Methods for enhancing the specificity and sensitivity of group A streptococcal immunoassays.

Abstract

The present disclosure provides methods and kits for detecting group A streptococcus in biological samples. More specifically, the present disclosure provides methods for enhancing the specificity and sensitivity of group A streptococcus immunoassays by including N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. Thus, the disclosed methods and kits are useful for reliable early diagnosis of streptococcal infection in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 169,555, filed April 1, 2021, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure provides a method for detecting an analyte in a biological sample. In particular, the present disclosure provides a method for enhancing the specificity and sensitivity of a Group A Streptococcus immunoassay by including N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. A kit for use in carrying out the method is also provided. [Background technology]

[0003] background The following description of the background of the present disclosure is provided solely to aid the reader in understanding the present disclosure and is not admitted to describing or constituting prior art to the present disclosure. Detection of pathogenic microorganisms in biological samples is crucial in clinical decision making, as treatment can vary considerably depending on the causative organism. Acute disease associated with S. pyogenes occurs primarily in the airways, bloodstream, or skin. Streptococcal disease is most often a respiratory infection (pharyngitis or tonsillitis) or skin infection (pyoderma). Acute S. pyogenes infections may manifest as pharyngitis (streptococcal pharyngitis), scarlet fever (rash), impetigo (infection of the superficial layers of the skin) or cellulitis (infection of the deeper layers of the skin). Invasive toxin-producing infections can result in necrotizing fasciitis, joint or bone infections, myositis, meningitis, endocarditis, and streptococcal toxic shock syndrome. Patients may also develop immune-mediated streptococcal sequelae, such as acute rheumatic fever and acute glomerulonephritis, following acute infection due to S. pyogenes, which occurs in 1-3% of untreated infections. These conditions and their symptoms may result from an abnormal immune response to group A streptococcal antigens, rather than bacterial outbreaks per se. Because of the incidence of rapidly progressive disease and the risk of serious sequelae in untreated infections, S. pyogenes remains a major health concern. Therefore, accurate and rapid identification of streptococcal infections can be crucial in order to provide prompt and appropriate treatment to patients. Summary of the Invention

[0004] overview The present disclosure provides devices, methods and improved diagnostic kits for detecting the presence of Group A Streptococcus (also referred to herein as "Strep A") in a biological sample. More particularly, the present disclosure provides an improved immunoassay in which Group A Streptococcus infection is detected with a reduced rate of false positive results and increased sensitivity (i.e., reduced false negative results) by the addition of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine to the assay. In one aspect, the disclosure provides a method for detecting at least one Group A Streptococcus species in a biological sample, comprising: (a) administering to the biological sample an antibody or antigen-binding fragment that specifically targets a Group A streptococcus specific antigen and an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG5-glucosamine, m-PEG6-glucosamine, m-PEG7-glucosamine, m-PEG8-glucosamine, m-PEG9-glucosamine, m-PEG10 ... and (b) detecting binding of the antibody or antigen-binding fragment to a group A streptococcus specific antigen, if present, in the biological sample, where detection of binding of the antibody or antigen-binding fragment to the group A streptococcus specific antigen indicates the presence of at least one group A streptococcus species in the sample. In some embodiments, the antibody is a monoclonal antibody. The group A streptococcus specific antigen may be selected from the group consisting of capsular polysaccharide ("C-substance"), cell wall peptidoglycan, lipoteichoic acid (LTA), M protein, pilus protein, fibronectin binding protein, and cell-associated streptokinase. Examples of group A streptococcus species include Streptococcus pyogenes or Streptococcus dysgalactiae.

[0005] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment comprises a reporter label. The reporter label may be, for example, a gold nanoparticle label, a chemiluminescent label, a radioactive label, a bioluminescent label, a fluorescent label, a chromogenic label, a spectroscopic label, a photochemical label, or an electrochemiluminescent label. Examples of fluorescent labels include, but are not limited to, Europium, TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, Monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, and the like. Green), TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Examples of chemiluminescent labels include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), and alkaline phosphatase. Examples of bioluminescent labels include aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of radioactive labels include, but are not limited to,3 H, 14 C. 32 P, 35 S, 36 Cl, 57 Co, 131 I and 186 Re is an example.

