SARS-CoV-2 infection serology

Understanding the serological characteristics of SARS-CoV-2 infection could help to improve diagnostic accuracy and aid in the development of vaccines for COVID-19.

Overview

• Introduction
• When can the antibody response be detected in COVID-19?
• Assay formats to characterize the serological response to SARS-CoV-2
• Products to support serological research
  • Lateral flow assays for the detection of SARS-CoV-2
  • Lateral flow assay development kit
  • Serologic ELISA kits for the detection of antibodies to SARS-CoV-2
  • Colloidal gold to support lateral flow assay development
• References

Introduction

Current diagnostics for SARS-CoV-2 infection rely on the detection of the viral RNA by real-time PCR (RT-qPCR). However, this has shown to yield a high number of false-negative results, is limited by requirements of extensive hands-on time from skilled workers and the need for specialist equipment, and results in longer diagnosis times ^1,2.

The detection of antibodies to SARS-CoV-2 offers a more robust and simpler method for the detection of the virus. As antibodies and protein antigens are more stable than RNA, how patient samples are transported and handled isn’t as critical for accurate detection, which reduces the number of potential false-negatives ^3,4. Serological assays detecting antibodies to SARS-COV-2 would therefore be useful to speed diagnosis, determine the immunity period for SARS-CoV-2, and re-admit people to work following potential infection ^1,2. This increased understanding of patient antibody responses could also provide additional insight for vaccine developers in trials.

The use of these assays could complement RNA detection strategies, providing information on the adapted immune response to SARS-CoV-2 infection, and aiding in infection control within the clinic and communities. Though many researchers are working to develop serological assays to detect SARS-CoV-2 infection, a thorough understanding of the antibody response in COVID-19 has restricted the speed of development in this field.

When can the antibody response be detected in COVID-19?

The standard humoral response to infection involves the early expression of IgM isotype antibodies, which matures into the expression of IgG isotype antibodies. Studies using sera from COVID-19 patients showed that the immune response to SARS-CoV-2 infection is slow, with the initial IgM antibody response peaking at 9 days post-exposure and the IgG antibody response peaking at 11 days, ^5,6 meaning that high sensitivity is particularly critical for any detection assay used in the early stages. Indeed, the sensitivity of antibody detection for all antibody isotypes increases from 7 days post-onset of disease symptoms (dpo) in all studies ^7-10.

The relative timecourse of the antibody response during SARS-CoV-2 infection ^5-12. Precise patient responses are reported to vary, thus delineating specific days for antibody response is still impractical based upon current literature.  

A wide range in the median rate of seroconversion and detection of COVID-19 targeted antibodies has been reported across the different study cohorts, ranging from 4 dpo of symptoms to 14 dpo ^7,9-11. IgM antibodies were shown to be present in some patients as early as 4 dpo ¹¹. IgA showed a similar seroconversion rate to IgM, with detectable levels present from 5 dpo ⁹. The greatest reported range in seroconversion rate is for IgG, with one study suggesting detectable IgG antibodies at 5 dpo ⁹, though the majority of studies report a seroconversion rate of 14 dpo ^9,10,12. Both IgM and IgG antibody isotypes were seen to be detected earlier and at higher titers in more severe cases of COVID-19 ¹³, which could explain some of the variance in the reported rates. Though increased age has been associated with severe COVID-19, the analysis of the humoral response shows no significant difference in the seroconversion rate of older patients or patients with increased viral load at earlier stages of the disease ¹⁴.

The potential diagnostic impact of analyzing the immune response in addition to the viral antigen is clear: one study showed that at the early stages of the disease, 5 dpo, IgM detection was positive in 93% of suspected cases with negative PCR results ¹⁰. In addition, while the overall detection sensitivity for RT-PCR testing was 52%, combining this with serological assay for COVID-19 increased the total sensitivity to 99% ¹⁵.

Assay formats to characterize the serological response to SARS-CoV-2

The most common assay formats for the detection of antibodies are ELISA and lateral flow immunoassays (LFAs), and both have their place in preventing the spread and analysis of infectious diseases, such as COVID-19.

The current standard nasopharyngeal swab required for RT-PCR assay is intrusive and uncomfortable for individuals and requires specialized staff to perform the swab. The use of alternative samples, such as saliva, could improve participation in testing, with many pursuing both ELISA and LFA assays that offer the required sensitivity and specificity with such sample matrices.

ELISA

An analysis of the sensitivity of different assay formats to IgG or IgM antibodies for SARS-CoV-2, showed the pooled sensitivity of ELISAs to be 84.3% (75.6-90.9%, 95% CI) and of LFAs to be 66% (49.3-79.3%) ¹⁶. One direct comparison across ELISA, LFA and chemiluminescent assay formats using the same patient samples showed ELISA to be the most sensitive assay for the seropositivity of SARS-CoV-2 antibodies ¹⁴.

Displaying the most sensitive and early detection for SARS-CoV-2, ELISAs are being developed for use with many sample types and have shown utility in the accurate detection of SARS-CoV-2 antibodies from as early as 4 dpo ¹¹.

Lateral flow assay

To help tackle the COVID-19 pandemic, the World Health Organisation has recommended that rapid point of care diagnostics for use at the community level, such as LFAs, are a research priority ¹⁷. LFAs offer benefits of rapid results, cost-effective testing and the ability for non-specialists to perform testing. Widely used for point of care (POC) and consumer-testing, LFAs can offer qualitative, semi-quantitative or quantitative analysis of a variety of target analytes, such as pathogens or biomarkers.

