRC Program Grant (#1071916) to KK. KK was supported by NHMRC Senior Research Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship (#1137285) and KS by an NHMRC Investigator grant (#1177174). THON is supported by NHMRC EL1 Fellowship (#1194036). LH is supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship from The University of Melbourne. JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. XJ is supported by China Scholarship Council-University of Melbourne joint Scholarship. CES has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (#792532). CES and KS received funding from the Doherty Collaborative Seed Grant. KK and AWC were supported by the University of Melbourne Dame Kate Campbell Fellowship. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.


SARS-CoV-2 Serology Testing in the Setting of Vaccination

To enable an effective vaccination strategy, we advocate for the use of accessible, automated, high-throughput SARS-CoV-2 serology testing to help confirm efficacy and promote public health. Siemens Healthineers formulated a position paper based on input from experts in the infectious disease, immunology and vaccine development fields and the currently available body of literature.

“In clinical practice, my patients who have either had the Covid-19 infection or have been vaccinated have been extremely interested in measuring their antibody response. I explain to them that this is the best surrogate we currently have to predict if they will likely be protected from future infection. Having had the vaccine myself and subsequently testing my own blood, I was immensely relieved to see I had developed anti-spike protein antibodies. It felt like I had put on an anti-Covid-19 Kevlar suit.”

Latinis Rheumatology, LLC, Kansas City (Harrisonville, MO and Leawood, KS)

"The position statement that you have here is timely. We need to have this discussion and preparation ahead of vaccinating the general population. Serology status is incredibly important due to asymptomatic carriers, and as these vaccines are introduced the need for robust neutralizing antibody testing would become even more important to understand where we are both before and after vaccination."

University of Cambridge (UK), Department of Medicine/CITIID

“As an immunologist and clinician, I certainly see the value of serology testing in understanding the immune response to vaccines, as outlined in the position paper. With every Covid-19 vaccine targeting the spike protein, it just makes sense that measuring the immune response against spike protein is the focus going forward with serology testing. Further, scientifically I think we’re going to see a quantitative threshold for Covid-19 antibody levels that becomes apparent in which the Covid-19 immune response weakens enough that the risk of infection returns. In this respect, measuring Covid-19 antibodies after a natural infection or after vaccination will determine if someone has reached that protective threshold and remeasuring antibody levels over time will determine the need to reimmunize. In some cases, if people who have had Covid-19 infection or those who have been immunized didn’t produce sufficient antibodies, they may need to be revaccinated sooner rather than later.”

Latinis Rheumatology, LLC, Kansas City (Harrisonville, MO and Leawood, KS)

“In clinical practice, my patients who have either had the Covid-19 infection or have been vaccinated have been extremely interested in measuring their antibody response. I explain to them that this is the best surrogate we currently have to predict if they will likely be protected from future infection. Having had the vaccine myself and subsequently testing my own blood, I was immensely relieved to see I had developed anti-spike protein antibodies. It felt like I had put on an anti-Covid-19 Kevlar suit.”

Latinis Rheumatology, LLC, Kansas City (Harrisonville, MO and Leawood, KS)

"The position statement that you have here is timely. We need to have this discussion and preparation ahead of vaccinating the general population. Serology status is incredibly important due to asymptomatic carriers, and as these vaccines are introduced the need for robust neutralizing antibody testing would become even more important to understand where we are both before and after vaccination."

University of Cambridge (UK), Department of Medicine/CITIID

“As an immunologist and clinician, I certainly see the value of serology testing in understanding the immune response to vaccines, as outlined in the position paper. With every Covid-19 vaccine targeting the spike protein, it just makes sense that measuring the immune response against spike protein is the focus going forward with serology testing. Further, scientifically I think we’re going to see a quantitative threshold for Covid-19 antibody levels that becomes apparent in which the Covid-19 immune response weakens enough that the risk of infection returns. In this respect, measuring Covid-19 antibodies after a natural infection or after vaccination will determine if someone has reached that protective threshold and remeasuring antibody levels over time will determine the need to reimmunize. In some cases, if people who have had Covid-19 infection or those who have been immunized didn’t produce sufficient antibodies, they may need to be revaccinated sooner rather than later.”

