ANTIBODY PAIRS FOR USE IN A RAPID INFLUENZA B DIAGNOSTIC TEST

Abstract

Novel antibody pairs for use in an Influenza B diagnostic test.

Claims

1. A matched antibody pair comprising the detection and capture antibodies in a lateral flow immunoassay to detect Nucleoprotein (NP) antigen of Influenza B present in a sample, wherein the antibodies of said matched antibody pair are independently capable of binding to an NP epitope in the region comprising amino acids 1-80 of Influenza B Nucleoprotein.

2. A matched antibody pair according to claim 1 wherein at least one of the antibodies is capable of binding to an NP epitope comprising SEQ. ID. No. 2 hereof.

3. A matched antibody pair according to claim 1 wherein at least the capture antibody is capable of binding to an NP epitope comprising SEQ. ID. NO: 2.

4. A matched antibody pair according to claim 1 wherein the detection antibody is Fitzgerald-fii 10-I55P, clone M02202, and the capture antibody is GenWay GWB-T00595, clone B265M.

5. A ternary complex comprising: (a) Influenza B Nucleoprotein (NP); (b) a detection antibody conjugated with or otherwise associated with a detectible signal; and (c) a capture antibody, optionally immobilized on a substrate, wherein (b) and (c) are capable of binding an epitope of (a) independently selected in each case from an NP epitope in the region comprising amino acids 1-80 of Influenza B Nucleoprotein.

6. A ternary complex according to claim 5 wherein (b) and (c) are capable of binding an epitope of (a) comprising SEQ. ID. No 2 hereof, whereby said ternary complex is formed.

7. A ternary complex according to claim 5 wherein (b) and (c) are capable of binding to an epitope of (a) comprising SEQ. ID. NO: 2.

8. A ternary complex according to claim 5 wherein (b) and (c) are, respectively, Fitzgerald-fii 10-I55P, clone M02202, and the capture antibody is GenWay GWB-T00595, clone B265M.

9. A substrate comprising immunochromatographic material having immobilized thereon the ternary complex according to claim 5.

10. A lateral flow immunoassay for detecting Influenza B Nucleoprotein (NP) in a sample, comprising: (a) a mobile phase comprising a detection antibody conjugated or otherwise associated with a detectible signal, said detection antibody being capable of binding NP to form a binary complex; and (b) a stationary phase comprising a substrate comprising immunochromatographic material having immobilized thereon a capture antibody, said capture antibody being capable of binding said NP of the binary complex to form a ternary complex, whereby a detectible signal generated by said ternary complex is indicative of the presence of NP in the sample, said capture and detection antibodies being capable of binding an NP epitope in the region comprising amino acids 1-80 of Influenza B Nucleoprotein.

11. A lateral flow immunoassay according to claim 10 wherein at least one of the antibodies is capable of binding to an NP epitope comprising SEQ. ID. No. 2 hereof.

12. A lateral flow immunoassay according to claim 10 wherein at least the capture antibody is capable of binding to an NP epitope comprising SEQ. ID. NO: 2.

13. A lateral flow immunoassay according to claim 10 wherein the detection antibody is Fitzgerald-fii 10455P, clone M02202, and the capture antibody is GenWay GWB-T00595, clone B265M.

14. A method for detecting Influenza B in a sample using a lateral flow immunoassay comprising a mobile phase and a stationary phase, said method comprising (a) introducing said sample to the mobile phase of said assay, (b) contacting Influenza B Nucleoprotein (NP) in said sample with at a detection antibody that is conjugated or otherwise associated with a detectible signal to form a binary complex comprising said detection antibody and NP in said mobile phase, and (c) contacting said mobile phase with a stationary substrate comprising immunochromatographic material having immobilized thereon a capture antibody, said capture antibody being capable of binding said NP of the binary complex to form a ternary complex, whereby a detectible signal generated by said ternary complex is indicative of the presence of NP in the sample, wherein said capture antibody and said detection antibody are capable of binding an NP epitope in the region comprising amino acids 1-80 of Influenza B Nucleoprotein.

