CME INDIA Presentation by Dr. S. K. Gupta, MD (Med), FICP, CFM (France) Clinical Asst. Professor GS Medical College, CCSU, Uttar Pradesh India. Visiting Consultant, Max Super Specialty Hospital, New Delhi; and Dr. Sonal Saxena, Director, Professor and HOD Dept. of Microbiology, MAMC, New Delhi; Special Thanks: Dr. Raman Sardana, Secretary-Hospital Infection Society India, Delhi for various Inputs; East Delhi Physicians Association.

Practical Information on Antibody Tests: Who Needs Them, Where to Get Them.

Why do people get Antibody Testing done?

• To Evaluate the Antibody development following the vaccination, in order to boost confidence of having protective cover in Covid Era.
• To decide if Booster dose is required after the disease/vaccine?
• To diagnose the past occurrence of Covid -19 in doubtful situations.
• Before Plasma donation -Now this reason is no more Valid as plasma therapy has been trashed.
• As a part of vaccine trials, Study protocols.

Let us analyse the above situations one by one:

Does a positive antibody test mean protective immunity blanket against COVID-19?

• A positive antibody test does not necessarily mean that one is immune to future SARS-CoV-2 infection, as the virus can Mutate riding over the immunity and also Antibody tests can be wrongly positive.

Can Antibody test be Falsely positive?

• Yes, there is also a chance that the positive result is wrong, known as a false positive.
• False positive tests may occur:
• Because antibody tests may detect coronaviruses other than SARS-CoV-2, such as those that cause the common cold.
• Chances of False positivity increase in isolated individual cases while chances of true positivity increase in setting of pandemics after mass vaccination.

If Positive Antibody Test doesn’t guarantee protection, how do researchers come know that vaccines are effective?

• Initial evidence is gained from large vaccine trials (phase 3). But Real Evidence comes from society (Phase 4) when pandemic starts to wane off after vaccination of huge population cohorts leading to development of Herd immunity.
• In current pandemic, there are many success stories where pandemic has been successfully contained by mass vaccination such as Israel, UK, USA, UAE.

Why individual antibody protection doesn’t work but same protection works in Herd immunity?

A: See the image to understand it better.

• When an individual is protected but society at large is susceptible virus circulates in society and gets a chance to mutate, ultimately overriding the immunity of an individual.
• But when larger number of people are immunised virus gets little chance to get transmitted form an infected person to others so virus reservoir doesn’t grow and ultimately gets contained.

Is it not a herculean task to aim for Herd immunity?

• Yes, it is. But sustained Govt and Public efforts archive results. Truly Herd immunity is like a mirage but it is still worth pursuing.

I am still not convinced and like to have an antibody test for my confidence.

• Over anxious approach may not help. As you don’t rush for antibody testing of your kids after DPT polio measles Vaccination. So is the Sars-Cov-2. Results of vaccination drive through mass vaccination. To have true immunity you should motivate others around you to get vaccinated. Secondly, if antibody report comes (false) negative, it will turn confidence into confusion with little possibility of revaccination.

Antibody testing before a vaccine dose after Covid or before booster (second) dose of vaccine?

• As per guidelines all those who have recovered from Covid should go for vaccination because antibody response after Covid is highly variable in individuals. Those who suffered from mild illness may develop inadequate short lasting immune response. The immunity development after vaccination is well studied and mapped phenomenon. Getting a jab 4 to 6 weeks (up to 6 months) after the full recovery from Covid is strongly advised without going for any antibody level testing.
• Those who have received first dose of any vaccine may also not be helped by anti-body testing before second dose as it is well known that some people develop demonstrable levels of antibodies only after booster dose.

Can antibody tests be used to diagnose COVID-19?

• No. An antibody test does not detect the presence of the SARS-CoV-2 virus to diagnose COVID-19. These tests can return a negative test result even in infected patients (for example, if antibodies have not yet developed in response to the virus) or may generate false positive results (for example, if antibodies to another coronavirus type are detected), so they should not be used to evaluate if one is currently infected or contagious (ability to infect other people).
• Presence of antibody in someone with no past history of symptoms of Covid doesn’t rule out false positive result but can be true positive report as a result of asymptomatic Covid.

Who should go for antibody titres?

• Immunocompromised people like advanced Cancer, Sick HIV patients, organ transplant recipient’s immunosuppressive treatment, patients on prolonged steroid therapy etc may be taken for antibody titres testing in consultation with their health care provider to decide for repeat higher dose of vaccination with adjustment of immunosuppressive therapy.

What is the optimal time of getting antibody testing?

• Antibody (IgG) testing is best done 3-4 weeks after the first dose and 2 to 3 weeks after the second dose. Testing done before or after the above window may give false negative results

Types of antibody tests available in market, their sensitivity, cost and interpretation.

Learn about various antigens in Virus which lead to antibody formation:

SARS CoV-2 genome encodes for four major structural proteins See Figure: for easy understanding:

Spike (S)

• S protein plays an essential role in viral attachment, fusion, entry, and transmission. It comprises of:
  • S1 subunit responsible for virus–receptor binding. 
  • A S2 subunit responsible for virus–cell membrane fusion.
• S1 is further divided into an N-terminal domain (NTD) and a receptor-binding domain (RBD).
• Envelope (E), Non-immunogenic.
• Membrane (M), Non- immunogenic.
• Nucleocapsid (N)] immunogenic Antibodies against N Ag seen only after Covaxin injection/Natural infection because it is whole virus vaccine while Pfizer Covishield etc. code only for S Protein.

What do serological tests detect?

Serological assays detect antibodies specific to viral proteins/domains.

