WO2022123470A1

WO2022123470A1 - Saliva based diagnostic assays for detection of sarscov2 receptor binding domain (rbd) neutralizing antibodies

Info

Publication number: WO2022123470A1
Application number: PCT/IB2021/061482
Authority: WO
Inventors: Satishchandran CHANDRASEKHAR; Gopi KADIYALA; Uday Saxena
Original assignee: Reagene Biosciences Pvt. Ltd.
Priority date: 2020-12-10
Filing date: 2021-12-09
Publication date: 2022-06-16

Abstract

The present invention provides rapid, inexpensive assays that use saliva to measure neutralizing antibody levels thus bypassing the need for blood. The ability to use saliva in the assays offers a significant advantage over the currently used blood-based assay kits. Applicants have designed three assays, a 1) fast flow device based on dot blot principle for single patient use, a 2) 96 well ELISA assay for use in hospitals or path labs for multiple saliva samples and an 3) LFA format assay for at home use.

Description

Saliva based diagnostic assays for detection of SarsCoV2 receptor binding domain (RBD) neutralizing antibodies

RELATED APPLICATIONS

This application is related to and takes priority from Indian Provisional Application 202041053792 dated 10^th December 2020 (10/12/2020) and is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides rapid, inexpensive assays that use saliva to measure neutralizing antibody levels thus bypassing the need for blood. The ability to use saliva in the assays offers a significant advantage over the currently used blood-based assay kits.

BACKGROUND OF THE INVENTION

Covid-19 first emerged in December 2019 in Wuhan, China. The cause of the disease was confirmed as a new kind of coronavirus, this virus is transmitted through air from people to people. The infection has quickly spread to most of countries around the world.

Covid-19 virus causes over 2% mortality, there is 7 to 14 days incubation period. The current primary diagnostic method for the presence of virus is majorly by RT-PCR of the viral RNA. There also are tests to detect the virus through what are called “antigen tests”. Antigen tests detect one or more of the proteins in the viral particle. Both RT-PCR and viral antigen tests are currently used widely in multiple formats and several variations of the two test themes. The antigen tests primarily use a test that detects the viral NP protein (Nucleoprotein), due to its abundance (1,000 copies per virion). The surface glycoprotein (S protein) of the virus is also used to detect the virus, albeit, less desirable due to decreased sensitivity, since -100 copies are present in each virion particle. Alternatively, researchers have developed methods to detect antibodies in persons who have been infected, as a measure of past exposure.

The viral coat primarily consists of S protein, the M (membrane) protein and E (envelope 5 protein). The S protein embedded in the viral membrane which is represented by about 100 copies per virion is responsible for initial interactions with the ACE-2 (Angiotensin Converting Enzyme) on the human cell surface (viral receptor). The interaction between the S protein and ACE-2 occurs through the RBD (Receptor Binding Domain) of the S protein, and disruption of this interaction is crucial to preventing viral entry, infection and disease severity. Presence of antibodies to the RBD is therefore virus neutralizing and protective. This was borne out in a recent publication demonstrating the correlate of protection being the virus neutralizing antibodies against the RBD of the virus (McMahan et al. Nature, December 4, 2020. https://doi.org/101038/s41586-020-03041-6).

Infection or vaccination typically produces specific IgM antibody initially, detectable at 2 to 5 days after the infection or vaccination, while specific IgG antibody appears after 5 to 7 days. Compared to other test, this test is more convenient with low cost, it can be performed in field, mobile hospital and laboratory. IgM is the first type of antibody made in the body as the first response to an infection by the immune system. It is the largest antibody found in the body and less abundant (5 to 10%) than the other antibodies. IgM exists as a pentamer consisting of 20 identical heavy and light chains. There are ten antigen binding sites for the IgM. However, due to the conformational constraints of the IgM, only five sites are available for antigen binding. While IgM is somewhat selective, its selectivity is not sufficient enough to be protective, however, its presence indicates beginnings of an immune response in an individual. IgG is another type of antibody produced by white blood cells and are found in all body fluids. It is the most abundant antibody found in blood (80%). IgG are produced at later stages of the infection and remain in the body for longer periods to fight against repeated infections. IgG exist as monomers with two antigen binding sites for each antibody. IgG is more specific or selective to a particular antigen compared to IgM, and affords protection from infection in vaccines and those previously infected. Nevertheless both IgM and IgG can be neutralising in nature against the antigen.