[0006] In some embodiments of the methods disclosed herein, the biological sample is a bodily fluid, such as urine, saliva, sputum, mucus, a throat swab sample, blood, blood components, such as plasma or serum, amniotic fluid, semen, wound fluid, vaginal fluid, tears, spinal fluid, lavage fluid, and other bodily fluids. Biological samples include, for example, swab specimens from the cervix, urethra, nasal passages, and throat. In some embodiments, the biological sample is collected via the use of a throat swab. In some embodiments, the biological sample is collected via a swab of the throat, tongue, cheek, teeth, gums, or nasal passages. Additionally or alternatively, in some embodiments of the methods of the present technology, binding of the antibody or antigen-binding fragment to a group A streptococcus specific antigen is detected by enzyme-linked immunosorbent assay (ELISA) or enzyme multiplied immunoassay technique (EMIT). Additionally or alternatively, in some embodiments, the biological sample is obtained from a subject suffering from or suspected of having a bacterial infection. In particular embodiments, the subject is a human.

[0007] In one aspect, the disclosure provides a device for detecting the presence of group A streptococcus in a sample, the device comprising a matrix having (i) a sample receiving zone for receiving a sample containing or suspected of containing a group A streptococcus specific antigen, (ii) a labeling zone containing a detection antibody or detection antigen binding fragment for specifically labeling the antigen as it passes therethrough, and (iii) a capture zone having means for specifically binding the labeled antigen thereon, the sample receiving zone, the labeling zone and the capture zone being arranged on the matrix in a liquid flow path, and at least one of the sample receiving zone, the capture zone and the labeling zone comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, the detection antibody is a polyclonal antibody. In some embodiments, the detection antibody is a monoclonal antibody. In some embodiments of the device, the detection antibody or detection antigen-binding fragment comprises a gold nanoparticle label, a chemiluminescent label, a radioactive label, a bioluminescent label, a fluorescent label, a chromogenic label, a spectroscopic label, a photochemical label, or an electrochemiluminescent label. Additionally or alternatively, in some embodiments, the means for specifically binding labeled antigen to the capture zone is a capture antibody or capture antigen-binding fragment. In particular embodiments, the capture antibody is a polyclonal antibody. In some embodiments, the capture antibody is a monoclonal antibody.

[0008] In another aspect, the disclosure provides a method for selecting a patient for antibiotic therapy, in some embodiments, the method includes detecting the presence of at least one Group A Streptococcus species in a biological sample obtained from the patient using any of the devices disclosed herein, and administering antibiotic therapy to the patient. In another embodiment, the method comprises the steps of (a) contacting a biological sample obtained from a patient with an antibody or antigen-binding fragment that specifically targets a group A streptococcus specific antigen and an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine, and (b) administering antibiotic therapy to the patient when binding between the antibody or antigen-binding fragment and the group A streptococcus specific antigen is detected in the sample. In some embodiments of the methods disclosed herein, the antibiotic therapy comprises one or more of penicillin, ampicillin, sulbactam, amoxicillin, clavulanic acid, clindamycin, erythromycin, macrolides, and cephalosporins. In some embodiments, the macrolide antibiotic is selected from the group consisting of azithromycin (Zithromax), clarithromycin (Biaxin), erythromycin (E-Mycin, Eryc, Ery-Tab, PCE, Pediazole, Ilosone), and roxithromycin. Examples of cephalosporins include Cefacetrile (e.g., Cephacetrile), Cefadroxil (e.g., Cefadroxyl; Duricef), Cefalexin (e.g., Cephalexin; Keflex), Cefaloglycin (e.g., Cephaloglycin),Cephalonium (e.g., cephalonium), Cephaloridine (e.g., cephaloradine), Cefalotin (e.g., cephalothin; Keflin), Cefapirin (e.g., cephapirin; Cefadryl), Cefatrizine, Cefazafl ur), Cefazedone, Cefazolin (e.g., cephazolin; Ancef, Kefzol), Cephradine (e.g., cephradine; Velosef), Cefroxadine, Ceftezole, Cefaclor (e.g., Ceclor, Distaclor, Keflor) ), Raniclor), Cefonicid (e.g., Monocid), Cefprozil (e.g., cefproxil; Cefzil), Cefuroxime (e.g., Zefu, Zinnat, Zinacef, Ceftin, Biofuroxsym, Xorimax), Cefuzonam, Cefmetazole (e.g., efmetazole, cefotetan, cefoxitin, loracarbef (e.g., Lorabid), cefbuperazone, cefmetazole (e.g., Zefazone), cefminox, cefotetan (e.g., Cefotan), cefoxitin (e.g., Mefoxin),Cefotiam (e.g., Pansporin), Cefcapene, Cefdaloxime, Cefdinir (e.g., Sefdin, Zinir, Omnicef, Kefnir), Cefditoren, Cefetamet, Cefixime (e.g., Fixx, Zifi, Suprax), Cefmenoxime (e.g., efmenoxime, cefodizime, cefotaxime (e.g., Claforan), cefovecin (e.g., Convenia), cefpimizole, cefpodoxime (e.g., Vantin, PECEF, Simplicef), cefteram, ceftamere (e.g., Enshort), ), Ceftibuten (e.g., Cedax), Ceftiofur (e.g., Naxcel, Excenel), Ceftiolene, Ceftizoxime (e.g., Cefizox), Ceftriaxone (e.g., Rocephin), Cefoperazone (e.g., Cefobid), Ceftazidime (e.g., Ceftazidime), tazidime (e.g. Meezat, Fortum, Fortaz, Latamoxef (e.g. moxalactam), Cefclidine, Cefepime (e.g. Maxipime), Cefluprenam, Cefoselis, Cefozopran, Cefpirome (e.g. Cefrom),Cefquinome, Flomoxef, Ceftobiprole, Ceftaroline, Ceftolozane, Cefaloram, Cefaparole, Cefcanel, Cefedrolol, Cefempidone, Cefetri Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftioxide, Cefuracetim, and Nitrocefin.