Available Serological assays detect antibody:

1. S1 and/or S2 subunits.
2. Against RBD.
3. Against N protein.

What is Functional class of Antibodies?

Antibodies may be Binding or Neutralizing.

1. There are three types of antibodies Anti N, Anti S and Anti RBD which may be Binding or Neutralizing.
2. Neutralizing antibodies (NAbs) are detected against many fragments in S-proteins (S1-NTD, RBD, or S2) and are considered key to recovery and protection against viral disease.

Which Vaccines produce what type of antibody?

1. m-RNA (Moderna/Pfizer): Anti RBD
2. Covishield: Anti Trimeric Spike Protein
3. Covaxin: Anti S and Anti N
4. Sputnik: Anti S

 All approved vaccines produce binding as well as neutralizing antibodies.

What are various methods of Antibody detection?

1. Chemiluminescence immunoassay(CLIA).
2. ELISA: Enzyme linked immosorbant Assay.
3. Radioimmunoassay (RIA).

Which is better method?

CLIA scores over other two methods.

• Qualitative Testing vs Quantitative Testing.
• Quantitative detection measures the actual titres of antibodies. However, no inter-kit standardization has yet been possible. Results on two different platforms may vary. Also, results of two different company testing kits using same platform may be different.
• While Qualitative antibody testing just detects presence or absence of antibodies in a given serum.

Some values written in Qualitative test report cause confusion of Quantitative test watch out

What do numerical figures in Qualitative Test mean?

• OD RATIO optical density ratio, COV -Cut off Value, IV index Value.
• These are ELISA test run time calibration units/value and don’t correlate significantly with Ab titres in serum.

How to interpret the assay report?

• Spend two minutes to read the fine print in report. You have spent money.
• Qualitative or Quantitative.
• Type of Kit used in assay Diasorin, Abbott, Quant on Architect, Euroimmun, Elecsys etc are reputed kits.
• Platform used in assay. CLIA scores over others.
• Higher Ab titres neither suggest higher immunity nor longer lasting immunity. But higher values are loved by all.

SARS-CoV-2 Neutralizing Antibodies

• Neutralizing antibodies (NAb) are a subset of antibodies that independently block viral entry into host cells. They are primarily of the IgG isotype.
• Important to consider the possibility that
  1. Not everyone will develop neutralizing antibodies to SARS-CoV-2.
  2. When neutralizing antibodies are developed, we still don’t know whether they are fully protective.
  3. What titre is needed to have full protective immunity or
  4. How long the titres of Nab persist.

How can we determine if the antibodies being detected by assay is capable of neutralizing SARS-CoV-2?

• Neutralizing antibodies are classically detected using Plaque Reduction Neutralization Tests (PRNTs). However, PRNTs require the use of live virus and must be performed in biosafety level 3 (BSL3) facilities. Furthermore, this type of test is challenging and time consuming to perform.
• A few studies have indicated a correlation between the antibodies detected by  serology assay and neutralizing action, but the data is not robust.

Rough Correlation with Serological values and Neutralizing Ab titres: Must be Pondered

• European commission had recommended an NT titre target of 1:320 or more for COVID-19 convalescent plasma.
• As per one study Rough correlates of NAb with Anti S antibody for some kits:
• Wantai ELISA titre <1:80, Euroimmun IgG titers <1:640 or Diasorin Anti-SARS-CoV-2 values <30 AU/mL correlated with poor Neutralizing Antibody titres in one study. (Negative Predictive Value).

Antibody testing -In Various Labs around

(Personal communique, reports from these labs, Web based advisory from various labs.)

• Most of the next-door standalone labs are doing Qualitative antibody testing because of Cost, Affordability and Ease of doing.
• Most Centralised/Reputed labs including SRL, Metropolitan, Thyrocare, LAL Path Lab, Max Lab etc. are doing both Quantitative as well as Qualitative analysis.
• Watch out the choice of test before you order.
  • Dr. Lal Path Lab is also doing Neutralizing Antibody Testing. It is a qualitative test done on EIA platform. Lal’s lab does not disclose the kit (company) used to detect the neutralizing antibody. Test costs Rs 4000/- Sample collected twice a week TAT is 48 hours.
  • LAISON® SARS-CoV-2 S1/S2 IgG assay (Diasorin, Italy) Highly reputed test kit based upon Chemiluminescence (CLIA) technology. This is a quantitative test. This test detects neutralizing antibodies which show 94.4% positive agreement to Plaque Reduction Neutralization test (PRNT). It is being used by some labs including Supratech, Dr. Dang’s Lab and Indraprastha Apollo Hospital Delhi etc.
  • SARS-CoV-2 IgG II Quant ON ARCHITECT- RBD Ab. It is a chemiluminescent microparticle immunoassay (CMIA) used for the qualitative and quantitative determination of IgG ab.  Quantitative measurement of IgG antibodies against the spike receptor-binding domain (RBD) of SARS-CoV-2
  • A wide range of COVID-19 vaccines esp. mRNA Pfizer vaccines utilizes spike protein and the RBD domain of the S1 subunit.
  • 1mg Lab and Apollo Hosp using SARS-CoV-2 IgG II Quant ON ARCHITECT kit. Quantitative Test. Cost ~ Rs.500.
• Abbot SARS CoV-2 IgG
  • The assay is designed to detect IgG antibodies to the nucleocapsid protein of SARS-CoV-2 in serum/plasma of patients of COVID-19 or after vaccination by Covaxin.

CME INDIA Tail Piece

A. Two Recent Indian Studies

B. Immunity

C. Delta Plus issues

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