DESC:FIELD OF THE INVENTION
The invention relates to a method for rapid detection of antibodies against SARS-CoV-2 in a biological sample. More specifically, the method involves novel recombinant nucleospike fusion protein for rapid detection of antibodies against SARS-CoV-2 in a biological sample by bio-layer interferometry assay.
BACKGROUND OF THE INVENTION
Coronaviruses (CoVs) are a group of highly diverse, enveloped, positive-sense, and single-stranded RNA viruses. They are known to cause diseases of respiratory, enteric, hepatic, and neurological systems in both humans and animals. Coronaviruses are zoonotic, i.e. can be transmitted between animals and people.
The novel coronavirus designated as severe acute respiratory syndrome-Coronavirus-2 (SARS-Cov-2), has caused the worst pandemic of 21st century so far affecting 213 countries and territories around the world. Pneumonia of unknown cause detected in Wuhan, China was first reported to the WHO Country Office in China on 31 December 2019; and by the 30th of January, WHO had declared SARS-2-CoV, the virus that causes the disease Covid-19, a public health emergency of international concern. As of May 2021, 167,492,769 cases and 3,482,907deaths have been reported worldwide. In India, more than 27,367,935 cases and 315,263 deaths have been reported so far. The severity of the spread is basically due to person-to-person transmission from infected individuals with no or mild symptoms.
The basic structure of SARS-CoV-2 is similar to any other coronavirus comprising of spike glycoprotein (S), membrane protein (M), envelope proteinand nucleocapsid protein (N). SARS-CoV-2 spikes are composed of trimmers of spike glycoprotein. The spike glycoprotein has two functional domains: S1 and S2. S1 domain is responsible for the binding with its receptor angiotensin-converting enzyme 2 (ACE2) on host cells. S2 domain is the transmembrane subunit that facilitates viral and cellular membrane fusion for entry into host cell. Therefore, the SARS-CoV-2 uses its spike glycoprotein for entry into host cells by binding to ACE2 receptor. The spike glycoprotein is the main antigenic component in the SARS-CoV which is an important target of host defense system for producing antibodies, and neutralizing the virus. Hence, the spike glycoprotein is a common target for vaccine and therapeutic development. The nucleocapsid protein is the most abundant protein in SARS-CoV-2 and is required for coronavirus RNA synthesis, and has RNA chaperone activity that may be involved in template switch. It is a highly immunogenic phosphoprotein and rarely changes/mutates. The nucleocapsid protein of SARS-CoV-2 is often used as a marker in diagnostic assays.
Reverse transcriptase polymerase chain reaction (RT-PCR) is one of the best ways to detect presence of viral nucleic acid in biological samples to detect and diagnose infections by RNA viruses including SARS-CoV-2. Quantitative RT–PCR (qRT–PCR) enables high throughput and reliable analysis. The test can be done on respiratory samples obtained by various methods, including a nasopharyngeal swab or sputum sample, and on saliva. Testing using qRT–PCR approaches for detection of the SARS-CoV-2 usually takes only 4–6?hours. However, the main limitation is, the typical turnaround time for screening and diagnosing patients with suspected SARS-CoV-2 being >24?h including the time needed to ship samples overnight to reference laboratories. Further, it takes too much time, energy, and many trained personnel to run the tests. Another major limitation of this method is its dependency on the location from which the sample is collected from the patients’ body. It is seen that in the cases of SARS-CoV-2 infection, in the first week of the infection the virus is detected in the upper respiratory tract, saliva etc. However, after one week, the virus may disappear from the upper tract and reach lungs. This disappearance can result in negative results if the sample is collected from upper respiratory tract, though the patient is still infected and is spreading the disease. In such cases further confirmatory tests are required.
Another diagnostic approach is the antigen testing, wherein the presence of viral antigen in the biological samples (nasal swabs, saliva, blood etc.) is tested in a quantitative, semi-quantitative, or qualitative assay. The antigen testing requires monoclonal antibodies specific for the viral antigens. An assay using a combination of different monoclonal antibodies against more than one viral antigen may result in more effective diagnosis. Easy antigen testing assays which can be visualized with naked eye can be developed. However, antigen testing in early stages may be difficult because it requires high viral count to work effectively and the results have poor accuracy, as this testing does not amplify the viral antigen. Moreover, the nasal and throat swabs of asymptomatic people may not have enough antigen particles to be detected as they have very little if any nasal or saliva discharge; even though these asymptomatic people can still spread the virus. Hence if test results are negative, it may require further confirmation by RT-PCR testing.
Another diagnostic approach would be to devise blood tests for antibodies against the SARS-CoV-2 virus, the serological tests. Part of the immune response to infection is the production of antibodies including IgM and IgG antibodies. Dual ELISA tests can be used to detect specific IgM and IgG against the virus in the blood of infected patients. Further, a fluorescent immunoassay system can measure quantitatively or semi-quantitatively the concentration of the IgM/IgG.
ELISA tests can be used to detect specific IgM, IgA and IgG against the virus in the blood of infected patients. However, the quantity of circulating antibodies detectable by these assays will typically require 4-6 days for IgM antibodies, >6 days for IgA antibodies and 5-10 days for IgG post infection. However, the major
limitation in the serological assays for SARS-CoV-2 is that a few infected people develop more antibodies towards viral spike glycoprotein, whereas others develop more antibodies towards nucleocapsid proteins (Geurtsvan Kessel et al., 2020; Long et al., 2020; Okba et al., 2020). Most of the available antibody detection methods rely on the spike protein, hence leaving a void in identification of people who have antibodies against nucleocapsid protein. Further, these tests may not be useful for detection of the infection in the initial stages as IgM in SARS-CoV-2 infection is known to appear and increase in an infected person after few days. Nevertheless, serological testing is one of the fastest and best methods to identify people who were recently infected or exposed, even if they are no longer shedding the virus. Individuals will typically have detectable antibodies for weeks to months after infection regardless of severity, mild or no clinical signs were present. Serological tests can be used in a situation like COVID-19 pandemic for large population testing, enabling identification followed by subsequent isolation of potential virus spreaders. Low-cost, rapid assays upon exit/entry of any person in a location is more reliable than existing thermal assays, especially useful at airports assisting international travel. However, the major limitation in the serological assays for SARS-CoV-2 is that few infected people develop more antibodies towards viral spike glycoprotein, whereas others develop more antibodies towards nucleocapsid proteins. Assays to detect different kinds of antibodies requires different antigen particles together and development of such assays may be expensive.
Taking into account the limitations of the existing prior art, the present invention provides a rapid serological diagnostic method for detecting total antibodies (IgM/IgG/IgA) against SARS-CoV-2.