[0009] In another aspect, the disclosure provides a kit comprising polyclonal and / or monoclonal antibodies targeting a Group A streptococcus specific antigen, at least one of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine, and instructions for use. Also disclosed herein is a kit comprising a device including a matrix having (i) a sample receiving zone for receiving a sample containing or suspected of containing a group A streptococcus specific antigen, (ii) a labeling zone containing a detection antibody for specifically labeling the antigen as it passes therethrough, and (iii) a capture zone having means thereon for specifically binding the labeled antigen, wherein the sample receiving zone, the labeling zone and the capture zone are arranged on the matrix in a liquid flow path, and a container containing an extraction reagent, wherein at least one of the extraction reagent, the sample receiving zone, the capture zone and the labeling zone comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In certain embodiments, N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine is deposited on the sample receiving zone. [Brief description of the drawings]

[0010] [Figure 1A] Figure 1 shows the effect of N-propionyl-D-glucosamine on false positives in the Veritor™ Strep A assay when analyzing test samples. N-propionyl-D-glucosamine was added to the extraction reagent and mixed with porcine mucin, a surrogate for human mucin from saliva, for specificity, or with Strep A extract for sensitivity. [Figure 1B] 1 shows the effect of N-propionyl-D-glucosamine on the sensitivity of the Veritor™ Strep A assay upon analysis of test samples. [Figure 1C]FIG. 1 shows the effect of N-propionyl-D-glucosamine on the sensitivity and false positives of the Veritor™ Strep A Assay when analyzing test samples. [Diagram 2] 1 shows the effect of N-propionyl-D-glucosamine on false positives in the Veritor™ Strep A assay due to saliva sample rrrr. [Figure 3A] 1 shows the effect of N-propionyl-D-glucosamine in the conjugate spray solution (on the conjugate pad) on the sensitivity and specificity of the Veritor™ Strep A assay. [Figure 3B] 1 shows the effect of N-propionyl-D-glucosamine in the conjugate spray solution (on the conjugate pad) on the specificity of the Veritor™ Strep A assay. [Figure 4] FIG. 1 shows the effect of N-propionyl-D-glucosamine in the conjugate spray solution (on the conjugate pad) on false positives in the Veritor™ Strep A assay caused by saliva. [Figure 5A] The effect of Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide on the sensitivity and specificity of the Veritor™ Strep A assay is shown. In this experiment, different concentrations of Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide were mixed with Strep A antigen-containing samples or mucin samples before adding them to the sample wells of a lateral flow test device. [Figure 5B] FIG. 1 shows the effect of Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide on the sensitivity and specificity of the Veritor™ Strep A assay. [Figure 6] The effect of 2-N-butanoyl-D-glucosamide on the sensitivity and specificity of the Veritor™ Strep A assay is shown in the experiment, in which different concentrations of 2-N-butanoyl-D-glucosamide were mixed with Strep A antigen-containing samples or mucin samples before they were added to the sample wells of a lateral flow test device. [Figure 7]The effect of dimeric bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide added to samples on the performance of two lots of Strep A devices that previously showed mucin cross-reactivity is shown. In this experiment, different concentrations of bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide were mixed with Strep A antigen-containing samples or mucin samples before adding them to the sample wells of a lateral flow test device. [Figure 8] The effect of adding Bis(2-(D-2-deoxyl-glucosaminyl))-PEG3-amide to the conjugate pad and sample pad soaking solutions on the performance of the Strep A device is shown. In this