Rapid Assay Methods and Kits for Detecting Neutralizing Antibody for Sars-Cov-2 Using Lateral Flow Assay and Enzyme-linked Immunosorbent Assay
20230194529 · 2023-06-22
Assignee
Inventors
Cpc classification
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
International classification
G01N33/543
PHYSICS
Abstract
A novel assay which can differentiate a neutralizing antibody from non-neutralizing antibody which can be easily visualized, for example, by a portable UV lamp, among other visualization techniques. This assay can produce results in about 30 minutes and can be performed by untrained individuals in a non-laboratory environment. Also described is an ELISA method for determining if a human possesses at least one type of neutralizing antibody against SARS-Cov-2.
Claims
1. The method for determining if a human possesses at least one type of neutralizing antibody against SARS-Cov-2 as claimed in claim 2, wherein steps a)-f) are performed sequentially in order as set forth.
2. A lateral flow assay method for determining if a human possesses at least one type of neutralizing antibody against SARS-Cov-2, comprising steps of: a) incubating a body fluid with at least one chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 or a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase for a period of time to form Mixture 1; b) adding an additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment to the Mixture 1 formed in step (a) and incubating for a period of time to form Mixture 2; c) contacting the Mixture 2 with anti-mIgG; d) visualizing whether the anti-mIgG produces a visible signal emitted by the horseradish peroxidase or a visualizable tag attached to the at least one chimeric protein comprising the receptor binding domain of the variant of SARS-Cov 2; and f) if the anti-mIgG produces a visible signal, concluding that the body fluid does not contain at least one type of neutralizing antibody against SARS-Cov-2.
3. (canceled)
4. The method of claim 2, further comprising a steps of adding rabbit IgG attached to a visualizable tag to the Mixture 2 prior to step (c), and contacting the Mixture 2 from step (d) with anti-rIgG and subsequently determining the anti-rIgG produces a signal in order to indicate proper operation of the method and use of a sufficient amount of sample in the method.
5. The method of claim 2, in which steps (c) and (d) are carried out sequentially with step (c) performed prior to step (d).
6. The method of claim 2, in which steps (c) and (d) are carried out simultaneously.
7. The method of claim 2, wherein the body fluid is whole blood or serum; the period of time in step (a) is 10-15 minutes; the period of time in step (b) is 10-15 minutes; and the receptor binding domain of the chimeric protein is conjugated to horseradish peroxidase.
8. The method of claim 2, wherein the body fluid is whole blood or serum; and the period of time in step (a) is 10-15 minutes; the period of time in step (b) is 10-15 minutes.
9. The method of claim 4, wherein the rabbit IgG has SEQ ID NO: 5 and the visualizable tag attached to the rabbit IgG is nanogold particles.
10. A kit for use in a method for determining if a human has at least one type of neutralizing antibody against SARS-Cov-2 comprising: a. a container containing at least one chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 or a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase; b. an additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment; c. a unit comprising the following elements laid out in the following successive order: (i) a sample pad onto which the at least one chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 or a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase, and a body fluid is loaded; (ii) a conjugate pad onto which the additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment is loaded; (iii) a test pad in contact with the sample pad and including a test line in which anti-mIgG is immobilized; (iv) an optional absorption pad in contact with the test pad to facilitate flow of liquid from the sample pad through the test pad; and (v) an optional backing card onto which elements (i)-(iv) are mounted; and d. instructions for use of the kit for determining if a human has at least one type of neutralizing antibody against SARS-Cov-2.
11. The kit of claim 10, further comprising rabbit IgG attached to a visualizable tag for loading onto the test pad, and an anti-rIgG control line on the test pad downstream of the test line.
12. The kit of claim 10, wherein the receptor binding domain of the at least one chimeric protein is attached to gold nanoparticles and the visualizable tag attached to the rabbit IgG is gold nanoparticles.