Currently, with the introduction of vaccines, large numbers of asymptomatic infected individuals and those who have recovered from infection, it is important to learn if these persons have mounted or continue to have an immune response against the virus and have antibodies that neutralize the virus. Essentially, the questions that warrants answers are; “Is the person responsive to vaccine”, “Is the individual protected from vaccine or prior infection”? If so, “for how long?” Will there be a need for another booster shot? It is now known that the immune response to the vaccine wanes over a period of months and a booster dose is being prescribed to offset the decrease in neutralizing antibi response to the vaccine.

SarsCov2 virus using its spike protein receptor binding domain (RBD) enters human cells such as lung cells by binding to the human cell surface ACE2 receptor. Most vaccines are therefore designed to immunize the individual against the RBD domain of the viral S protein with the intent of raising neutralizing antibodies which will negate the binding of the viral RBD to human cell ACE2 receptor and thus prevent entry of the virus. With the recent discovery that presence of neutralizing antibodies is key to protection, there is an urgent need to develop simple and rapid tests to screen large population, asymptomatic, symptomatic and previously infected people for these antibodies - i.e. those that will block the interaction of SarsCov2 RBD domain to the human ACE2 receptor.

In addition to blood, neutralizing antibodies have also been now observed in human saliva and mirror the antibody profile in the blood.

The present invention provides provides rapid, inexpensive assays that use saliva to measure neutralizing antibody levels thus bypassing the need for blood. Being able to measure the neutralising antibodies in saliva is a significant advantage of the invention because 1) drawing blood is not required 2) saliva offers a convenient sample to obtain from an individual 3) has the potential to be used in “at home” diagnostic tests and 4) minimal equipment and medical intervention needed to obtain samples.

Given that the vaccines have been successfully launched there is a need for a selective and sensitive test for detecting the presence of neutralizing antibodies against COVID-19 virus in a subject. In this invention we describe three separate assays (1) dot blot, 2) ELISA; and 3) Lateral Flow assay. All three diagnostic invented use the principle of competition of neutralising antibodies in saliva with RBD.

DESCRIPTION OF FIGURES

Figure 1. ACE2 concentration curve with fixed amount of biotinylated RBD (1 pg/ml) Figure 2. Binding of range of biotinylated RBD concentrations added to ACE2 (1 pg/ml) Figure 3. Concentration curve using biotinylated RBD (0.1 to 1 pg/ml) in the presence of 5 pg /ml unlabeled RBD.

Figure 4. Neutralizing activity (percent inhibition of biotinylated RBD binding to ACE2) in saliva of individuals who are unvaccinated or vaccinated against SarsCoV2. KM, SK, MR and US are individuals in the study.

Figure 5. Reversal of neutralizing activity of saliva from vaccinated individuals (KM, saliva is undiluted) and of US (after buffer dilution of saliva) showing decrease in neutralizing activity and thereby specificity of the effect. Figure 6. Reversal of neutralizing activity of saliva from vaccinated individuals (RP, US) by buffer dilution of saliva showing specificity. Saliva from unvaccinated individuals SK and NS shows no neutralizing activity as expected

Figure 7. Dot blot assay on commercially available fast flow spot device.

Figure 8.Typical ELISA format used in the studies to demonstrate saliva activity.

Figure 9. Binding concentration curve of anti-IgY to RBD protein coated on ELISA plate showing linearity of binding.

Figure 10. Binding of anti-IgY to native RBD and delta RBD coated on ELISA plate.

Figure 11. Neutralizing activity by saliva from vaccinated individual with anti-RBD IgY demonstrating competition for binding to native RBD coated on the ELISA plate Figure 12. Neutralizing activity by saliva from vaccinated individual with anti-RBD IgY demonstrating competition for binding to Delta RBD coated on the ELISA plate.

Figure 13. Lateral Flow Immunoassay prototype.

Figure 14. Lateral flow assay of 1) Biotinylated RBD binding to ACE-2 at control line (C) showing broad stain indicating optimal binding 2) Reduced (stain) binding of Biotinylated RBD to ACE in presence of saliva from vaccinated individual at Test line (T) indicating neutralizing activity.