experiment, both the conjugate pad and the sample pad were pretreated with solutions containing different concentrations of Bis(2-(D-2-deoxyl-glucosaminyl))-PEG3-amide. Once the anti-Strep A detection conjugate was sprayed onto this pretreated conjugate pad, different test devices were made by mixing the different conjugate pads with different sample pads containing different amounts of Bis(2-(D-2-deoxyl-glucosaminyl))-PEG3-amide. These devices were tested with Strep A antigen-containing positive samples or mucin-containing samples that would cause a false positive signal against the control device that did not contain any Bis(2-(D-2-deoxyl-glucosaminyl))-PEG3-amide on either the sample pad or the conjugate pad. [Figure 9]The effect of adding different concentrations of bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide to the conjugate pad soaking solution on the ability of Strep A detection assay is shown. The results in Figure 8 imply that high concentrations of bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide in either the sample pad soaking solution or the conjugate pad soaking solution can effectively eliminate the false positive signal caused by mucin, but also inhibit the true signal. In this experiment, bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide was added to the conjugate pad soaking solution at concentrations from zero to 12.5 mM. Strep A antigen-containing positive samples or mucin-containing samples that would cause false positives were added to a lateral flow device made with a conjugate pad treated with different concentrations of bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide. The results demonstrate that lower concentrations of the dimer can effectively eliminate mucin cross-reactivity without significantly affecting true sensitivity. [Figure 10] The effect of m-PEGn(4,6,10)-glucosamine in samples on the performance of the Strep A assay is shown. In this experiment, different concentrations of m-PEGn(4,6,10)-glucosamine were mixed with Strep A antigen-containing samples or mucin samples before adding them to the sample wells of a lateral flow test device. The results show that these agents are effective in eliminating mucin cross-reactivity without significantly affecting the sensitivity of the Strep A assay. [Figure 11] The effect of m-PEG5000-glucosamine on the ability of the Strep A detection assay is shown. The results show that m-PEG5000-glucosamine does not reduce the false positive signal caused by mucin, but instead increases the false positive signal in the presence of mucin. However, in the absence of mucin, no false positive signal was observed when m-PEG5000-glucosamine was added to the extraction solution (GAS 1+2). [Figure 12]1 shows the structures of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, and m-PEG10-glucosamine compositions disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description Streptococcus pyogenes (group A streptococcus) is a gram-positive, non-motile, non-spore-forming bacterium that occurs in chains or pairs of cells, with individual cells being round to ovoid cocci with a diameter of 0.6-1.0 μm. S. pyogenes is one of the most frequent pathogens of humans. Approximately 5-15% of normal individuals harbor this bacterium, usually in the respiratory tract, yet remain asymptomatic. As a resident flora, S. pyogenes can infect when defenses are compromised or when the organism is capable of penetrating constitutive defenses. The cell surface of S. pyogenes is primarily responsible for many of the bacterium's virulence determinants, especially those associated with colonization and evasion of phagocytosis, as well as the host's immune response. Bacterial surfaces are incredibly complex and chemically diverse. Antigenic components include capsular polysaccharide (C-substance), cell wall peptidoglycan and lipoteichoic acid (LTA), as well as various peripheral proteins such as M protein, pilus proteins, fibronectin-binding proteins (e.g. protein F) and cell-associated streptokinase.