13-15. (canceled)
16. An ELISA method for determining if a human possesses at least one type of neutralizing antibody against SARS-Cov-2, comprising steps of: a) incubating a body fluid with at least one chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase for a period of time to form Mixture 1; b) adding Mixture 1 to a plate coated with an additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment; c) capturing unbound chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase on the plate, and removing chimeric protein comprising a receptor binding domain of a variant of SARS-Cov-2 conjugated with horseradish peroxidase that is bound to neutralizing antibody by washing with phosphate buffer; and d) visualizing horseradish peroxidase activity by adding 10-Acetyl-3,7-dihyroxyphenoxazine; e) if no signal indicative of horseradish peroxidase activity is visible, concluding that the body fluid contains at least one type of neutralizing antibody against SARS-Cov 2; and f) if a signal indicative of horseradish peroxidase activity is visible, concluding that the body fluid does not contain at least one type of neutralizing antibody against SARS-Cov 2.
17. The method of claim 2, wherein the receptor binding domain of the at least one chimeric protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
18. The method of claim 17, wherein the comprising ACE2 fused with a mouse immuoglobulin G1 fragment has SEQ ID NO: 4.
19. The method of claim 2, wherein the receptor binding domain of the at least one chimeric protein is conjugated to gold nanoparticles.
20. The kit of claim 10, wherein the receptor binding domain of the at least one chimeric protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
21. The kit of claim 20, wherein the additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment has SEQ ID NO: 4.
22. The kit of claim 11, wherein the rabbit IgG has SEQ ID NO: 5.
23. The ELISA method of claim 16, wherein the receptor binding domain of the at least one chimeric protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and the additional chimeric protein comprising ACE2 fused with a mouse immunoglobulin G1 fragment has SEQ ID NO: 4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0073] In one aspect, the present invention provides a method for rapid detection of neutralizing antibody against SARS-CoV-2, such as neutralizing IgG antibody against SARS-CoV-2. SARS-CoV-2 expresses a unique spike protein that interacts with human angiotensin-converting enzyme 2 (ACE2) for invading human respiratory epithelial cells. Studies have shown that the neutralizing IgG antibody that binds to the receptor binding domain (RBD) of the spike protein that interacts with ACE2 is capable of reducing or neutralizing the virulence of SARS-CoV-2. Currently, multiple variants of SARS-CoV-2 are circulating globally. The United Kingdom (UK) identified a variant called B.1.1.7 carrying a mutation in the spike protein (N501Y) that affects the conformation of RBD. In South Africa (SA), another variant called B.1.351 sharing mutations in B.1.1.7 as well as additional mutation in the spike protein (E484K) that can provide resistance to current Covid-19 vaccine.
[0074] In order to detect the neutralizing antibody that binds to the RBD of the spike protein that inhibit interaction of spike protein with human ACE2, we generated Chimeric proteins in which the variants of RBD spike protein is fused with horse radish peroxidase (HRP). For example, Chimeric Protein 1 (SARS-CoV-2-RBD-GFPuv) is used to bind to this receptor binding domain (RBD) of the spike protein. Chimeric protein 1 includes a marker such as a fused green fluorescence protein (GFPuv) for the visualization of the assay result.
[0075] Chimeric protein 1 includes an original RBD fused with the HRP. Chimeric protein 2 includes the UK variant RBD fused with the HRP, Chimeric protein 3 includes the SA variant RBD fused with the HRP. We generated another Chimeric Protein 3 (ACE2-mIgG1Fc) in which the extracellular domain of the ACE2 is fused with constant regions of mouse immunoglobulin G1 (IgG).
[0076] In one embodiment, the method of the invention is carried out using a lateral flow assay format. In lateral flow assay, body fluids will be pre-incubated the RBD-HRP chimeric proteins, followed by the ACE2-mIgG1Fc. In the presence of neutralizing antibody, the neutralizing antibody will bind to the RBD-HRP, so the entire neutralizing antigen-antibody complex will be captured at the anti-hIgG line (Positive line). In the absence of neutralizing antibody, the RBD-HRP will bind to the ACE2-mIgG1Fc, so the entire protein-protein complex will be captured at the anti-mIgG line (negative line). The horse radish peroxidase activity within RBD-HRP chimeric proteins will be visualized by adding a substrate (ADHP, 10-Acetyl-3,7-dihyroxyphenoxazine) which generate visible colorimetric signal as well as fluorescent signal at 570 nm.
[0077] The amount of RBD-HRP chimeric proteins captured by neutralizing antibody may be empirically determined and compared to a standard to identify whether the neutralizing antibody is present at protective levels.