Latinis Rheumatology, LLC, Kansas City (Harrisonville, MO and Leawood, KS)

“In clinical practice, my patients who have either had the Covid-19 infection or have been vaccinated have been extremely interested in measuring their antibody response. I explain to them that this is the best surrogate we currently have to predict if they will likely be protected from future infection. Having had the vaccine myself and subsequently testing my own blood, I was immensely relieved to see I had developed anti-spike protein antibodies. It felt like I had put on an anti-Covid-19 Kevlar suit.”

Latinis Rheumatology, LLC, Kansas City (Harrisonville, MO and Leawood, KS)

Serology tests can inform vaccination utilization and status of vaccine response at multiple junctures:

  • Data to establish a threshold for protection or immunity
  • Post-vaccination initial response 1
  • Duration of vaccination response 2

Appropriate characteristics of a serology assay in the assessment of need to vaccinate and vaccine response:

  • Quantitative results
  • Spike protein receptor-binding domain (S1 RBD) neutralizing IgG antibody detection
  • Very high specificity (≥99.5%)

Neutralization of the SARS-CoV-2 virus with S1-RBD

Neutralizing Antibodies: Why the Spike Protein?

SARS-CoV-2 serology assays that utilize the receptor-binding domain (RBD) of the S1 spike antigen detect neutralizing antibodies that block the virus entry into cells. 3-8 S1-RBD-specific assays are likely to prove advantageous over S1 and whole spike, especially if using a quantitative assay, as neutralizing versus binding antibodies might be expected to be enriched and therefore better correlate to immunity.

The utilization of the S1-RBD is aligned with the multiple vaccines in development that target or include the SARS-CoV-2 S1 RBD, with the goal of producing neutralizing (and therefore likely protective) antibodies in vaccinated subjects. 9 The spike protein and particularly the RBD are the most common target of vaccine designs.


SARS-CoV-2 Multiplex Serology Assays

ProcartaPlex Serology Assays

The proteins that serve as primary antigens to stimulate an immune response producing IgA, IgM, and IgG antibodies during SARS-CoV-2 infection include the nucleocapsid (N), the spike (S) protein, and sub-regions of the spike protein such as the receptor binding domain (RBD) and the S1 regions. The Nucleocapsid (N) protein has the highest homology (90%) between SARS-CoV-1 and SARS-CoV-2 (1). Serology test kits available during the early phase of the SARS-CoV-2 pandemic were developed to detect antibodies against the Nucleocapsid, which displayed significant cross-reactivity, and thus higher false positive readings for subjects exposed to SARS-CoV-1 (1). The SARS-CoV spike (S) protein assembles into a trimerized structure to form a crown-like (hence corona) appearance and is composed of a S1 and S2 subunit. Within S1, the receptor binding domain (RBD), which is located in the C-terminal subdomain, has higher identity (74%) between SARS-CoV and SARS-CoV-2 than the N-terminal domain, consistent with the view that SARS-CoV-2 may use ACE2 as its receptor for entry into host cells like SARS-CoV (2). The RBD has been identified as one of the immunodominant sites of the SARS-CoV-2 spike protein, with antibodies against the spike protein correlating well with neutralization. In addition, it is important to test serologic cross-reactivity with endemic and seasonal coronaviruses to rule out false-positive results. The Human Coronavirus Ig Total 11-Plex ProcartaPlex Panel enables screening of four SARS-CoV-2 antibodies (Spike trimer, S1 subunit, RBD, and Nucleocapsid), six coronavirus strains (SARS-CoV-1, MERS, CoV-NL63, CoV-KHU1, CoV-229E, CoV-OC43 ), and one negative control in a single well using Luminex xMAP technology. Simultaneous detection of anti-SARS-CoV-2 antibodies and related coronavirus antibodies in one assay can save time to provide a complete, holistic data set using plasma or serum samples.