OBJECT(S) OF THE INVENTION
Accordingly, the present invention takes into account the drawbacks of the prior art and provides an invention with the main object of the invention providing a novel recombinant nucleospike fusion protein for rapid detection of antibodies against SARS-CoV2, a method for rapid detection of antibodies against SARS-CoV-2 in a biological sample, and a diagnostic kit for performing the same.
Another object of the invention is to provide a novel recombinant nucleospike fusion protein to develop biosensors, diagnostic assays, vaccines, therapeutic agents etc. for SARS-CoV-2.
Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 in a biological sample by bilayer interferometry enabling rapid and high throughput screening, and detection within 120 seconds, more specifically detection of antibodies against SARS-CoV-2 within 60 seconds.
Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 using recombinant nucleospike fusion protein which is highly sensitive because it detects total antibodies including IgM, IgG, and IgA against SARS-CoV-2 in biological samples such as serum, saliva, sputum etc.
Yet another object of the invention to provide an in vitro method for rapid detection of antibodies against SARS-CoV-2 using recombinant nucleospike fusion protein enabling population level testing or seroepidemiology of countries, races, etc.

SUMMARY OF THE INVENTION
In one of the embodiments of the present invention, the invention provides a novel recombinant nucleospike fusion protein of SARS-CoV-2 of Seq. ID 1 encoded by DNA of Seq. ID 2, capable of binding to antibodies against nucleocapsid protein, and spike glycoproteins of SARS-CoV-2, which has application in development of biosensors, diagnostics assays, vaccines, and therapeutic compositions. The detection of antibodies against SARS-CoV-2 in a biological sample indicates infection of patient with SARS-CoV-2 from whom the biological sample is derived.
In another embodiment, the invention provides an in vitro method for rapid detection of antibodies against SARS-Cov-2 in a biological sample by biolayer interferometry comprising the steps of
(a) immobilizing a bio-layer interferometry biosensor’ tip with a purified recombinant ligand to detect antibodies against SARS-Cov-2;
(b) washing the biosensor obtained in step (a) to remove excess unbound molecules of recombinant ligand;
(c) exposing the washed biosensor to a biological sample; and
(d) measuring and analyzing biomolecular interactions between the biosensor and analytes of the biological sample by biolayer interferometry method,
wherein,
the recombinant ligand is recombinant nucleospike fusion protein of SARS-CoV-2 represented by Seq. ID 1 which has affinity for antibodies against SARS-CoV-2; and
Seq. ID 1 is encoded by Seq. ID 2.
In another embodiment of the invention, the invention provides a bio-layer interferometry assay for detecting antibodies against SARS-CoV-2 in a biological sample, which is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized ligand/protein on the biosensor tip, and an internal reference layer. When the immobilized ligand on the biosensor tip binds to an analyte for which the ligand has affinity, it causes a shift in the interference pattern. This shift is proportional to number of molecules of ligand bound to analyte, hence more the binding of ligand and analyte, the more is the shift. Therefore, this method is not only qualitative but also quantitative in nature. In the present invention, Seq. ID 1 is immobilized on a biolayer interferometry biosensors’ tip. Seq. ID 1 is a 1405 amino acid long fusion protein comprising of spike glycoprotein sequence of SARS-CoV2 from amino acid 1-970, a linker sequence from amino acids 971-974, nucleocapsid protein sequence SARS-CoV2 from amino acid 975-1392; and a His-tag tail sequence from amino acids 1393-1405. The biosensors’ tip with immobilized Seq. ID 1 is exposed to a biological sample; if the biological sample has antibodies against SARS-CoV2, the subsequent biomolecular interactions between the Seq. ID 1 on biosensors’ tip and antibodies in the biological sample will result in shift of interference pattern thus enabling in vitro detection of antibodies against SARS-CoV2 in the biological sample. Further, the quantification of the amount of antibodies against SARS-CoV2 by analysis of the shift of interference will enable determination of stage and progression of the infection by SARS-CoV2.
In another embodiment of the present invention, the invention provides an in vitro method which is more sensitive for detection of total antibodies against SARS-CoV2. Seq. ID 1 is fusion of nucleocapsid protein and spike glycoprotein of SARS-CoV2 which enables detection of antibodies against both the proteins. Known serological assays use either one of the proteins to detect one kind of antibodies only which makes the assay less sensitive.
Further, the present invention enables detection of total antibodies against SARS-CoV-2 with high specificity, compared to known tests such as ELISA tests.
Further, the method for detection of antibodies against SARS-CoV-2 in a biological sample using bio-layer interferometry biosensor with immobilized Seq. ID 1 is rapid and can be achieved within 120 seconds, more specifically, the detection of antibodies against SARS-CoV-2 is achieved within 60 seconds. This makes the method useful for high throughput screening which is highly desirable for handling situations of epidemics and pandemics like that of Covid-19.

