This document discusses SARS-CoV-2 neutralizing antibody testing. It begins by introducing the speaker, Dr. Bikash Kumar Chaudhury. The webinar outline includes an overview of SARS-CoV-2, immunity, serological tests for SARS-CoV-2, neutralizing antibodies specifically, and tests offered at Vijaya Diagnostic Centre. It describes neutralizing antibodies' role in blocking viral entry and their importance for immunity. Interpretation of serological test results is also discussed, noting limitations in determining prior infection timing, shedding, or protection from reinfection.

1. SARS-COV-2 Neutralizing Antibody Test
(Testing in the Setting of Post Recovery/ Vaccination)

2. Today’s Speaker
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Dr. Bikash Kumar Chaudhury is Medical
Biochemist at Vijaya Diagnostic Centre in
Hyderabad.
He completed his MD in Biochemistry at
Guwahati Medical College and Hospital and is
also certified Assessor by the National
Accreditation Board for Testing and Calibration
Laboratories (NABL), and is currently the Head
of Department at Vijaya Diagnostic Centre
Limited.
Dr. Bikash Kumar Chaudhury, MD (Biochemistry)
Head of Department (Biochemistry)

3. Outline of Today’s Webinar
• Overview of SARS-CoV-2: The Virus
• Immunity
• Serologic Tests for SARS-CoV-2
• Neutralizing Antibodies to SARS-CoV-2
• Serological tests at Vijaya Diagnostic Centre
• Commencement of Neutralizing Antibody Test
• Summary

4. SARS-CoV-2: The Virus
• Coronaviruses belong to the
Coronaviridae family in the
Nidovirales order
• Coronaviruses are enveloped
viruses, minute in size (65–
125 nm in diameter) and contain
a single-stranded RNA as a
nucleic material, size ranging
from 26 to 32kbs in length

5. SARS-CoV-2: The Virus

6. Coronavirus Emergence
• Coronaviruses are a large family of
viruses that are known to cause
illness ranging from the common cold
to more severe diseases such as
Middle East Respiratory Syndrome
(MERS) and Severe Acute
Respiratory Syndrome (SARS).
• 2019-novel coronavirus (COVID-19)
was identified in Wuhan, China. This
is a new coronavirus that has not
been previously identified in
humans.

7. What do we mean by immunity ?
• Immunity is the ability to resist a disease or an infection.
• Immunity to viral infection is a combined outcome of both
cellular and humoral (antibody) immune responses. While
cellular immune response is often difficult to measure,
antibody production serves as a hallmark of humoral
immune response.

8. Timing of Antibody Response to SARS-CoV-2
• New virus = no pre-existing antibodies or
immunity
• We are still learning about our immune
response to SARS-CoV-2
– Many develop Abs ~1-2 weeks after
symptoms
• Due to delay in seroconversion,
Abs do not play a routine role in
diagnosis
– >95% of patients are Ab positive after
2 weeks
• Some patients may not
seroconvert
- Immunostatus
- Assay dependent?
- Severity of illness?
– IgM declines 5-7 weeks post onset
– IgG remains positive for ≥10 weeks
post onset
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IgM Antibodies
Antibody Detection
IgG Antibodies
Molecular Detection
Viral RNA
Figure modeled after Sethuraman N, et. al. JAMA. 2020; E1-E3
Symptom
Onset
Infection
Week -1 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

9. Serologic Tests for SARS-CoV-2
>200 commercially available serologic tests for
anti-SARS-CoV-2 antibody detection!
(More antibody tests for SARS-CoV-2 than for any other
infectious disease)

10. • Format
– Lateral flow assays
– Enzyme immunosorbent assays
– Chemiluminescent immunoassays
• Specimen type
– Serum, Plasma,
– Finger stick/venous whole blood (LFAs)
• Immunoglobulin class detected
– IgM
– IgG
– IgA
– Total Ab
• SARS-CoV-2 antigen used
– S1 and/or S2 of Spike protein
– Receptor binding domain (RBD)
– Nucleocapsid – most abundant viral protein
https://www.ncbi.nlm.nih.gov/books/NBK554776/
Variations in SARS-CoV-2 Serologic Test Designs
CDC COVID-19 Guidelines (May 23, 2020):
- No advantage testing for IgG, IgM & IgG or Total
- Testing for IgA not recommended!

11. What are Neutralizing Antibodies?
• Neutralizing antibody is a subset of antibody that can inhibit viral
replication and represents a major mechanism of humoral immunity
against viral infection.
• The protective function of neutralizing antibodies is mainly mediated by
the blocking of the interaction between the virus and its host cells,
resulting in the inhibition of viral entry to cells, thus, prevents viral
infection. Because of the mechanism of action, it is not surprising that
most neutralizing antibodies are against viral surface proteins.

12. What do we know about neutralizing antibodies to
sars-cov-2?
• The virus causing COVID-19, SARS-CoV-2, uses the Spike protein (S) to bind to the
receptor on host cells to trigger cell entry and infection.
• S protein consists of S1 and S2 subunits, and S1 interacts with the host cells via the
Receptor Binding Domain (RBD).
• Monoclonal antibodies to S1 protein that exhibit neutralizing activities are being
developed as potential therapeutics for COVID-19.
• Neutralizing antibodies to the S protein are the key active ingredients of
convalescent plasma used to treat severe COVID-19 patients
• In addition, almost all the COVID-19 vaccines currently under development target the
S protein with the goal as to induce neutralizing anti-S antibodies.
• Preliminary vaccine studies showed a correlation between neutralizing antibody
titers with protective efficacy in animal models.

14. All these observations suggest that
S protein is a primary target for
neutralizing antibodies against
SARS-CoV-2, and anti-S neutralizing
antibodies play a key role in COVID-
19 immunity
What do we know about neutralizing antibodies to
sars-cov-2? (Cont…)

15. The Role of Neutralizing Antibodies in
Protective Immunity
• Protective immunity is multifaceted!
• Antibodies can be binding or neutralizing
– Binding (non-neutralizing) Abs
• Produced at high levels, but unable to independently prevent
infection
• Bind and flag pathogen as ‘invader’
• Good markers of prior infection
– Neutralizing Abs (NAbs)
• NAbs binds to the virus leading to loss of infectivity and blocking
viral entry into host cells
• Functions independent of other immune system components
• Testing for NAbs was challenging till now

17. Interpretation of Results from Antibody Tests
for SARS-CoV-2
• Negative Result:
– Likely no prior infection or exposure to the virus
• Individuals tested too soon following infection or immunosuppressed patients may be
negative
• Small percentage of individuals may not seroconvert
• Positive Result:
– Suggests recent or past infection
• May be impacted by the local/regional prevalence
– What these results do not (yet) tell us:
• When the patient was infected
• Whether they are shedding virus (live or dead)
• Whether patients/individuals are protected against re-infection
– Cannot use positive results to guide decisions regarding adherence to social distancing
recommendations or use of personal protective equipment

19. Uses For SARS-CoV-2 Serologic Testing
• Diagnosis?
• Limited utility. Can be offered as an adjunct for those who present late or
have suspected false negative upper respiratory samples and a lower
respiratory sample cannot be collected
• Epidemiologic Studies?
– Useful, if:
• Assay has adequate specificity (>99.5%)
• Used to screen high pretest probability populations
• Identification of Convalescent Plasma Donors?
– Yes
• Evaluation of immune response to vaccines?
– Yes
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