Figure 1. Screening results from 39 Covid PCR (+) and 168 healthy PCR (-) controls for SARS-CoV-2 antigens. Results show antibody levels detected for spike timer, S1, RBD, and Nucleocapsid antigens correspond to the expected results of high antibody levels for PCR (+) and low antibody levels for PCR (-) samples.


References

Naji H (2020) The emerging of the 2019 novel coronavirus 2019-nCoV. Eur J Med Health Sci. https://doi.org/10.24018/ejmed.2020.2.1.169

Chan JFW, Li KSM, To KKW et al (2012) Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect 65:477–489. https://doi.org/10.1016/j.jinf.2012.10.002

Lim YX, Ng YL, Tam JP, Liu DX (2016) Human coronaviruses: a review of virus–host interactions. Diseases. https://doi.org/10.3390/diseases4030026

Jones BA, Grace D, Kock R et al (2013) Zoonosis emergence linked to agricultural intensification and environmental change. Proc Natl Acad Sci 110:8399–8404. https://doi.org/10.1073/pnas.1208059110

Kahn JS, McIntosh K (2005) History and recent advances in coronavirus discovery. Pediatr Infect Dis J 24:S223–S227. https://doi.org/10.1097/01.inf.0000188166.17324.60

Fehr AR, Perlman S (2015). Coronaviruses: an overview of their replication and pathogenesis. In Coronaviruses. Methods in molecular biology. Humana Press, New York, pp 1–23

Sun C, Chen L, Yang J et al (2020) SARS-CoV-2 and SARS-CoV spike-RBD structure and receptor binding comparison and potential implications on neutralizing antibody and vaccine development. bioRxrv. https://doi.org/10.1101/2020.02.16.951723

Schmaljohn AL (2013) Protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts. Curr HIV Res 11:345–353. https://doi.org/10.2174/1570162x113116660057

Bosch BJ, De HCAM, Rottier PJM (2004) Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent. J Virol 78:7369–7378. https://doi.org/10.1128/JVI.78.14.7369

Fan H, Ooi A, Tan YW et al (2005) The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties. Structure 13:1859–1868. https://doi.org/10.1016/j.str.2005.08.021

Jiang S, Hillyer C, Du L (2020) Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol 41:355–359. https://doi.org/10.1016/j.it.2020.03.007

di Gabriella M, Cristina S, Concetta R et al (2020) SARS-Cov-2 infection: response of human immune system and possible implications for the rapid test and treatment. Int Immunopharmacol 84:106519. https://doi.org/10.1016/j.intimp.2020.106519

Lin Q, Zhu L, Ni Z et al (2020) Duration of serum neutralizing antibodies for SARS-CoV-2: lessons from SARS-CoV infection. J Microbiol Immunol Infect. https://doi.org/10.1016/j.jmii.2020.03.015

Vashist SK (2020) In vitro diagnostic assays for COVID-19: recent advances and emerging trends. Diagnostics 10:202. https://doi.org/10.3390/diagnostics10040202

Zhou P, Lou YX, Wang XG et al (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273. https://doi.org/10.1038/s41586-020-2012-7

Park TJ, Hyun MS, Lee HJ et al (2009) A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome. Talanta 79:295–301. https://doi.org/10.1016/j.talanta.2009.03.051

Park TJ, Lee S-K, Yoo SM et al (2011) Development of reflective biosensor using fabrication of functionalized photonic nanocrystals. J Nanosci Nanotechnol 11:632–637. https://doi.org/10.1166/jnn.2011.3269

Woo PCY, Lau SKP, Wong BHL et al (2004) Longitudinal profile of immunoglobulin G (IgG), IgM, and IgA antibodies against the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in patients with pneumonia due to the SARS coronavirus. Clin Diagn Lab Immunol 11:665–668. https://doi.org/10.1128/CDLI.11.4.665

Siracusano G, Pastori C, Lopalco L (2020) Humoral immune responses in COVID-19 patients: a window on the state of the art. Front Immunol 11:1049. https://doi.org/10.3389/fimmu.2020.01049