BRIEF DESCRIPTION OF THE DRAWING(S)
The object of the invention may be understood in more details and more particularly description of the invention briefly summarized above by reference to certain embodiments thereof which are illustrated in the appended drawings, which drawings form a part of this specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective equivalent embodiments.
Figure 1A is a schematic elucidation of basic principle of bio-layer interferometry assay and its analysis;
Figure 1B is a schematic elucidation of analysis of bio-layer interferometry sensogram using biosensor with ligands at different stages such as equilibration, ligand binding, washing, and analyte binding;
Figure 2 is a graphical representation of binding rates of ligands - nucleocapsid ligand (N), S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch, Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+S1), S1 and part of S2 subunit of spike protein (S-16-986), and Seq. ID 1 (rNS), with analytes (total antibodies against SARS-CoV-2) in bio-layer interferometry assay;
Figure 3A is a bio-layer interferometry sensorogram depicting ligand-analyte binding rates of bio-layer interferometry biosensor immobilized with Seq. ID 1 (ligand) with antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum sample with dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times;
Figure 3B is a graphical representation of ligand-analyte binding rates of bio-layer interferometry biosensor immobilized with Seq. ID 1 (ligand) and antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum sample with dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times;
Figure 4 is a bio-layer interferometry sensorogram depicting real-time ligand-analyte binding of bio-layer interferometry biosensor immobilized with Seq. ID 1 (ligand) and antibodies (analyte) against SARS-CoV-2 in SARS-CoV-2 RT-PCR +ve serum samples and RT-PCR –ve serum samples;
Figure 5A is a graphical representation comparing ligand-analyte binding rates of Seq. ID 1 (BLI-Seq. ID 1) using bio-layer interferometry assay to detect total antibodies against SARS-CoV2, and binding rates of ligand-analyte in commercially available kits of ELISA for IgG antibodies, and that of IgM antibodies against Spike S1-RBD of SARS-CoV-2 in RT-PCR +ve and RT-PCR –ve plasma samples;
Figure 5B is a graphical representation of receiver operating characteristic curves (ROC) area showing relation between sensitivity and specificity of each ligand-analyte binding assay - BLI-Seq. ID 1, ELISA IgG (Spike S1-RBD), and ELISA IgM (Spike S1-RBD), where BLI-Seq. ID 1 binding rates showed 1.000, confidence interval in between 1.000 to 1.000, and p values <0.0001, ELISA - IgG showed only 0.9023 and confidence interval in between 0.8100 to 0.9945, and ELISA - IgM showed only 0.9070 and confidence interval in between 0.8243 to 0.9898; Line indicates cut off point;
Figure 6A is a graphical representation comparing ligand-analyte binding rates of BLI-Seq. ID 1 to detect total antibodies, and ELISA assay using gamma irradiated whole viral particle of SARS-CoV-2 as antigen to detect IgG antibodies in RT-PCR +ve and RT-PCR –ve plasma samples;
Figure 6B is a graphical representation of correlation of binding rates of - BLI-Seq. ID 1, and ELISA IgG (whole viral particle) for each sample, where both assays showed a good correlation at R2=0.9581, Person r=0.9788 and p value <0.0001;
Figure 7A is graphical representation showing binding rates rNS- Seq. ID 1 by bio-layer interferometry assay using 40 plasma samples collected before COVID-19 pandemic in India (Nov 2019) and 3 plasma samples (RT-PCR +ve) collected after COVID-19 pandemic;

Figure 7B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity for discrimination of RT-PCR positive and plasma samples collected before COVID-19 pandemic showing 100% specificity (ROC area under the curve: 1.000, 95% confidence interval in between 1.000 to 1.000 and p values 0.004249);

Figure 8A is graphical representation showing binding rates rNS- Seq. ID 1 by bio-layer interferometry assay using 850 plasma samples collected from a community who were never diagnosed for SARS-CoV-2 (no COVID-19 symptoms) and 122 plasma samples collected from people diagnosed for SARS-CoV-2 using RT-PCR;

Figure 8B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity showing curve area of 0.9818, at 95% confidence interval in between 0.9663 to 0.9973 and p <0.0001.
Figure 9A depicts a binding curve (bio-layer interferometry sensogram) representing detection of total antibodies against SARS-CoV-2 in 20 µl blood sample collected from patients diagnosed for SARS-CoV-2 (2 RT-PCR +ve) and healthy volunteers (5 RT-PCR –ve);
Figure 9B depicts a binding curve (bio-layer interferometry sensogram) representing detection of total antibodies against SARS-CoV-2 in 10 µl plasma sample collected from the same patients diagnosed for SARS-CoV-2 (2 RT-PCR +ve) and same healthy volunteers (5 RT-PCR –ve); and
Figure 9C is a graphical representation comparing binding rates of ligand-analyte using bio-layer interferometry assay with RT-PCR +ve blood and plasma samples using the following ligands- nucleocapsid ligand (N), S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch, Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+S1), and Seq. ID 1 (rNS).

DETAILED DESCRIPTION OF THE INVENTION
The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.