DESCRIPTION OF THE INVENTION

With the introduction of vaccines against Sars CoV2 virus, it has now become important to measure whether they are effective in immunizing the vaccinated individual or not. Successful immunization should result in the vaccine mounting an antibody response to the antigen used in the individual. In case of SarsCov2 the antigen used in vaccine immunizations is the receptor binding domain (RBD) of the virus spike protein. The principle used is that the antibody developed against RBD will neutralize the binding of the virus to its host cell surface ACE2 receptor. This inhibition of the virus binding by antibodies (called neutralizing antibodies) to host cell essentially prevents its entry into host and thereby renders the virus harmless.

There have to be rapid and qualitative and quantitative diagnostic assays to quickly check if an individual has responded to the vaccine or not and to determine if additional doses and booster are needed. The success of any vaccine strategy depends on the immune response it elicits in the individual. Measuring neutralizing antibodies in blood is the most common way to check for vaccine response. However, that requires a trained professional, hospital visits and sterile environment. To meet this demand, applicants have designed three separate assays that can be adapted to a commercial diagnostic kit either at home or in hospitals. Specifically, applicants provide rapid and inexpensive assays that use saliva to measure neutralizing antibody levels thus bypassing the need for blood (although blood and other tissue fluids may be used as a sample instead of saliva). The ability to use saliva in the assays offers a significant advantage over the currently used blood-based assay kits.

The present invention provides at least three different methods for detection all of which use saliva as a sample 1) fast flow assay based on dot blot principle for single patient use,

2) ELISA assay for use in hospitals or pathology laboratories for multiple saliva samples and

3) LFA assay for at home use.

The present invention provides test assays for determining the presence of COVID-19 RBD antibodies in a subject, specifically in saliva. The method includes analysing saliva sample that is obtained from the subject, using SarsCoV2 spike protein of RBD binding to human ACE2 to determine a possible presence of COVID-19 anti- RBD antibody in saliva. If there are neutralising antibodies in saliva then the binding of RBD to ACE-2 will be inhibited/neutralized. The invention as described herein provides a method for detecting the presence of antibodies against COVID- 19 virus in a subject. The said method involves obtaining saliva sample from a subject to be tested for the presence of antibodies against COVID- 19 RBD. The sample from the subject as described herein includes saliva but can be adapted to serum, blood, plasma, sweat, body fluids or a mixture thereof.

The method includes analysing the sample that is obtained from the subject using a test kit comprising a COVID- 19 RBD protein and human ACE2 protein to determine a possible presence of COVID-19 anti-RBD antibody.

In a key aspect, the invention also provides a method for detecting the presence of neutralizing antibodies against COVID-19 virus in a subject. The method includes:

1.1. analysing a saliva sample obtained from said subject using COVID-19 RBD (“RBD”) protein and human ACE2 protein to determine a possible presence of neutralising antibodies that bind COVID-19 RBD protein which prevents binding of COVID-19 RBD protein to the human ACE2 protein and the said analysis indicates the presence of anti-COVID-19 RBD antibody, wherein the presence of neutralizing antibodies against COVID-19 RBD is indication that the said subject has neutralizing antibodies and is protected from infection.

1.2. analysing for the presence of neutralizing antibodies against COVID- 19 RBD as provided in step 1.1. In particular, the tests include determining the presence of antibodies to RBD of COVID-19 in three ways. The diagnostic test can be carried out in a 1) dot blot format or 2) competition ELISA or 3) LFA assay.

The invention in brief provides the following:

A method for detecting the presence of antibodies against COVID-19 RBD in a subject, said method comprising analysing a saliva sample obtained from said subject and using a COVID- 19 RBD to determine a possible presence of COVID- 19 RBD antibody; and wherein the presence of COVID-19 RBD antibody is indication that neutralizing antibody to COVID- 19 is present in said subject.