15. A method for detecting Influenza B according to claim 14 wherein at least one of the antibodies is capable of binding to an NP epitope comprising SEQ. ID. No. 2 hereof.

16. A method for detecting Influenza B according to claim 14 wherein at least the capture antibody is capable of binding to an NP epitope comprising SEQ. ID. NO: 2.

17. A method for detecting Influenza B according to claim 14 wherein the detection and capture antibodies are, respectively, Fitzgerald-fii 10-I55P, clone M02202, and GenWay GWB-T00595, clone B265M.

18. A method for diagnosing a patient afflicted with Influenza B comprising testing a biological sample from the patient in a lateral flow immunoassay according to claim 10, and determining the presence of Influenza B Nucleoprotein, wherein the presence of a detectible signal is indicative of Influenza B infection in said patient.

19. (canceled)

20. (canceled)

21. (canceled)

22. A method for monitoring the efficacy of therapeutic treatment of Influenza B in a patient by testing a biological sample from the patient in a lateral flow immunoassay according to claim 10, and determining the presence or absence of Influenza B Nucleoprotein, both prior to and following administration of a pharmaceutical active agent for treating Influenza B.

23. A medical device comprising the lateral flow immunoassay according to claim 10.

24. A kit comprising the lateral flow immunoassay according to claim 10 in a housing, and optionally, a reader device and/or instructions for use of the kit.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIG. 1 is a schematic view of a lateral flow immunoassay, as previously described.

[0085] FIG. 2 shows the amino acid sequence of NP antigen of human Influenza B virus (strain B/Lee/1940), based on UniProt Accession No. P03466 (SEQ ID NO:1).

[0086] FIG. 3 shows the peptide sequence (in 1-letter code, 3-letter code, and full names) of an NP binding epitope of human Influenza B (SEQ ID No: 2), corresponding to position amino acids 31-43 of SEQ. ID. NO. 1.

[0087] FIG. 4 shows wet dipstick dose response curves obtained in Example 1, which plot mean Rann score (n=2) against concentration (ng/ml) of purified NP antigen of Influenza B (Florida/4/2006) using the indicated capture and conjugate antibody pairs.

[0088] FIG. 5 is a dose response curve obtained in Example 2, which plots mean Rann Score (n=3) against concentration (ng/ml) of purified NP antigen of Influenza B (Florida/4/2006), using Antibody Pair (A) of the examples.

DETAILED DESCRIPTION OF THE INVENTION

[0089] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. By “specifically binds” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F.sub.ab, F.sub.ab′ and F.sub.(ab)2 fragments, scFvs, and F.sub.ab expression libraries.

[0090] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an antibody. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide.

[0091] The strength, or affinity, of binding can be expressed in terms of the dissociation constant (K.sub.d.) of the interaction, wherein a smaller K.sub.d represents a greater affinity. Immunological binding properties of selected antibodies can be quantified using methods well known in the art. An antibody of the present invention is said to specifically bind to an influenza epitope when the equilibrium binding constant K.sub.d. is ≤1 pM, preferably ≤100 nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. See U.S. Pat. No. 9,951,122, which is incorporated by reference.

[0092] The term “biological sample” is intended to refer to a composition comprising tissues, cells and/or biological fluids isolated from a subject, as well as tissues, cells and/or fluids present within a subject. Examples of biological samples may comprise saliva, sputum, nasal aspirate or swab, nasopharyngeal aspirate or swab, throat swab, and cheek scraping or swab. Also included within the usage of the term “biological sample” are compositions comprising blood or a fraction or component of blood including blood serum, blood plasma, or lymph.

[0093] The novel antibody combinations of the invention comprise at least two species of antibody, at least one of which is capable of functioning as a detection antibody when conjugated or otherwise associated with a signal moiety, and at least one of which is capable of functioning as a capture antibody when bound to or otherwise immobilized on an immunochromatographic strip.

[0094] The murine monoclonal antibodies described herein are substantially homogenous; have specificity and affinity for the NP protein, and are essentially non-cross-reactive with other viral proteins.