[0078] For instance, Chimeric protein 1 will bind to the neutralizing IgG antibody, so that the entire antigen-antibody complex will bind to the anti-hIgG (Positive line). The amount of Chimeric protein 1 captured by neutralizing antibody may be empirically determined and compared to a standard to identify whether the neutralizing antibody is present at protective levels.
[0079] Similarly, as another example, Chimeric Protein 2 (ACE2-mIgG1Fc) in which the extracellular domain of ACE2 is fused with constant regions of mouse immunoglobulin G1 (IgG) that will bind to anti-mIgG (negative line) for detection of SARS-CoV-2-RBD-GFPuv complex in human IgG is also used in the assay as an indicator of the absence or substantial absence of the neutralizing antibodies.
[0080] To be sure that the assay is working properly, rabbit IgG conjugate with nanogold particles (AuNP). The rabbit IgG conjugate will bind to the anti-rabbit IgG (control line) to indicate that the assay is working properly and that a sufficient amount of sample has been employed.
[0081] In the other embodiment, the method of the invention is carried out using an ELISA format. In ELISA, body fluids will be pre-incubated the RBD-HRP chimeric proteins to allow the binding of neutralizing antibody to the RBD-HRP. The mixture will be then added to the capture plate on which is pre-coated with the the ACE2-mIgG1Fc. The unbound RBD-HRP will be captured on the plate, while the neutralizing antibody and the RBD-HRP chimeric protein complex will be removed during washing. The horse radish peroxidase activity within RBD-HRP chimeric proteins will be visualized by adding a substrate (ADHP, 10-Acetyl-3,7-dihyroxyphenoxazine) which generate visible colorimetric signal as well as fluorescent signal at 570 nm.
[0082] In one embodiment, the method is conducted using Positive and Negative lines specifically designed to determine if the sample contains a protective level of neutralizing antibody. In this embodiment, when the sample contains a protective level of neutralizing antibody, there will be a significantly stronger visible signal at the Positive line. If the sample does not contain any neutralizing antibody or only a small amount of neutralizing antibody, there will be a significantly stronger visible signal at Negative line. When the sample contains some neutralizing antibody but less than the protective level of neutralizing antibody, there will be strong visible signals at both of the Positive and Negative lines.
[0083] In another aspect, the present invention relates to a kit for carrying out the above described methods of the present invention.
Specific Embodiments
[0084] If a protective level of neutralizing antibody is present in the sample of body fluid, SARS-CoV-2-RBD-HRP chimeric proteins (Chimeric protein 1, 2, and 3) will substantially bind or completely bind with the neutralizing human IgG antibody. This will prevent subsequent binding of ACE2-mIgG1Fc to SARS-CoV-2-RBD-HRP.
[0085] If a protective level of neutralizing antibody is not present in the sample of body fluid, the first incubation of SARS-CoV-2-RBD-HRP chimeric proteins (Chimeric protein 1, 2, and 3) with the body fluid sample will leave some free SARS-CoV-2-RBD-HRP chimeric proteins. This will allow the downstream ACE2-mIgG1Fc (Chimeric Protein 4) to bind to SARS-CoV-2-RBD-HRP chimeric proteins, resulting in a complex of the SARS-CoV-2-RBD-HRP chimeric proteins and ACE2-mIgG1Fc.
[0086] If a protective level of neutralizing antibody is present in the sample of body fluid, SARS-CoV-2-RBD-GFPuv (Chimeric Protein 1) will substantially completely bind or completely bind with the neutralizing human IgG antibody. This will prevent subsequent binding of ACE2-mIgG1Fc to SARS-CoV-2-RBD-GFPuv.
[0087] If a protective level of neutralizing antibody is not present in the sample of body fluid, the first incubation of SARS-CoV-2-RBD-GFPuv (Chimeric Protein 1) with the body fluid sample will leave some free SARS-CoV-2-RBD-GFPuv (Chimeric Protein 1). This will allow the downstream ACE2-mIgG1Fc (Chimeric Protein 2) to bind to SARS-CoV-2-RBD-GFPuv, resulting in a complex of the SARS-CoV-2-RBD-GFPuv and ACE2-mIgG1Fc.