The RBD of COVID-19 S-protein as provided in the present method comprises amino acid sequence of SEQ ID NO: 1: HMASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQ IAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIY QAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPG TNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTLE

• The protein sequence of RBD of COVID-19 S-protein is a subset of protein sequences that contain the neutralizing epitopes of COVID- 19 S-protein

• The method detecting the presence of antibodies against COVID-19 RBD in a subject wherein said steps comprise analysing for the presence of COVID- 19 RBD antibody by following the steps as below:

1) applying the saliva sample onto a nitrocellulose based fast flow assay based on dot blot principle; or

2) Using competition ELISA; or

3) Using saliva samples in LFA format

The fast flow assay (1) which is based on dot blot principle is particularly meant for single patient use whereas the ELISA assay in a 96 well format (2) is for use in hospitals or pathology labs for multiple saliva samples and the LFA format assay (3) is for “at home” use.

• The foregoing discussion of the invention in the form of Examples has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. All references cited herein are incorporated by reference in their entirety.

EXAMPLES

Example 1

DOT BLOT assay

1. ACE2 ( Tranalab Pvt Limited, India)

2. Biotinylated RBD and avidin (Commercially available kit) Buffers:

1. Wash buffer ( Commercially available)

2. Blocking buffer (3%FBS+0.01M PBS)

3. Biotin assay buffer (from kit) Procedure for DOT BLOT assay:

1. Cut strips of nitrocellulose membrane (3M).

2. Draw two etched lines on all the strips.

3. Deposit 10 pL 0.25 pg/ml ACE2 in between the lines.

4. Incubate for an hour at room temperature.

5. Add enough blocking buffer in order to fully immerse the strips in it. Pour off extra buffer and let the strips sit at room temperature for an hour. Make sure to add blocking buffer only after strips are completely dry and don’t let strips go dry after this step.

6. Add 10 pL 0.25 pg/ml B-RBD prepared in 0.01M PBS on the control strip, with or without 10 uL of antagonist on test strip.

7. After an incubation of an hour at 37°C, wash the strips with washing buffer three times, with each wash lasting ten minutes, perform the washing on a shaker.

8. Pour off any extra buffer after washing.

9. Add 20pL working solution of avidin per the kits instructions to all the strips. Protect from light.

10. Observe the colour development immediately and quantitate the spot intensity using ImageJ software

Dot Blot assay examples 1. ACE2 concentration curve in binding of RBD

We designed a rapid and inexpensive dot blot assay as described above to measure the binding of RBD to ACE2 and the effect of neutralizing antibodies on the binding. To optimize the binding of biotinylated RBD to ACE2, we spotted a range of ACE-2 concentrations (0.1-1 pg/ml) on the nitrocellulose membrane strip and then examined the binding of biotinylated RBD using the protocol reported above. As shown in Figure 1, the binding of RBD was linear within the concentrations of ACE2 used with a regression value (R2) of 0.98 suggesting robust binding of RBD to ACE2.

2. Optimal concentration of RBD binding to ACE2

We next determined the optimal concentrations of biotinylated RBD binding to ACE2. For this we tested the binding of a range of concentrations of biotinylated RBD to ACE2 blotted on the membrane. Figure 2 shows that the binding increased linearly within the range of biotinylated RBD tested (0.1-0.8 pg/ml) with R2 value of 0.99. These studies demonstrate that the dot blot is a viable assay to study the binding of RBD to ACE2.

3. Competition by unlabeled RBD of biotinylated RBD binding to ACE2

To further ascertain the specificity of the binding of biotinylated RBD to ACE2 in our assay we then tested the competition of binding using excess unlabeled RBD. A concentration curve using biotinylated RBD (0.1 to 1 pg/ml) in the presence of 5 pg /ml unlabeled RBD was performed. As shown in Figure 3, there was varying amount of inhibition with almost complete inhibition of biotinylated RBD binding to ACE2 by unlabeled RBD at lower concentrations of biotinylated RBD.

4. Using dot blot assay to test saliva samples for neutralizing antibodies

After optimizing the conditions as describe above, we then examined the ability of saliva samples to neutralize the binding of biotinylated RBD to ACE2 spotted on membrane. The idea here is that individuals whose saliva has neutralizing antibodies against RBD will inhibit the binding. Saliva samples were tested from following individuals KM, NR, SK, US1 and US2.