[0095] The terms “Nucleoprotein” or “NP” as employed herein shall be understood to refer to the viral protein in its monomeric as well as its oligomeric (e.g., dimers, trimers, tetramers, etc.), especially homo-oligomeric, forms.

[0096] Certain murine anti-Influenza B nucleoprotein monoclonal antibodies are commercially available from a variety of suppliers, including Antibodies-Online, Meridian Life Science, GenWay, Fitzgerald, and Hytest, and were used to prepare the novel antibody pairs of the invention.

[0097] Said isolated and purified monoclonal antibodies can be prepared by generally known techniques, e.g., by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975).

[0098] The invention is also contemplated to include NP-epitope binding fragments of said monoclonal antibodies, as well as humanized forms of said monoclonal antibodies and NP-binding fragments thereof.

[0099] NP protein binding epitopes of certain of the murine mAbs were mapped to the area close to the N-terminus of Influenza B Nucleoprotein, thus, for example, within the region comprising (e.g., consisting of one or more residues between) position amino acids 1-80; preferably amino acids 1-50; e.g., 20-50, or 25-50, even more preferably 30-45, of full-length Nucleoprotein of Influenza B.

[0100] In one embodiment, at least one of the antibodies of the combinations of the invention has affinity for, and specifically binds, the epitope disclosed as SEQ ID NO: 2, a peptide comprising residues 31-43 (PIIKPATLAPPSN) of Influenza B virus, accessioned P04665 (SEQ. ID. NO. 1).

[0101] In a preferred embodiment, at least the capture antibody has affinity for, and specifically binds, the epitope disclosed as SEQ ID NO: 2.

[0102] In a further embodiment, both capture and detection antibodies are characterized by binding affinity for SEQ. ID NO. 2.

[0103] Epitope mapping of certain of the commercially available NP monoclonal antibodies listed on Table 1 by known methods (see, e.g., H. M. Geysen et al., Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 3998-4002, July 1984) using 93 overlapping peptides corresponding to the length of the NP antigen (strain B/Lee/1940) (SEQ ID NO:1), revealed that 2 of the mAbs (mAb InB12 and B265M) had an affinity and specificity (i.e. ≥0.4 OD.sub.492 nm measured by ELISA) to the NP epitope sequence indicated in Table 1:

TABLE-US-00001 TABLE 1 NP Epitope Reference in Function Vendor Product # Clone Isotype Sequence Figure 4 Capture Acris 3IF18-InB12 InB12 IgG2b SEQ. ID. 2 Acris Mab InB12 cature Conjugate Acris 3IF18-InB12 InB12 IgG2b SEQ. ID. 2 Acris Mab InB12 (B) conjugate Capture GenWay GWB- B265M IgG2b SEQ. ID. 2 GenWay GWB- T00595 TOO595 capture Capture/ Fitzgerald-fii 10-I55O M62151 IgG2b Fizgerald 10-1550 Conjugate capture Conjugate Fitzgerald-fii 10-155O M62151 IgG2b Fitzgerald 10-155O (B) conjugate Capture Fitzgerald-fii 10-I55P M02202 IgG1 Fitzgerald 10-155P capture Conjugate Fitzgerald-fii 10-155P M02202 IgG1 Fitzgerald 10-155P (B) conjugate Capture Fitzgerald-fii 10-I55Q M12212 IgG2b Fitzgerald 10-155Q capture Conjugate Hytest RIF17 R2/3 IgG2a Hytest RIF 2/3 (B) conjugate

[0104] At least one of the detection and capture antibodies of the invention has affinity for, and specifically binds, the epitope having SEQ. ID. NO:2.

[0105] In a preferred embodiment of the invention, each of the detection and capture antibodies has affinity for, and specifically binds, the epitope having SEQ. ID. NO: 2.

[0106] A preferred antibody pair of the invention comprises:

[0107] (a) as detection antibody, the mAb which is commercially available from Fitzgerald-fii as mAb 10-155P, clone M02202, preferably conjugated to colloidal gold; and

[0108] b) as capture antibody, the mAb which is commercially available from GenWay as mAb GWB-T00595, clone B265M.