Zhang N, Wang L, Deng X et al (2020) Recent advances in the detection of respiratory virus infection in humans. J Med Virol 92:408–417. https://doi.org/10.1002/jmv.25674

van der Hoek L (2007) Human coronaviruses: what do they cause? Antivir Ther 12:651–658

Carlos WG, Dela Cruz CS, Cao B et al (2020) Novel Wuhan (2019-nCoV) coronavirus. Am J Respir Crit Care Med 201:P7–P8. https://doi.org/10.1164/rccm.2014P7

Lam CWK, Chan MHM, Wong CK (2004) Severe acute respiratory syndrome: clinical and laboratory manifestations. Clin Biochem Rev 25:121–132

Wong TW (2006) ‘“Will the SARS epidemic recur?”’ A retrospective analysis of the experts’ opinions. J Epidemiol Community Heal 60:87

Rabaan AA (2017) Middle East respiratory syndrome coronavirus: five years later. Expert Rev Respir Med 11:901–912. https://doi.org/10.1080/17476348.2017.1367288

Hu B, Ge X, Wang L-F, Shi Z (2015) Bat origin of human coronaviruses. Virol J 12:221. https://doi.org/10.1186/s12985-015-0422-1

Chan JF-W, Kok K-H, Zhu Z et al (2020) Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 9:221–236. https://doi.org/10.1080/22221751.2020.1719902

Lu H, Stratton CW, Wei Y (2020) The Wuhan SARS-CoV-2—What’s next for China. J Med Virol 92:546–547. https://doi.org/10.1002/jmv.25738

Kwong KCNK, Mehta PR, Shukla G, Mehta AR (2020) COVID-19, SARS and MERS: a neurological perspective. J Clin Neurosci. https://doi.org/10.1016/j.jocn.2020.04.124

World Health Organization (2020) WHO Coronavirus Disease (COVID-19) Dashboard. https://covid19.who.int/

Zheng J (2020) SARS-CoV-2: an emerging coronavirus that causes a global threat. Int J Biol Sci 16:1678–1685. https://doi.org/10.7150/ijbs.45053

Lu R, Zhao X, Li J et al (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395:565–574. https://doi.org/10.1016/S0140-6736(20)30251-8

Wong HYF, Lam HYS, Fong AH-T et al (2020) Frequency and distribution of chest radiographic findings in COVID-19 positive patients. Radiology. https://doi.org/10.1148/radiol.2020201160

Kim S-H, Ko J-H, Park GE et al (2017) Atypical presentations of MERS-CoV infection in immunocompromised hosts. J Infect Chemother 23:769–773. https://doi.org/10.1016/j.jiac.2017.04.004

Zeng Q, Chen L, Cai X et al (2003) Chest X-ray and CT in the diagnosis of SARS. Chin J Radiol 37:600–603

Zhu X, Wang X, Han L et al (2020) Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19. medRxiv. https://doi.org/10.1101/2020.03.17.20037796

Hirotsu Y, Mochizuki H, Omata M (2020) Double-Quencher Probes improved the detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by one-step RT-PCR. medRxiv. https://doi.org/10.1101/2020.03.17.20037903

Qiu G, Gai Z, Tao Y et al (2020) Dual-functional plasmonic photothermal biosensors for highly accurate severe acute respiratory syndrome coronavirus 2 detection. ACS Nano. https://doi.org/10.1021/acsnano.0c02439

Diao B, Wen K, Chen J et al (2020) Diagnosis of acute respiratory syndrome coronavirus 2 infection by detection of nucleocapsid protein. medRxiv. https://doi.org/10.1101/2020.03.07.20032524

Layqah LA, Eissa S (2019) An electrochemical immunosensor for the corona virus associated with the Middle East respiratory syndrome using an array of gold nanoparticle-modified carbon electrodes. Microchim Acta 186:224. https://doi.org/10.1007/s00604-019-3345-5