Vaccinated (KM and MR)

Unvaccinated (SK)

Individual before (US1) and after vaccination (US2) As can be seen in Figure 4 anti-RBD IgY resulted in the most significant inhibition as expected. Saliva from vaccinated individuals identified as KM and MR similarly showed neutralizing activity in blocking the binding of biotinylated RBD to ACE2 on nitrocellulose strip.

Saliva from an individual who was not vaccinated and thus not expected to have any neutralizing activity failed to block binding.

An individual identified as US, whose saliva was tested before vaccination (US1) showed no neutralizing activity. However, two weeks after vaccination, saliva sample US2 from the individual showed strong neutralizing activity. These data demonstrate that the dot blot test is sensitive enough to detect neutralizing activity in saliva.

To further validate the saliva findings, applicants further performed additional experiments - with the US2 sample, we diluted the saliva to see if we lose the neutralizing activity. As shown in Figure 5, dilution of US2 five times and 10 times did show loss of activity indicating that the activity was real and not an artifact. Similarly, further dilutions with another individual sample (RP, vaccinated) was performed and the results were consistent with presence of neutralizing antibodies in vaccinated individual RP. SK and NS were individuals who were unvaccinated and their saliva showed no neutralizing activity (Figure 6).

5. The dot blot system of the present invention was tested using an actual fast-flow spot device that is commercially viable. Applicants tested whether anti-RBD IgY could neutralize the binding of biotinylated RBD. The control spot was more intense than the spot in which anti-RBD IgY was added demonstrating the adaptability of dot blot in a commercial type fast flow spot device (Figure 7).

Example 2

ELISA Assay Examples

Method

1. Coat a 96 well plate with 100 pL of 2pg/ml RBD (prepared in IX PBS).

2. Incubate overnight at 4°C.

3. Wash the plate three times with 400 pL of 0.1 % PBST.

4. Tap the plate gently on the tissue paper to remove any remaining buffer.

5. Add 200 pL of 3% BSA to all the wells.

6. Incubate at room temperature for 1 hour. 7. Wash the plate three times with 400pof 0.1 % PBST.

8. Tap the plate gently on the tissue paper to remove any remaining buffer.

9. Add 100 pL buffer to negative control well in duplicates, 100 p L of Ipg/mLAnti RBD-IgY to positive control wells in duplicates in the presence or absence of saliva samples.

10. Add lOOpL of lug/mLAnti RBD-IgY plus samples (AntiRBD -IgY prepared in saliva samples instead of PBS) to sample wells in duplicates.

11. After 30 minutes and three washes, incubate with HRP labelled anti-IgY antibody and let it sit 30 minutes.

12. Wash the plate 3 times, add HRP substrate and read OD at 454 nm.

1. ELISA assay prototype

Applicants also developed a 96 well ELISA assay for detection of neutralizing antibodies in saliva. This ELISA was developed in using a neutralizing IgY antibody to RBD. The IgY bind strongly to plated RBD. The principle of the assay was to measure the binding of this IgY in the presence or absence of saliva which would be the source of competing antibodies. 96 well ELISA offers the advantage of being able to screen multiple samples and is well suited for a hospital or pathology lab setting. ELISA assay developed herein is along the principle shown in Figure 8. Purified RBD (antigen) will be coated on the ELISA plate and then sample (saliva) mixed with neutralizing IgY will be added. The plate is then washed HRP couple anti-IgY antibody will be added followed by color detection by adding HRP substrate. This is a competition ELISA, if the human saliva has neutralizing antibodies, it will compete with anti-RBD IgY and reduce their binding.

2. Neutralizing anti-RBD IgY interacts with RBD

Applicants first demonstrated that the anti-RBD protein IgY from immunized chicken eggs binds to RBD (which will then lead to neutralization). As shown in Figure 9, addition of anti-RBD IgY to RBD coated wells increased linearly with increasing concentrations (0 to 4 pg/ml) with R2 value of 0.94.

Applicants next compared the binding of neutralizing IgY to native RBD and delta RBD. As shown in Figure 10, the anti-RBD IgY bound to both RBD and interestingly bound more to delta RBD as evidenced by higher color formation (measured as absorbance at OD450 nm) of HRP labelled anti-IgY RBD.