[0012] Rapid diagnostic methods for microbial infections are needed to provide timely results to guide clinical treatment. Some of the most effective of these rapid methods have been immunologically based. Monoclonal and polyclonal antibodies against microbial specific antigens have been developed and used in immunoassays to identify specific microorganisms in biological samples. For example, immunoassays for the identification of group A streptococcus antigens in human samples are useful for early detection of S. pyogenes infections so that appropriate antibiotic therapy can be initiated. Group A streptococcus in pharyngeal exudates can be identified by polyclonal or monoclonal antibodies against antigens specific for group A streptococcus. One such test is described in U.S. Pat. No. 5,770,460, which provides a one-step lateral flow assay for group A streptococcus specific antigens. However, tests that rely on pharyngeal swabs are often complicated by a high false positive rate. Although the instructions for use of throat swab tests specifically instruct users to avoid contact with the tongue, cheeks and / or teeth with the swab, inadvertent contact nevertheless often occurs.

[0013] Epithelial cells from the tongue, cheeks and / or teeth may contain molecular mimics of one or more components of the S. pyogenes cell wall, and polyclonal antibodies specific for group A streptococcus may bind to and "recognize" epitopes on epithelial cells of subjects who are not infected with or do not have group A streptococcus, resulting in false positive results. In particular, salivary mucins and other glycosylated proteins found in the oral cavity contain the sugar, N-acetyl-D-glucosamine, as a glycan component. N-acetyl-D-glucosamine is considered the immunodominant component of the group A streptococcus (GAS) carbohydrate antigen epitope. As a result, the presence of these glycoproteins in throat swab specimens can cause false positive results in immunoassays designed to detect group A streptococcus. Immunoassays for group A streptococcus commonly use rabbit polyclonal antibodies as reagents, which typically contain a mixture of clones of variable affinity and specificity. Previous studies have reported that the addition of N-acetyl-D-glucosamine improves assay specificity and results in reduced interference (false positives) due to salivary proteins. However, this effect is accompanied by a significant decrease in assay sensitivity. See US 2013 / 0196337. Highly specific and sensitive immunoassays with reduced rates of both false positives and false negatives are needed to provide rapid detection of group A streptococcus infections.

[0014] The present disclosure demonstrates that when N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine are included in the reagents of a lateral flow assay, they preferentially bind antibody clones with low specificity for intact GAS antigen epitopes, thereby preventing those antibodies from cross-reacting with mucins and other glycoproteins. As demonstrated herein, inclusion of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine in a lateral flow immunoassay eliminates or significantly reduces the assay signal due to the presence of mucin while retaining high sensitivity for Group A Strep antigens.

[0015] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Where a range of values ​​is given, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range should also be understood to be specifically disclosed to the tenth of the unit of the lower limit. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is also encompassed by this disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded in the range, and each range in which either or neither or both are included in the smaller range is also encompassed by this disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also within the scope of this disclosure.

[0016] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise. Thus, for example, reference to "a cell" includes a plurality of cells. As used herein, the term "antibody" collectively refers to immunoglobulin or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, e.g., mammals, e.g., humans, goats, rabbits, and mice, as well as non-mammalian species, e.g., shark immunoglobulins. As used herein, "antibody" (including intact immunoglobulins) and "antigen-binding fragments" refer to an immunoglobulin that specifically binds to a molecule of interest (or a group of highly similar molecules of interest) and has a binding constant at least 10 times higher than the binding constant for other molecules (e.g., in a biological sample). 3 M -1 Large, at least 10 4 M -1 Greater than or at least 10 5 M -1 The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0017] More specifically, an antibody refers to a polypeptide ligand that contains at least a light or heavy immunoglobulin chain variable region that specifically recognizes and binds to an epitope of an antigen. H ) region and the light chain variable (V LThe V domain is made up of heavy and light chains with variable regions called V domains. H Area and V L The regions are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (these regions are also known as "domains"). Together, the heavy and light chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference). The Kabat database is now maintained online. The sequences of the framework regions of the various light or heavy chains are relatively conserved within a species. The antibody framework regions, which are the combined framework regions of the constituent light and heavy chains, largely adopt a β-sheet conformation, with the CDRs forming loops that connect and in some cases form part of the β-sheet structure. In this way, the framework regions act to form a scaffold that provides for positioning the CDRs in the correct orientation by non-covalent interactions between the chains.