Meyer B, Drosten C, Müller MA (2014) Serological assays for emerging coronaviruses: challenges and pitfalls. Virus Res 194:175–183. https://doi.org/10.1016/j.virusres.2014.03.018

Liu L, Liu W, Zheng Y et al (2020) A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients. medRxiv. https://doi.org/10.1101/2020.03.06.20031856

Cheng MS, Toh C-S (2013) Novel biosensing methodologies for ultrasensitive detection of viruses. Analyst 138:6219–6229. https://doi.org/10.1039/C3AN01394D

Huang X, Wei F, Hu L et al (2020) Epidemiology and clinical characteristics of COVID-19. Arch Iran Med 23:268–271. https://doi.org/10.34172/aim.2020.09

Abbasi J (2020) The promise and peril of antibody testing for COVID-19. JAMA. https://doi.org/10.1001/jama.2020.6170

Ksiazek TG, Erdman D, Goldsmith CS et al (2003) A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348:1953–1966. https://doi.org/10.1056/NEJMoa030781

Leung DTM, Tam FCH, Ma CH et al (2004) Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid. J Infect Dis 190:379–386. https://doi.org/10.1086/422040

Tan Y-J, Goh P-Y, Fielding BC et al (2004) Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers. Clin Diagn Lab Immunol 11:362–371. https://doi.org/10.1128/CDLI.11.2.362

Wu H-S, Chiu S-C, Tseng T-C et al (2004) Serologic and molecular biologic methods for SARS-associated. Emerg Infect Dis 10:304–310. https://doi.org/10.3201/eid1002.030731

Amanat F, Stadlbauer D, Strohmeier S et al (2020) A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv. https://doi.org/10.1101/2020.03.17.20037713

Khan S, Nakajima R, Jain A et al (2020) Analysis of serologic cross-reactivity between common human coronaviruses and SARS-CoV-2 using coronavirus antigen microarray. bioRxiv. https://doi.org/10.1101/2020.03.24.006544

Lin D, Liu L, Zhang M et al (2020) Evaluations of serological test in the diagnosis of 2019 novel coronavirus (SARS-CoV-2) infections during the COVID-19 outbreak. medRxiv. https://doi.org/10.1101/2020.03.27.20045153

Zhang P, Gao Q, Wang T et al (2020) Evaluation of recombinant nucleocapsid and spike proteins for serological diagnosis of novel coronavirus disease 2019 (COVID-19). medRxiv. https://doi.org/10.1101/2020.03.17.20036954

Maache M, Komurian-Pradel F, Rajoharison A et al (2006) False-positive results in a recombinant severe acute respiratory western blot assay were rectified by the use of two subunits (S1 and S2) of spike for detection of antibody to SARS-CoV. Clin Diagn Lab Immunol 13:409–414. https://doi.org/10.1128/CVI.13.3.409

Xiang J, Yan M, Li H et al (2020) Evaluation of enzyme-linked immunoassay and colloidal gold-immunochromatographic assay kit for detection of novel coronavirus (SARS-Cov-2) causing an outbreak of pneumonia (COVID-19). medRxiv. https://doi.org/10.1101/2020.02.27.20028787

Hoy CFO, Kushiro K, Yamaoka Y et al (2019) Rapid multiplex microfiber-based immunoassay for anti-MERS-CoV antibody detection. Sens Bio Sens Res. https://doi.org/10.1016/j.sbsr.2019.100304

Aydin S (2015) A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 72:4–15. https://doi.org/10.1016/j.peptides.2015.04.012

Wang Y-D, Li Y, Xu G-B et al (2004) Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot. Clin Immunol 113:145–150. https://doi.org/10.1016/j.clim.2004.07.003

Carattoli A, Di BP, Grasso F et al (2005) Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS. J Med Virol 76:137–142. https://doi.org/10.1002/jmv.20338

Woo PCY, Lau SKP, Wong BHL et al (2005) Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 43:3054–3058. https://doi.org/10.1128/JCM.43.7.3054

Fukushi S, Fukuma A, Kurosu T et al (2018) Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA. J Virol Methods 251:22–29. https://doi.org/10.1016/j.jviromet.2017.10.008