In one of the embodiment of the invention, the invention provides a novel recombinant nucleospike fusion protein of SARS-CoV-2 of amino acid sequence of Seq. ID 1.The invention also provides the nucleotide sequence of Seq. ID 2 as which encodes Seq. ID 1.
Seq. ID 1 is a 1405 amino acid long fusion protein comprising of spike glycoprotein sequence from amino acid 1-970, a linker sequence from amino acids 971-974, nucleocapsid protein sequence from amino acid 975-1392; and a His-tag tail sequence from amino acids 1393-1405. The spike glycoprotein sequence in the Seq. ID 1 corresponds to amino acids 16-985 of Chain A spike glycoprotein of SARS-CoV-2 (NCBI accession no: QHD43416.1) corresponding to subunit 1 and part of subunit 2 of spike protein; and nucleocapsid protein sequence in Seq. ID 1 corresponds to amino acids 2-419 of nucleocapsid protein of SARS-CoV-2 (NCBI accession no: QHD43417.1). C-terminal His-tag of recombinant nucleospike fusion protein enables purification, and immobilization onto affinity based biosensors, chips etc.
In another embodiment of the invention, the invention provides application of Seq. ID 1 to detect SARS-CoV-2 infection in a patient. Seq. ID 1 comprises of highly immunogenic domains of spike glycoprotein and nucleocapsid protein of SARS-CoV-2. Hence, Seq. ID 1 is capable to bind to total antibodies produced against SARS-CoV-2, thus enabling indirect detection of SARS-CoV-2 infection in patients from whom the biological samples were obtained.
Systemic immune response to infection results in the production of antibodies including IgM, IgA and IgG antibodies. ELISA tests can be used to detect only specific IgM, IgA and IgG against the virus in the blood of infected patients. However, the quantity of circulating antibodies detectable by these assays will typically require 4-6 days for IgM antibodies, >6 days for IgA antibodies and 5-10 days for IgG post infection.
Taking into account the limitations of the existing diagnosis methods, the invention provides an in vitro rapid antibody detection method based on bio-layer interferometry total antibody assay. BLI-TAA is a label-free optical analytical technology for measuring biomolecular interactions by analyzing the interference pattern of white light reflected from two surfaces. The binding between a ligand immobilized on the biosensor and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in absorption properties with a wavelength shift, which is a direct measure of the change in thickness of the biological layer.
Accordingly, in another embodiment of the invention, the invention provides an in vitro method for rapid detection of total antibodies against SARS-CoV-2 in a biological sample of SARS-CoV-2 of Seq. ID 1 by biolayer interferometry. The sensitivity of the biolayer interferometry assay using Seq. ID 1 as ligand is highly enhanced as Seq. ID 1 acts as antigen which would be having affinity to all kinds of antibodies IgG, IgM, and IgA, and the assay does not only bind to IgG or IgM alone antibodies as in the case of ELISA tests. In the present assay, virus-specific ligand - Seq. ID 1 is immobilized on the sensor tip. When the biosensor with Seq. ID 1 comes in contact with a patient’s plasma or whole blood containing the antibodies against SARS-CoV-2 (analyte), a shift in wavelength is observed indicating presence of antibodies against SARS-CoV-2. This testing method takes less than a minute to detect the presence of SARS-CoV-2 antibodies during and after infection. If there is no shift in wavelength it indicates absence of SARS-CoV-2 antibodies, hence no infection by SARS-CoV-2.
EXAMPLE 1
LIGANDS USED FOR RAPID DETECTION OF TOTAL ANTIBODIES AGAINST SARS-CoV-2 IN A BIOLOGICAL SAMPLE
The present invention relates to a novel recombinant nucleospike fusion protein of SARS-CoV-2 represented by Seq. ID 1 which has affinity for antibodies against SARS-CoV-2 and its use in rapid detection of total antibodies against SARS-CoV-2 by bio-layer interferometry assay. To analyze the efficiency and efficacy of the Seq. ID 1, experiments were conducted comparing Seq. ID 1 ligand (recombinant nucleospike fusion protein-rNS) with other antigens of SARS-CoV-2 viral particles. Ligands corresponding to antigens of SARS-CoV-2 viral particles used for experiments are provided below:
i. nucleocapsid ligand (N) - 2-418 amino acid stretch of NCBI accession no: QHD43417.1,
ii. S1 subunit spike protein ligand of (S1) - 16-681 amino acid stretch of NCBI accession no: QHD43416.1,
iii. Equimolar mixture of nucleocapsid ligand and S1 subunit spike protein ligand in 1:1 ratio (N+S1),
iv. S1 and part of S2 subunit of spike protein (S-16-986) - 16-986 amino acid stretch of NCBI accession no: QHD43416.1, and
v. Seq. ID 1 (rNS) – containing16-985 amino acid stretch of spike glycoprotein of NCBI accession no: QHD43416.1 corresponding to subunit 1 and part of subunit 2 of spike protein; and 2-419 amino acid stretch of nucleocapsid protein of NCBI accession no: QHD43417.1.
Spike S1 subunit of SARS-CoV-2 contains a receptor-binding domain that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Spike protein plays an important role in the induction of neutralizing-antibodies and T-cell responses, as well as protective immunity. Hence, S1 subunit (S1) corresponding to 16-681 amino acid stretch of NCBI accession no. QHD43416 was used for experimental studies as a purified ligand.
The S2 subunit (686–1273 residues) of spike protein plays a key role in mediating virus–cell fusion and its integration into host cells. A portion of S2 subunit 682-985 amino acid stretch was also a part of the rNS of Seq. ID 1, hence, the ligand S (16-986) corresponding to 16-986 amino acid stretch with S1 subunit and part of S2 subunit was used for experimental studies.
Nucleocapsid protein of SARS-CoV-2 is also known to act as an antigen efficient in eliciting a serological response in COVID-19 positive patients. A study that used ELISA to measure only antibodies to the nucleocapsid protein found that patients become seropositive 10–18 days after the onset of symptoms (Guo et. al., 2020). Another study showed that nucleocapsid antibodies emerge before spike antibodies (Burbelo et.al., 2020). Hence, Nucleocapsid protein of SARS-CoV-2 (N) corresponding to 2-418 amino acid stretch of NCBI accession no. QHD43417 was used for experimental studies as a purified ligand.
Since it is known that antibodies against both spike and nucleocapsid proteins are generated in a serological response in a patient, S1 subunit (S1) and Nucleocapsid protein (N) of SARS-CoV-2 were purified and mixed in a ratio of 1:1 and the mixture (N+S1) was used for experimental studies as a purified ligands.