[0088] In one specific embodiment, the novel lateral flow assay kit of the invention may contain the anti-human IgG antibody immobilized (printed) at the Positive line of the assay device and the anti-mouse IgG antibody immobilized (printed) at the Negative line of the assay device. The complex of SARS-CoV-2-RBD-HRP chimeric proteins and human neutralizing IgG will be captured at the Positive line formed by the anti-human IgG antibody. The complex of SARS-CoV-2-RBD-HRP chimeric proteins and ACE2-mIgG1Fc will be captured at the Negative line formed by the anti-mouse IgG antibody. The horse radish peroxidase activity signal produced by the substrate (ADHP) can be visualized as a colorimetric signal or fluorescent signal.
[0089] In another specific embodiment, the novel lateral flow assay kit of the invention may contain the anti-human IgG antibody immobilized (printed) at the Positive line of the assay device and the anti-mouse IgG antibody immobilized (printed) at the Negative line of the assay device. The complex of SARS-CoV-2-RBD-GFPuv and human IgG will be captured at the Positive line formed by the anti-human IgG antibody. The complex of SARS-CoV RBD-GFPuv and ACE2-mIgG1Fc will be captured at the Negative line formed by the anti-mouse IgG antibody. The GFPuv signal produced by the fluorescent marker can be visualized using a UV or wood lamp.
[0090] If samples contain protective levels of neutralizing antibody, there will be a visible signal only at the Positive line. If samples do not contain any neutralizing antibody, there will be visible signal only at Negative line. If samples contain some neutralizing antibody but not a protective level, there will be visible signals at both Positive and Negative lines. When test results show a visible signal only at the Positive line, this corresponds to protection from SARS-COV-2.
[0091] Schematic illustrations of the assay are shown in
[0092] “Neutralizing antibody” as used herein refers to an antibody that specifically binds to the RBD of the spike protein of SARS-COV-2, and defends a cell from SARS-COV-2 by neutralizing the ability of the RBD of the spike protein of SARS-COV-2 to bind to the ACE2 receptor. Neutralization renders SARS-COV-2 non-infectious and non-pathogenic. Neutralizing antibodies are part of the humoral response of the immune system against the virus. Neutralizing antibodies prevent the virus particle from interacting with the ACE2 receptor of host cells thereby preventing infection of the host cells.
[0093] “Body fluids” are liquids originating from inside the body of a living human. Body fluids include fluids that are excreted or secreted from the body. Exemplary body fluids include: [0094] Aqueous humour and vitreous humour [0095] Bile [0096] Blood serum or whole blood [0097] Breast milk [0098] Cerebrospinal fluid [0099] Cerumen (earwax) [0100] Endolymph and perilymph [0101] Female ejaculate [0102] Gastric juice [0103] Mucus (including nasal drainage and phlegm) [0104] Peritoneal fluid [0105] Pleural fluid [0106] Saliva [0107] Sebum (skin oil) [0108] Semen [0109] Sweat [0110] Tears [0111] Vaginal secretion [0112] Vomit [0113] Urine
[0114] Any of the foregoing body fluids can be tested using the assay apparatus of the present invention. However, in most cases the tested body fluid is typically whole blood, saliva, or blood serum.
[0115] The novel LFA apparatus of the present invention comprises the following elements set forth in successive order (see
[0120] The sample pad and test pad can be made from a permeable material, e.g., nitrocellulose, glass fiber, capable of transporting an aqueous solution by capillary action, wicking, or simple wetting. The backing card (the black element in
[0121] Exemplary LFA Protocol
[0122] A body fluid is mixed with the RBD-HRP chimeric proteins (Chimeric Protein 1, 2, and/or 3) in a container (e.g. a plastic well) and incubated for about 10 to 15 minutes to form Mixture 1. Subsequently, Chimeric Protein 4 is added to Mixture 1 and incubated for an additional 10 to 15 minutes to form Mixture 2.
[0123] Mixture 2 and rabbit IgG conjugated with AuNP are applied to the sample pad shown in
[0124] An optional absorption pad can be located downstream of the test pad and the test pad and absorption pad are provided without contacting surfaces so that Mixture 2 can flow from the test pad to the absorption pad. After Mixture 2 passes through the Negative line, it moves to the absorption pad which may be composed of sorbent or a super sorbent material. The purpose of absorption pad is to assure that Mixture 2 is drawn through the Positive and Negative lines. Thus, the absorption pad is preferably sized to ensure that all of Mixture 2 can reach at least the Negative line during the test.
[0125] The Positive and negative lines are printed with a predetermined amount of immobilized goat anti-hIgG and anti-mIgG, respectively. Any suitable conventional immobilization technique may be employed to print or apply the Positive and Negative lines.