Zhao J, Yuan Q, Wang H et al (2020) Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin Infect Dis. https://doi.org/10.1093/cid/ciaa344

González-Martínez MÁ, Puchades R, Maquieira Á (2018) Immunoanalytical technique: enzyme-linked immunosorbent assay (ELISA). Modern techniques for food authentication. Academic Press, New York, pp 617–657

García-González E, Aramendía M, Álvarez-Ballano D et al (2016) Serum sample containing endogenous antibodies interfering with multiple hormone immunoassays. Laboratory strategies to detect interference. Pract Lab Med 4:1–10. https://doi.org/10.1016/j.plabm.2015.11.001

Guan M, Chan KH, Peiris JSM et al (2004) Evaluation and validation of an enzyme-linked immunosorbent assay and an immunochromatographic test for serological diagnosis of severe acute respiratory syndrome. Clin Diagn Lab Immunol 11:699–703. https://doi.org/10.1128/CDLI.11.4.699

Guan M, Chen HY, Foo SY et al (2004) Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to Severe Acute Respiratory Syndrome (SARS) coronavirus in SARS patients. Clin Diagn Lab Immunol 11:287–291. https://doi.org/10.1128/CDLI.11.2.287

Liu X, Shi Y, Li P et al (2004) Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol 11:227–228. https://doi.org/10.1128/CDLI.11.1.227

Che X-Y, Qiu L-W, Liao Z-Y et al (2005) Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43. J Infect Dis 191:2033–2037. https://doi.org/10.1086/430355

Smith-Norowitz TA, Kusonruksa M, Wong D et al (2012) Long-term persistence of IgE anti-influenza A HIN1 virus antibodies in serum of children and adults following influenza A vaccination with subsequent H1N1 infection: a case study. J Inflamm Res 5:111–116. https://doi.org/10.2147/JIR.S34152

Martins-Gomes C, Silva AM (2018). Western blot methodologies for analysis of in vitro protein expression induced by teratogenic agents. Teratogenicity testing. Methods in molecular biology. Humana Press, New York, pp 191–203

Mahmood T, Yang P-C (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4:429–434. https://doi.org/10.4103/1947-2714.100998

Qiu D, Tannock GA, Barry RD, Jackson DC (1992) Western blot analysis of antibody responses to influenza virion proteins. Immunol Cell Biol 70:181–191. https://doi.org/10.1038/icb.1992.23

Castejon MJ, Yamashiro R, Oliveira CAF, Veras MASM (2017) Performance validation of western blot for anti-HIV antibody detection in blood samples collected on filter paper (DBS). J Bras Patol e Med Lab 53:5–12. https://doi.org/10.5935/1676-2444.20170002

Kurien BT, Scofi RH (2015) Western blotting: an introduction. Methods in molecular biology. Humana Press, New York, pp 17–30

He Q, Chong KH, Chng HH et al (2004) Development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol 11:417–422. https://doi.org/10.1128/CDLI.11.2.417

Wang Y, Chang Z, Ouyang J et al (2005) Profiles of IgG antibodies to nucleocapsid and spike proteins of the SARS-associated coronavirus in SARS patients. DNA Cell Biol 24:521–527. https://doi.org/10.1089/dna.2005.24.521

Guan M, Chen HY, Tan PH et al (2004) Use of viral lysate antigen combined with recombinant protein in western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome. Clin Diagn Lab Immunol 11:1148–1153. https://doi.org/10.1128/CDLI.11.6.1148

Odell ID, Cook D (2013) Immunofluorescence techniques. J Investig Dermatol 133:e4. https://doi.org/10.1038/jid.2012.455

Dilnessa T, Zeleke H (2017) Cell culture, cytopathic effect and immunofluorescence diagnosis of viral infection. J Microbiol Mod Technol 2:102

Bossuyt X, Cooreman S, De Baere H et al (2013) Detection of antinuclear antibodies by automated indirect immunofluorescence analysis. Clin Chim Acta 415:101–106. https://doi.org/10.1016/j.cca.2012.09.021