Seq. ID 1 ligand, rNS, consisting of spike protein ranging from 16-986 amino acids (which includes Full length S1 (670 amino acids)subunit and Part of S2 subunit (300 amino acids)S2 Heptad region-1) of SARS-CoV-2, and Nucleocapsid protein (418 amino acids) inframe to S2 domain with Serine Glycine linker (SGSG).
EXAMPLE 2
PREPARATION OF BIO-LAYER INTERFEROMETRY BIOSENSORS WITH SEQ. ID 1 AND OTHER LIGANDS
A. Cloning of DNA sequences encoding ligands
DNA encoding S1 subunit (S1) of spike protein corresponding to 16-681 amino acid stretch of NCBI accession no. QHD43416 was cloned in into pcDNA3.1 (+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
DNA encoding S1 subunit and part of S2 subunit (S (16-986)) of spike protein corresponding to 16-986 amino acid stretch of NCBI accession no. QHD43416 was cloned in into pcDNA3.1 (+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
DNA encoding Nucleocapsid protein (N) corresponding to 2-418 NCBI accession no. QHD43417 was cloned in pET28a(+), and pcDNA3.1(+) with a secretory signal nucleotide sequence at the 5’ terminal C-terminal His-tag.
Seq. ID 2 encoding Seq. ID 1, recombinant nucleospike fusion protein (rNS), was be cloned in into pcDNA3.1(+) in between NheI and XhoI restriction sites with a secretory signal nucleotide sequence at the 5’ terminal, hence Seq. ID 1 is expressed with an N-terminal secretory signal peptide of sequence MGVKVLFALICIAVAEA which enables secretion of Seq. ID 1 outside host expressing cells, and C-terminal His-tag. This enables easy purification of Seq. ID 1 from the culturing media and purification.
B. Cell lines and culture:
FS-293 and CHO-S mammalian cells were used for expression of DNA vectors for ligands – S1, S (16-986), N, and rNS. FS-293 cells were maintained in chemically defined FS-293 expression media and CHO-S maintained in chemically defined FS-CHO media respectively.
C. Transfection and Affinity purification:
For transfecting 293/CHO-S cells in 50 ml of media at density of 1X106 cell/ml of growth medium, on the day of transfection, complex mixture was prepared at DNA to Polyethyleneimine (PEI) max ratio of 3:1 diluted in OptiMEM media (Invitrogen, USA) with careful mixing by pipetting (15 times) followed by incubation for 10 minutes at room temperature. Secreted spike protein (S), nucleocapsid protein (N), and recombinant nucleospike fusion protein of Seq. ID 1 (rNS) were purified from filtered cell supernatants using passed over NiNTA resin in phosphate buffered solution (PBS) pH 7.5 10mM Imidazole as binding buffer and PBS pH 7.5 200 mM Imidazole as elution buffer.
D. Immobilization of Ligands on Anti-His Biosensor
The bio-layer interferometry biosensor used for this purpose has anti-His antibodies on its tip. His-tag of purified S1, S (16-986), N, and rNS of Seq. ID 1 were used for binding of Seq. ID 1 to the biosensors’ tip by antibody-antigen affinity. Purified S1 and N ligands were mixed in 1:1 ratio and used as ligand mixture for binding on to the biosensors’ tip.
C-terminal His tag of ligands were immobilized on bio-layer interferometry biosensor using inline protocol according manufactured instructions and then washed with PBS buffer. After removal of the excess unbound ligands from the biosensor, the biosensor was ready to be used for analysis of detection and quantification of SARS-CoV-2 in a biological sample.
A biological sample can be blood sample, serum sample taken from blood, sputum, saliva swab, nasal swab, throat swab etc. derived from patients diagnosed of SARS-CoV-2 infection by RT-PCR (RT-PCR +ve) and uninfected controls confirmed by RT-PCR tests (RT-PCR -ve).
EXAMPLE 3
METHOD OF PERFORMING BIO-LAYER INTERFEROMETRY ASSAY
A. General principle for bio-layer interferometry assay
As described in Figure 1A, the basic principle of bio-layer interferometry assay is based on affinity of ligand on the tip of biosensor and analyte in the biological sample. A biosensor with only immobilized ligand will display a certain interference pattern for white light reflected from two surfaces: a layer of immobilized ligand on the biosensor tip, and an internal reference layer. However, when the ligand is bound to an analyte (biomolecular interactions between ligand and analyte), it would increase the thickness of the biosensors’ tip which results in shift of interference pattern for white light. This shift of interference pattern is analyzed as presence of analyte in the biological sample; and the extent of shift of interference pattern determines the quantity of analyte present in the biological sample. The interference pattern of white light by the biosensor is taken in real time.
For analyzing a biological sample for antibodies against SARS-CoV-2, the bio-layer interferometry biosensor with anti-His was used for immobilization of C-terminal His-tag ligands – S1, S (16-986), N, N+S1, and rNS of Seq. ID 1, and the bio-layer interferometry biosensor with immobilized ligands was exposed to the biological sample. If any antibodies against SARS-CoV-2 in the biological sample bind to the ligand on the biosensor tip it would result in a shift of interference pattern of white light.
B. Method for analysis of biological sample for antibodies against SARS CoV-2
Biological samples such as plasma or whole blood were obtained from infected patients who had tested positive for SAR-CoV2 by RT-PCR (RT-PCR +ve) and uninfected patients had tested negative for SAR-CoV2 by RT-PCR (RT-PCR -ve).
i. Sample collections:
All blood samples were collected by health professionals by abiding to human ethical approvals by Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, TS, India. Blood was collected from RT-PCR +ve patient and healthy volunteers (RT-PCR -ve) in I3-EDTA vacutainers according to manufacturer protocol. Plasma was separated by centrifugation at 3000 gX for 10 minutes. Clear plasma was collected and stored at -80 °C until further process.
ii. Method for Analysis
The method for analysis is as follows:
1. hydrating anti-His bio-layer interferometry biosensor in PBS for 30 seconds;
2. dipping of anti-His bio-layer interferometry biosensor in a preparation of NiNTA purified C-terminal His-tagged ligand protein for immobilizing the ligand on the tip of the biosensor for 1 minute;
3. washing the biosensor obtained in step 1 with PBS for 30 seconds to remove excess unbound ligand;
4. allowing ligand-analyte binding by dipping biosensor with immobilized ligand into a biological sample for 120 seconds; and
5. acquiring data of interference pattern (biolayer interferometry sensograms) of white light by biosensor in real-time during steps 1,2, 3 and 4, and analyzing the data.
The ligands used for conducting the assay were one of the following: S1, S (16-986), N, N+ S1 in 1:1 ratio, or rNS of Seq. ID 1.