[0109] The antibody pairs of the invention are particularly useful as detection and capture reagents in an LFIA of the sandwich type, as described above.

[0110] It will be evident that the term “sandwich” (or “sandwich-type”) as employed herein is intended to refer to an immunoassay in which the paired detection and capture antibodies are capable of binding to different, or the same, epitopes of optionally oligomerized NP analyte.

[0111] The following examples are provided by way of illustration only by means of various particular embodiments and are in no way to be considered limitative of the invention.

Materials and Methods.

[0112] The following antibody pairs were tested:

TABLE-US-00002 TABLE 2 mAb Pairs Detection Capture (A) Hytest RIF 2/3 (B) conjugate Fitzgerald 10-1550 capture (B) Hytest RIF 2/3 (B) conjugate Fitzgerald 10-155P capture (C) Hytest RIF 2/3 (B) conjugate Fitzgerald 10-155Q capture (D) Hytest RIF 2/3 (B) conjugate GenWay GWB-TOO595 capture (E) Fitzgerald 10-1550 (B) conjugate Acris Mab InB12 capture (F) Fitzgerald 10-155P (B) conjugate GenWay GWB-TOO595 capture (G) Acris Mab InB12 (B) conjugate Fitzgerald 10-155Q capture
Detection antibody-gold conjugate. Conjugates of the detection antibody with 40 nm colloidal gold particles (HD.GC40.OD10) supplied by BBI Solutions OEM Ltd., were prepared by known techniques. The conjugates were diluted with PBS 1% Tween 20 to form a suspension having optical density (OD) of 1.
NP standards were prepared by serial dilutions of recombinant antigen of Influenza B (Florida/4/2006) in PBS 1% Tween 20 pH 7.2 to a concentration of 1-50 nanograms per milliliter.

Example 1. Half (i.e. “Wet”) Dipstick Dose Response of Detection Antibody Conjugate to NP Antigen of Influenza B (Florida/4/2006)

[0113] Capture antibody was striped onto a nitrocellulose membrane card, 2.5×30 cm, at a rate of 0.1 microliters per second using an Isoflow reagent dispensing module. The card was allowed to dry at ambient temperature overnight in a humidity controlled dry room, and then laminated with a 21-millimeter wide cellulosic fiber wick (222 Ahlstrom membrane). The card was cut to a 5-millimeter wide strip using a kinematic slitter (Kinematic Matrix 2360), yielding 5-millimeter wide half lateral flow dipsticks (“half-sticks”).

[0114] 20 μl of each of the NP stock solution and the detection antibody-gold conjugate suspension were added to a 96-well plate and incubated for 5 minutes. The half-stick samples were placed in the wells, and allowed to stand until all liquid was absorbed.

[0115] The results of visual scoring of the color intensity of the test line of the samples against a Rann scale of 0 (no visible color on the test line) to 2 (visible color on the test line) to 11 (very intense color on the test line), are shown on FIG. 4.

[0116] As shown in FIG. 4, even without optimization or amplification, the Fitzgerald 10-1550 and Hytest RIF17 2/3 antibody pair (A) can detect down to 1 ng/ml influenza B (Florida/4/2006) NP viral antigen.

Example 2. Evaluation of Antibody Pair (F) in “Dry Down” Format

[0117] Nitrocellulose cards were striped with 1.0 mg/ml of capture antibody, and allowed to dry overnight at ambient temperature in a humidity controlled environment. A conjugate pad on the membrane card was sprayed with detection antibody colloidal gold conjugate.

[0118] Using standard techniques, devices were run with 60 μl of each of the NP standards for 20 minutes.

[0119] The results of visual scoring of the test line against a Rann scale as previously described are shown in FIG. 5.

[0120] Antibody Pair (F), i.e. GenWay GWB-T00595 (capture) and Fitzgerald-fii 10-155P(B) (conjugate), was shown to detect NP antigen of Influenza B (Florida/4/2006) down to 1 ng/ml.