Zhu H, Hu S, Jona G et al (2006) Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray. Proc Natl Acad Sci 103:4011–4016. https://doi.org/10.1073/pnas.0510921103

Reusken C, Mou H, Godeke GJ et al (2013) Specific serology for emerging human coronaviruses by protein microarray. Eurosurveillance 18:20441. https://doi.org/10.2807/1560-7917.ES2013.18.14.20441

Chan PKS, Ng K-C, Chan RCW et al (2004) Immunofluorescence assay for serologic diagnosis of SARS. Emerg Infect Dis 10:530–532. https://doi.org/10.3201/eid1003.030493

Manopo I, Lu L, He Q et al (2005) Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV. J Immunol Methods 296:37–44. https://doi.org/10.1016/j.jim.2004.10.012

Koczula KM, Gallotta A (2016) Lateral flow assays. Essays Biochem 60:111–120. https://doi.org/10.1042/EBC20150012

Wong RC, Tse HY (2008) Lateral flow immunoassay. Humana Press, New York

Bahadır EB, Sezgintürk MK (2016) Lateral flow assays: principles, designs and labels. Trends Anal Chem 82:286–306. https://doi.org/10.1016/j.trac.2016.06.006

Zhao L, Sun L, Chu X (2009) Chemiluminescence immunoassay. Trends Anal Chem 28:404–415. https://doi.org/10.1016/j.trac.2008.12.006

Cai X-F, Chen J, Hu J-L et al (2020) A peptide-based magnetic chemiluminescence enzyme immunoassay for serological diagnosis of corona virus disease 2019 (COVID-19). J Infect Dis. https://doi.org/10.1101/2020.02.22.20026617

Qu J, Wu C, Li X et al (2020) Profile of IgG and IgM antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clin Infect Dis. https://doi.org/10.1093/cid/ciaa489

Turner APF (2013) Biosensors: sense and sensibility. Chem Soc Rev 42:3184–3196. https://doi.org/10.1039/c3cs35528d

Caygill RL, Blair GE, Millner PA (2010) A review on viral biosensors to detect human pathogens. Anal Chim Acta 681:8–15. https://doi.org/10.1016/j.aca.2010.09.038

Lakshmipriya T, Gopinath SCB (2019) An introduction to biosensors and biomolecules. In Nanobiosensors for biomolecular targeting. Elsevier, New York, pp 1–21

Hsueh P-R, Hsiao C-H, Yeh S-H et al (2003) Microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome, Taiwan. Emerg Infect Dis 9:1163–1167. https://doi.org/10.3201/eid0909.030367

Peiris JSM, Lai ST, Poon LLM et al (2003) Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361:1319–1325. https://doi.org/10.1016/S0140-6736(03)13077-2

Shi Y, Yi Y, Li P et al (2003) Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay. J Clin Microbiol 41:5781–5782. https://doi.org/10.1128/JCM.41.12.5781

Okba NMA, Müller MA, Li W et al (2020) SARS-CoV-2 specific antibody responses in COVID-19 patients. Emerg Infect Dis. https://doi.org/10.1101/2020.03.18.20038059

Perera RAPM, Mok CKP, Tsang OTY et al (2020) Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020. Eurosurveillance 25:2000421. https://doi.org/10.2807/1560-7917

EUROIMMUN AG (2020) Anti-SARS-CoV-2 ELISA (IgG) Instruction for use

InBios International Inc. (2020) InBios SCoV-2 Detect TM IgG ELISA Instructions for Use

Beijing Wantai Biological Pharmacy Enterprise Co. Ltd. (2020) WANTAI SARS-CoV-2 Ab ELISA

Bio-Rad Laboratories Inc. (2020) Platelia SARS-CoV-2 Total Ab

Shanghai Fosun Long March Medical Science Co. (2020) Serology Test Evaluation Report for “Fosun COVID-19 IgG/IgM Rapid Antibody Detection Kit” from Shanghai Fosun Long March Medical Science Co., Ltd.