[0126] Suitable sorbents which can be used in the absorption pad can include commercial materials of the type available, for example, from The Dow Chemical Company of Midland, Mich., and the Chemical division of American Colloid, Arlington Heights, Ill. These materials can absorb many times their weight in water and are commonly used in disposable diapers. They typically comprise lightly crosslinked polyacrylate salts, typically alkali metal salts.
[0127] The RBD-HRP chimeric protein (Chimeric protein 1, 2, and/or 3) contains a horse radish peroxidase activity which can be visualized by adding a substrate (ADHP) that generates a colorimetric signal or fluorescent signal. Alternatively, the RBD-HRP chimeric protein (Chimeric protein 1, 2, and/or 3) contains a GFPuv label which can be visualized by using a lamp or UV light. When color appears at the Positive line, the presence of neutralizing antibody is indicated, whereas when color appears at the Negative line, the absence of the neutralizing antibody is indicated. A color chart can optionally be provided for aiding in interpretation of the results of the assay.
[0128] The label, tag or marker used in the RBD-HRP chimeric proteins may be, for example, horse peroxidase activity (HRP) or Green Fluorescent Protein UV variant (GFPuv). However, other labels such metal sol, such as colloidal gold, and other types of colored or fluorescent particles known to be useful as marker substances in immunoassay procedures can also be used instead of the HRP or GFPuv. See, for example, U.S. Pat. No. 4,313,734, Feb. 2, 1982, to Leuvering, the disclosure of which is incorporated herein by reference. For details and engineering principles involved in the synthesis of colored particle conjugates see Horisberger, Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and scanning Electron Microscopy, Biol. Cellulaire, 36, 253-258 (1979); Leuvering et al, Sol Particle Immunoassay, J Immunoassay 1 (1), 77-91 (1980), and Frens, Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions, Nature, Physical Science, 241, pp. 20-22 (1973).
[0129] Other assay methods besides LFA methods can also be used to carry out the method of the present invention, including, for example ELISA assays.
Example 1 Making the Chimeric Protein 1
[0130] The original receptor binding domain of the SARS-CoV-2 spike protein gene was amplified by polymerase chain reaction from the plasmid obtained from the University of Georgia. This source plasmid is also available from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources).
[0131] To express the horse radish peroxidase (HRP) in the human cell line, the codon usage of the gene encoding horseradish peroxidase (GenBank: M3715) was optimized by the codon usage frequency table for human (Athey, J., Alexaki, A., Osipova, E. et al. A new and updated resource for codon usage tables. BMC Bioinformatics 18, 391 (2017) (doi.org/10.1186/s12859-017-1793-7). The codon optimized HRP gene was synthesized (Blue Heron) and amplified by polymerase chain reaction. Two gene fragments were joined by overlap extension polymerase chain reaction and cloned into pLenti6 V5 expression plasmid (Invitrogen) for expression in HEK293T cells. The sequence of Chimeric Protein 1 is shown in
Example 2 Making the Chimeric Protein 2
[0132] The gene encoding the UK variant RBD of spike protein was synthesized (Blue Heron) and amplified by polymerase chain reaction. This gene fragment was in-frame fused with the codon optimized horse radish peroxidase gene fragment by overlap extension polymerase chain reaction and cloned into pLenti6 V5 expression plasmid (Invitrogen) for expression in HEK293T cells. The sequence of Chimeric Protein 2 is shown in
Example 3 Making the Chimeric Protein 3
[0133] The gene encoding the SA variant RBD of spike protein was synthesized (Blue Heron) and amplified by polymerase chain reaction. This gene fragment was in-frame fused with the codon optimized horse radish peroxidase gene fragment by overlap extension polymerase chain reaction and cloned into pLenti6 V5 expression plasmid (Invitrogen) for expression in HEK293T cells. The sequence of Chimeric Protein 3 is shown in
Example 4 Making of Chimeric Protein 4
[0134] The ACE2 receptor gene was amplified from the A549 cell line. The mouse Fc region of IgG1 is from a mouse B cell. Two gene fragments will be joined by overlap extension polymerase chain reaction and cloned into pLenti6 V5 expression plasmid (Invitrogen) for expression in HEK293T cells. The sequence of Chimeric Protein 4 is shown in