An LFA test strip is very simple; involving the chromatographic separation of a sample across a nitrocellulose membrane and the identification of a specific analyte by binding antibodies conjugated to gold nanoparticles on the test strip to yield a signal. Difficulties in the development of LFAs commonly arise in transitioning the assay to the test format, with extensive optimization often required to ensure stable and consistent binding of the antibodies to the gold nanoparticles on the test strip.

The current LFAs for the serological analysis of SARS-CoV-2 infection do not show sufficient sensitivity for patient application, though there are many under development ¹⁶.

Serological assay development for the analysis of SARS-CoV-2 infection is being scaled up globally, to increase diagnostic capacity and support vaccine development through a more complete understanding of the antibody responses observed in patient samples.

At Abcam, we have a range of solutions to help researchers in the serological characterization of SARS-CoV-2 infection and the development of LFAs for COVID-19, including LFA assay kits for SARS-CoV-2 antibody detection, a universal lateral flow assay kit and gold conjugation kit to allow rapid proof-of-concept, and highly consistent and scalable gold nanoparticles for LFA development and scale-up.

Products to support serological research

Lateral flow assays for the detection of SARS-CoV-2

Lateral flow assay development kit

Abcam's Universal Lateral Flow Assay Kit enables the simple and rapid development of proof-of-concept sandwich lateral flow immunoassays. It does so by removing the need to spray down capture antibodies on the test strips and to make gold conjugates with passive absorption, which both typically require expertise, special equipment, hands-on time, large amounts of antibodies and rounds of optimization. Instead, any antibody pairs used can simply and rapidly be conjugated to the gold nanoparticles with our Lightning-Link and Gold conjugation technologies and combined with the universal strips, other reagents and buffers. The Universal Lateral Flow Assay Kit can also be used for antibody pair screening for assay development.

Serologic ELISA kits for the detection of antibodies to SARS-CoV-2

Colloidal gold products to support lateral flow assay development

Gold nanoparticles are ideally suited to lateral flow immunoassays due to their intense ruby red color, which makes them easy to visualize on the test strip. Abcam's colloidal gold has been developed using specialized techniques that enable the production of extremely uniform nanoparticles with a narrow size distribution, thereby minimizing assay variability. Our colloidal gold can be produced at high concentrations (>20OD) and large volumes (liters) without compromising quality.

The use of colloidal gold for lateral flow immunoassays requires passive adsorption of the antibody to the surface of the nanoparticle, a process that can be challenging and necessitates specialist knowledge. To overcome this we have developed GOLD conjugation kits. The 40nm lyophilized gold nanoparticles within the kit have a unique surface-coating that covalently binds the antibody or protein forming a highly stable conjugate. The conjugation is quick and easy with ready to use conjugates in 20 minutes.

If you are interested in the commercial use of our products, please contact us at partnerships@abcam.com.

These products are intended for research use only, not for use in diagnostic, therapeutic or any other purposes.

References

1. Nuccetelli M et al. SARS-CoV-2 infection serology: a useful tool to overcome lockdown? Cell Death Discovery 6, Article 8 (2020)
2. Hamid H et al. COVID-19 Pandemic and Role of Human Saliva as a Testing Biofluid in Point-of-Care Technology. Eur J Dent  Pre-publication doi: 10.1055/s-0040-1713020 (2020)
3. Kucirka LM et al. Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since Exposure. Ann Intern Med M20-1495 (2020)
4. Watson J et al. Interpretating a COVID-19 test result. BMJ 369:m1808 (2020)
5. Liu L et al. A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients. medRxiv doi: 10.1101/2020.03.06.20031856 (2020)
6. Li Z et al. Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis. J Med Virol. doi:10.1002/jmv.25727. Online ahead of print. (2020)
7. Xiang F et al. Antibody Detection and Dynamic Characteristics in Patients with COVID-19. Clin Infect Dis doi: 10.1093/cid/ciaa461 Online ahead of print. (2020)
8. Traugott M et al. Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Assays in Different Stages of Infection: Comparison of Commercial Enzyme-Linked Immunosorbent Assays and Rapid Tests. J Infect Dis. 222(3):362-366 (2020)
9. Guo et al. Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Clin Infect Dis. doi: 10.1093/cid/ciaa310. Online ahead of print. (2020)
10. Zhao J et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019.  Clin Infect Dis. doi: 10.1093/cid/ciaa344. Online ahead of print (2020)
11. Suhandynata RT et al. Longitudinal Monitoring of SARS-CoV-2 IgM and IgG Seropositivity to Detect COVID-19. J Appl Lab Med doi: 10.1093/jalm/jfaa079. Online ahead of print. (2020)
12. Ma H et al. Serum IgA, IgM, and IgG responses in COVID-19. Cell Mol Immunol. 17, 773–775 (2020)
13. Tan W et al. Viral Kinetics and Antibody Responses in Patients with COVID-19. medRxiv doi: 10.1101/2020.03.24.20042382 (2020)
14. Lou B et al. Serology characteristics of SARS-CoV-2 infection since the exposure and post symptoms onset. medRxiv doi: 10.1101/2020.03.23.20041707 (2020)
15. Kai-Wang To K et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis 20(5):565-574 (2020)
16. Espejo AP et al. Review of Current Advances in Serologic Testing for COVID-19. Am J Clin Path doi: 10.1093/ajcp/aqaa112 (2020)
17. WHO 2019 Novel Coronavirus Global Research and Innovation Forum: Towards A Research Roadmap [Accessed: 17/07/2020] (2020)