3. Saliva samples compete with neutralizing IgY Since the anti-RBD IgY are neutralizing antibodies to be used in the ELISA assay to compete with saliva antibodies, its ability to compete with RBD in saliva samples was further characterized. As shown in the results, the binding of anti-RBD IgY with either native or delta RBD in the presence or absence of saliva from vaccinated individual (KM) (Figures 10, 11) was examined. Saliva samples were able to neutralize the binding of IgY to either native RBD or Delta RBD similarly as in the absence of saliva suggesting that the ELISA can be used to detect neutralizing antibodies against native or delta RBD in saliva suggesting it can be a diagnostic assay test.

Example 3.

LFA Format assay Examples

Applicants also developed an assay that can be converted for typical home use Lateral Flow Assay. The assay will be designed as shown below (Figure 13). The components of LFA are

1. Sample dock where the saliva sample is applied

2. Conjugate release pad where gold labelled or biotinylated RBD is deposited

3. Sample and test line nitrocellulose strip where ACE2 (Test line) and absorbent pad to drain excess buffer

4. Saliva sample is added and due to lateral flow moves to conjugate pad to bind to biotinylated RBD and then onto ACE2 in test line. If there are antibodies to RBD in saliva then less biotinylated RBD will bind to ACE2 and give less color line.

Using the above LFA format applicants used saliva samples to detect presence of neutralizing antibodies: those that will prevent the binding of biotinylated RBD to ACE2 deposited on the Test line. The study was performed in two strips, one for control sample (biotinylated RBD only) and a second strip where anti-RBD IgY was added. Shown below are examples to support that saliva can also be used in a LFA format. The top LFA strip contains a control sample (no saliva added but only buffer) and the lower strip where anti-RBD IgY was added and neutralized the binding (Figure 14). Similar neutralizing activity was obtained using has saliva sample from a vaccinated individual (after 2 shots and 2 months) was able to reduce the line intensity suggesting the presence of neutralizing antibodies. These data suggest that saliva sample based LFA can be developed as a single at home use diagnostic.

Claims

CLAIMS We Claim:

1. A method of detecting the presence of antibodies against COVID 19 virus in a subject, the said method comprising detection of neutralizing antibodies against SarsCov2 spike protein RBD domain in a test sample of the said subject and wherein the presence of COVID-19 RBD antibody is indication of presence of neutralizing antibody to COVID- 19 in the said subject.

2. The method of detecting the presence of antibodies against COVID 19 virus as claimed in claim 1 wherein the test sample is selected from saliva, blood, serum, sweat, body fluids or a mixture thereof.

3. The method of detecting the presence of antibodies against COVID 19 virus as claimed in claim 1 wherein the test sample is saliva.

4. A method of detecting the presence of antibodies against COVID 19 virus in a subject, the said method comprises detection of antibodies by 1) dot blot assay, 2) competition ELISA, or 3) LFA assay.

5. A method of detecting neutralizing antibodies against delta strain of COVID 19 virus said method comprising detection of anti-RBD antibodies in a test sample of the said subject and wherein the presence of COVID-19 RBD antibody is indication of presence of neutralizing antibody to the delta strain of COVID-19 in the said subject.

6. The method of detecting neutralizing antibodies against delta strain of CO VID 19 virus as claimed in claim 5 wherein the test sample is saliva and the said method comprises detection of antibodies by 1) dot blot assay, 2) competition ELISA, or 3) LFA assay.

7. A method of testing the effectiveness of COVID 19 vaccines in an individual wherein the said method comprises testing of the individual’s saliva for neutralizing antibodies before and after vaccination.

8. A method of testing the need for a booster vaccine dose in an individual wherein the said method comprises testing of the individual’s saliva for the presence of neutralizing antibodies before and after every vaccine dose.

9. A method of testing the presence of recent COVID 19 infection in an individual wherein the said method comprises testing the presence of neutralizing antibodies in the saliva sample of the said individual. A method of testing the presence or absence of immune response to fight the viral infection caused by COVID-19 wherein the said method comprises testing the presence of neutralizing antibodies in the saliva sample of the said individual. The method of testing as claimed in claims 7-10 wherein the said method comprises use of saliva as the test sample and detection of neurtralizing antibodies against

SarsCov2 spike protein RBD domain by 1) dot blot assay, 2) competition ELISA, or 3) LFA assay.