The real-time data of interference pattern for all the steps of the method (steps 1-4) is provided within 300 seconds (6 mins) as depicted in Figure 1B. Whereas, once the biosensors are ready for use, the real-time data of interference pattern to detect antibodies against SARS-CoV-2 in a biological sample is provided within 120 seconds. Shift in interference pattern due to presence of antibodies against SARS-CoV-2 is detected within 60 seconds.

C. Comparison of binding rates of ligand with antibodies in biological sample
The binding efficiency of each ligand S1, S (16-986), N, the combination N+S1, and rNS of Seq. ID 1 were compared. As depicted in Figure 2, S1 ligand had the lowest binding rate, the longer spike ligand S (16-986) showed around1.5 fold better binding rate than S1, and N ligand showed around 2 fold better binding efficiency compared to S1 ligand alone. The combination of N and S1 ligands in equimolar ratio 1:1 (N+S1) showed significant increase in binding rate of around 3.25 fold increase in detection of antibodies in SARS-CoV-2 positive plasma compared to S1 ligand alone, and around 1.62 fold better efficiency than N ligand alone. A complete additive effect of combination of S ligand and N ligand was not achieved. However, rNS of Seq. ID 1 showed around 4.5 fold better efficiency than S ligand, only 3 fold better efficiency than S (16-986), 2.25 better efficiency than N ligand alone, and around 1.4 better efficiency than the equimolar combination N+S1.
This clearly showed that rNS of Seq. ID 1 has better sensitivity than spike protein (S1 or S-(16-986)), nucleocapsid protein, or even equimolar combination N+S1.

The commercial kits which are available for serological tests for SAR-CoV2 generally comprise of either spike ligands or nucleocapsid ligands. It is clear from the above experiment that these ligands alone are not sensitive enough and may easily provide false negative results for detecting antibodies against SAR-CoV2. Even though simple combination of mixing nucleocapsid protein and spike protein showed better binding rate efficiency but still does not provide a good additive effect. Whereas, the recombinant nucleospike (rNS) of Seq. ID 1showed significantly better binding rate efficiency than nucleocapsid protein and spike protein combination. This showed the improved efficacy of the recombinant Seq. ID 1.

D. Determination of optimal dilution of biological sample for Bio-layer Interferometry assay
The optimal dilution of biological sample for conducting bio-layer Interferometry assay using rNS of Seq. ID 1 was determined.
Plasma obtained from RT-PCR +ve patient was diluted using PBS in range of 10X to 200X and tested for ligand-analyte binding rates of bio-layer interferometry biosensor with rNS of Seq. ID 1 with antibodies (analytes) in diluted plasma sample. Table 1 provides details of ligand-analyte binding rates with different dilutions of RT-PCR +ve serum sample ranging between 10 to 200 times.
Definition: Ligand = Capture protein; Analyte = Plasma
Plasma dilution 1 to 200 1 to 150 1 to 100 1 to 75 1 to 50 1 to 25 1 to 10
Ligand to Analyte Binding rate 0.00 0.02 0.19 0.32 0.58 0.97 1.34
Buffer to Analyte 0.00 0.00 0.00 0.00 0.05 0.13 0.16
Ligand to Buffer 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Subtraction of Binding rate (Ligand to Analyte-Buffer to Analyte)
Plasma dilution 1 to 200 1 to 150 1 to 100 1 to 75 1 to 50 1 to 25 1 to 10
Ligand to Analyte Binding rate 0.00 0.02 0.19 0.32 0.53 0.84 1.18
Standard Deviation (3 samples) 0.00 0.01 0.00 0.00 0.02 0.04 0.10

As depicted in Table 1, and Figure 3A and Figure 3B, a dilution of plasma sample by 10 times (represented as 1 to 10 or 10X) provides sufficient binding efficiency between the biosensor with rNS of Seq. ID 1 (ligand) and the antibodies (analyte) against SARS-CoV-2 in the diluted plasma sample. Higher dilutions show reduced binding constants indicating the reduction in the number of analytes, further confirming that the biosensor with rNS of Seq. ID 1 is capable to detect antibodies (analyte) against SARS-CoV-2 in a diluted plasma sample.

E. Bio-layer interferometry assay using rNS of Seq. ID 1 as ligand to detect total antibodies in biological sample against SARS-CoV-2

Figure 4 provides bio-layer interferometry sensorogram using biosensor with rNS of Seq. ID 1 comparing interference pattern of 20 times diluted plasma sample of RT-PCR +ve patient with 20 times diluted plasma sample of seven negative control patients (RT-PCR -ve).The data of interference pattern is acquired in real-time data during hydration of biosensor depicted in first 30 seconds in the graph, binding of rNS Seq. ID 1 (ligand) for 120 seconds depicted from 30-150 seconds in the graph, washing the biosensor to remove unbound rNS Seq. ID 1 for 30 seconds depicted from 150-180 seconds, and exposing biosensor with diluted plasma sample of patient depicted from 180-300 seconds. The shift in interference pattern is clearly visible in plasma sample of SARS-CoV-2 infected RT-PCR +ve patient compared to all other interference pattern of RT-PCR –ve samples. The data were baseline subtracted prior to fitting performed using a 1:1 binding model and the ForteBio data analysis software. This clearly shows that the assay can easily be used to distinguish between SARS-CoV-2 infected and uninfected samples.

F. Comparison of Bio-layer interferometry assay using rNS of Seq. ID 1 as ligand and commercially available ELISA kits for detection antibodies in biological sample against SARS-CoV-2

Next, the bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand for detecting total antibodies against SARS-CoV-2 was compared with commercially available ELISA kits (SARS-CoV-2 Spike S1-RBD IgG & IgM ELISA Detection Kit from Genscript, China). A total of 17 RT-PCR -Ve and 31 RT-PCR +Ve were analysed for total antibodies by ‘bio-layer interferometry assay using rNS of Seq. ID 1, and IgG & IgM ELISA using Spike S1-RBD. As depicted in Figure 5A, Bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand for detecting total antibodies against SARS-CoV-2detected antibodies in all the 31 RT-PCR +Ve plasma samples, and the assay couldn’t detect any antibodies in RT-PCR -Ve plasma. Hence, there were no false positive or false negative results. Whereas, GenScript ELISA kit detected IgM in only 24 out of 31, and IgG in 29 out of 31 RT-PCR +ve samples thus giving around 25% false negative results in case of IgG and around 6.5% false negative results. GenScript assay also showed positivity in one sample for IgM and in 4 samples for IgG in RT-PCR -Ve plasma, thus giving around 6% false positive in IgM detection, and 23.5% false positive in IgG detection. This shows that Bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand is more sensitive and specific compared to the commercially available Covid-19or SARS-CoV-2 testing tests.
Further, an ROC curve analysis was performed on this data, and as depicted in Figure 5B, it was found that bio-layer interferometry assay using the rNS of Seq. ID 1 as ligand showed 100% specificity and sensitivity, whereas, GenScript kit showed ROC curve area of only 0.9023 for total IgG and confidence interval in between 0.8100 to 0.9945 and ROC curve area showed only of 0.9070 for total IgM and confidence interval in between 0.8243 to 0.9898.

To further test the sensitivity of the bio-layer interferometry assay using the rNS of Seq. ID 1, the assay was compared to a commercially available ELISA kit developed by National Institute of Virology, India using killed SARS-CoV-2 viral particles. Since the ELISA kit was developed using gamma irradiated whole viral particle as antigen, it is supposed to detect antibodies against SARS-CoV-2 in total, i.e antibodies against all antigens, and reported to have 95% specificity and sensitivity against SARS-CoV-2. A total of 486 samples which never tested for SARS-CoV-2 were analyzed by both assays and sensitivity was compared. As depicted in Figure 6A, a total 30 plasma samples were shown positive by ELISA kit and 32 plasma samples by bio-layer interferometry assay. Line indicated cut off point. As depicted in Figure 6B, both ELISA assay and bio-layer interferometry assay total antibody assay showed a good correlation at R2=0.9581, Person r=0.9788 and p value <0.0001. This suggested that bio-layer interferometry has more specificity and sensitivity in detecting antibodies. Further, a total of 4 positive samples analysed by bio-layer interferometry assay using the rNS of Seq. ID 1 differed with said ELISA kit ELISA kit. This difference could be due to the fact that the ELISA kit detects only anti-IgG and not anti-IgM antibodies against SARS-CoV-2. Detection of IgM antibodies is of paramount importance to diagnose infection in early stages. The bio-layer interferometry assay using the rNS of Seq. ID 1kit shows greater sensitivity and specificity in detection of IgM, IgA, and IgG antibodies against SARS-CoV-2 infection.

G. Testing of Specificity and Sensitivity of bio-layer interferometry assay using the rNS of Seq. ID 1
Bio-layer interferometry assay using the rNS of Seq. ID 1 was used for testing different kinds of samples.
Figure 7A depicts a graph representing the binding rate of 40 plasma samples collected beforeCOVID-19 pandemic in India (Nov 2019) and 3 plasma samples (RT-PCR +ve) collected after COVID-19 pandemic. Figure 7B depicts Receiver operating characteristic curve (ROC) analysis showing sensitivity versus specificity for discrimination of RT-PCR positive and plasma samples collected before COVID-19 pandemic showing 100% specificity (ROC area under the curve: 1.000, 95% confidence interval in between 1.000 to 1.000 and p values 0.004249).

A test was conducted with 972 plasma samples of which 850 plasma samples collected from a community who were never diagnosed for SARS-CoV-2 (no COVID-19 symptoms) and 122 plasma samples collected from people diagnosed for SARS-CoV-2 using RT-PCR. These samples were tested using Bio-layer interferometry assay using the rNS of Seq. ID 1 and binding rates and specificity of the assay was tested. As depicted in Figure 8A, zero samples of the 850 plasma samples of persons with no COVID-19 symptoms showed binding rate above =0.1nm which establishes very high specificity. Line indicates cut off point. Moreover, in 5 samples tested for RT-PCR +Ve didn’t show antibody against SARS-CoV-2 beyond the threshold levels. This could be due to early detection of viral infection by RT-PCR. Furthermore, as depicted in Figure 8B in overall specificity and sensitivity by ROC curve analysis demonstrated that curve area of 0.9818, at 95% confidence interval in between 0.9663 to 0.9973 and p <0.0001. This large set of data demonstrated that Bio-layer interferometry assay using the rNS of Seq. ID 1is very sensitive and specific to SARS-CoV-2 infection.
This suggested that the Bio-layer interferometry assay using the rNS of Seq. ID 1 is suitable for rapid detection of total antibodies against SARS-CoV2 at a large scale with high specificity and sensitivity rates enabling population level testing or seroepidemiology of countries, races, etc. Such tests in large communities etc. are usefully to determine herd immunity etc.
A common and important precaution taken by many countries today to curb spread of COVID-19 disease is prohibiting entry of people at country level, state level, or even district level. Conducting an RT-PCR of each person not makes the procedure cumbersome, and expensive, but it is time taking as well because RT-PCR results take 24-48 hrs. Therefore the need of the hour is development of techniques which can provide qualitative and quantitative results in an accurate manner and which are very fast.
The above experiment establishes that the Bio-layer interferometry assay using the rNS of Seq. ID 1provides test results within a total of 300-350 secs once the samples and biosensors are ready. It is useful to screen people at various checkpoints such as airports, railway stations, borders etc. to restrict the disease from entering the location and spread of the disease.
H. Use of bio-layer interferometry assay using the rNS of Seq. ID 1 for directly testing whole blood samples
To reduce the time for preparing of samples, direct blood samples (20µl) of 2SARS-CoV-2 RT-PCR +Ve and 5 RT-PCR -Ve samples were tested by comparing with respective to plasma samples for binding rate using bio-layered interferometry assay using rNS of Seq. ID 1. As depicted in the sensograms of Figure 9A and 9B, it is clearly visible that the sensogram using plasma sample perfectly matched and correlated with that of the blood sample analysis.
Further, efficiency of the bio-layered interferometry to detect total antibodies directly from whole blood samples compared to plasma was tested using ligands, N, S1, N+S1 (Equimolar ratio), and rNS of Seq. ID 1. As depicted in Figure 9C, binding rates of blood samples vs plasma samples of each ligand is almost similar. The data clearly concludes that bio-layered interferometry total antibody assay can performed in whole blood. However, care should be taken to prevent the lysis of RBCs while collecting the sample. This makes the procedure faster and easier, obviating the need of extracting plasma from blood. Further, the data again suggests that rNS of Seq. ID 1 shows highest sensitivity and efficiency than N or S1 alone, or even N+S1 equimolar ligand combination.

While certain exemplary embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of, and not restrictive on, the broad invention, and that this invention not be limited to the specific constructions and arrangements shown and described, since various other changes, combinations, omissions, modifications and substitutions, in addition to those set forth in the above paragraphs, are possible. Those skilled in the art will appreciate that various adaptations and modifications of the just described embodiments can be configured without departing from the scope and spirit of the invention.
,CLAIMS:CLAIMS
We claim:
1. An in vitro method for rapid detection of total antibodies against SARS-CoV-2 in biological sample using biolayer interferometry comprising the steps of:
(e) hydrating a bio-layer interferometry biosensor’ tip with a buffer solution;
(f) immobilizing a purified ligand to detect total antibodies against SARS-CoV-2 in a biological sample on the biosensor’ tip;
(g) washing the biosensor with immobilized purified ligand obtained in step (a) to remove excess unbound molecules of the ligand;
(h) exposing the washed biosensor to a biological sample; and
(i) acquiring data of interference pattern of white light by biosensor of biolayer interferometry assay in real-time during steps a, b, c, and d, where the shift of interference pattern from step c to d is indicative of presence of antibodies against SARS-CoV-2 in a biological sample;
wherein,
the ligand to detect total antibodies against SARS-CoV-2 in a biological sample is recombinant nucleospike fusion protein of Seq. ID 1;
Seq. ID 1 is encoded by Seq. ID 2;
the time taken for detection of antibodies against SARS-CoV-2 in a biological sample is around 300 seconds; and
measuring and analyzing shift in interference pattern of biosensor immobilized with Seq. ID 1 due to presence of antibodies against SARS-CoV-2 in a biological sample is achieved within 60 seconds.

2. The method as claimed in claim 1, wherein, the method detects IgMs, IgG, and IgAs against both nucleocapsid proteins and spike protein of SARS-CoV-2, making the method highly sensitive.

3. A novel recombinant nucleospike fusion protein capable of binding to antibodies against of SARS-CoV-2,
wherein,
the recombinant nucleospike fusion protein is represented by Seq. ID 1 of 1405 amino acid length consisting a spike glycoprotein fragment from amino acid 1-970, a linker sequence from amino acids 971-974, a nucleocapsid protein fragment from amino acid 975-1392, and a His-tag tail sequence from amino acids 1393-1405;
and
Seq. ID 1 is encoded by DNA of Seq. ID 2.

4. The novel recombinant nucleospike fusion protein as claimed in claim 3, wherein, the novel recombinant nucleospike fusion protein has application in development of biosensors, diagnostics assays, vaccines, and therapeutic compositions.