[0018] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas V LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to a target protein have a specific V H Area and V L Each CDR will have a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e. different binding sites for different antigens) have different CDRs. Although it is the CDRs that differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). As used herein, "immunoglobulin-related composition" refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or an antigen-binding fragment thereof specifically binds to an antigen. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide responsible for binding to an antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0019] As used herein, "association" refers to the coincidence of the onset or manifestation of a disease, condition, or phenotype. Association can be due to, but is not limited to, genes responsible for housekeeping functions whose alterations may provide the basis for various diseases and conditions, genes that are part of pathways involved in specific diseases, conditions, or phenotypes, and genes that directly contribute to the manifestation of a disease, condition, or phenotype. "Binding affinity" refers to the strength of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, while high affinity complexes generally contain antibodies that tend to remain bound to the antigen for extended periods of time. As used herein, "chromophore" refers to a moiety that has absorption properties, i.e., the ability to become excited when illuminated by any of a variety of photon sources. Chromophores can be fluorescent or non-fluorescent, and include dyes, fluorophores, luminescent molecules, chemiluminescent molecules, and electrochemiluminescent molecules, among others.

[0020] As used herein, the term "detection" refers to determining the presence of a target polypeptide in a sample. Detection does not require the method to provide 100% sensitivity and / or 100% specificity. As used herein, an "effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine" refers to an amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine that is sufficient to preferentially bind to an antibody (or antigen-binding fragment) clone that has low specificity for an intact GAS antigen epitope, such that the clone does not cross-react with mucins and other glycoproteins. As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody or antigen-binding fragment thereof. Epitopes 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. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, the epitope is a conformational or non-conformational epitope.

[0021] "Homology" or "identity" or "similarity" refers to the sequence similarity between two epitopes or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequence is occupied by the same nucleic acid base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence, meaning that when the two sequences are compared, when aligned, this percentage of bases (or amino acids) are identical. This alignment and the percentage of homology or sequence identity can be determined using software known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; Filter=none; Strand=both; Cutoff=60; Expectation=10; Matrix=BLOSUM62; Descriptions=50 sequences; Sort=by HIGH SCORE; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that share a specified percentage of homology and encode polypeptides that have the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity with each other, or less than 25% identity.

[0022] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having distinct antigenic specificities (e.g., an isolated antibody that specifically binds to a protein of interest is substantially free of antibodies that specifically bind to antigens other than the protein of interest). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of a protein of interest may have cross-reactivity with other related antigens, e.g., antigens from other species (e.g., species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. "Label" refers to any moiety that, when attached to a moiety described herein, e.g., a peptide, protein, or antibody, renders the moiety detectable using known detection methods, e.g., spectroscopic, photochemical, electrochemiluminescent, and electrophoretic methods. Various labels suitable for use in the present disclosure include labels that generate a signal by chemical or physical means, which is detectable by visual or instrumental means. Exemplary labels include, but are not limited to, gold nanoparticles, fluorophores, and radioisotopes. The label allows for direct detection of the labeled compound by an appropriate detector, e.g., a fluorometer or reflectance reader. The labels may include enzymes and substrates, chromogens, catalysts, fluorescent compounds, phosphorescent compounds, chemiluminescent compounds, and radioactive labels. Typically, visually detectable labels are used, thereby providing an instrumental (e.g., a spectrophotometer or reflectance reader) readout of the amount of analyte in the sample. Labels include enzymes, e.g., horseradish peroxidase, galactosidase (alpha and / or beta), alkaline phosphatase, and the like. Suitable substrates include 3,3',5,5'-tetramethylbenzidine (TMB) and 1,2 dioxetanes. Detection methods will depend on the label used and will be apparent to those skilled in the art. Examples of suitable direct labels include gold nanoparticles, radiolabels, fluorophores, chromophores, chelators, particles, chemiluminescent agents, and the like.

[0023] In some embodiments, the label is a direct label, i.e., a label that is detectable or produces a detectable signal by itself, or an indirect label, i.e., a label that is detectable or produces a detectable signal in the presence of another compound. A "labeled secondary antibody" refers to an antibody that is attached to a detectable label. The label enables the antibody to produce a detectable signal that is related to the presence of the analyte in the fluid sample. Examples of suitable radioactive labels include, but are not limited to, 3 H, 14 C. 32 P, 35 S, 36 Cl, 57 Co, 131 I and 186 Examples of suitable indirect labels include enzymes capable of reacting or interacting with a substrate (e.g., those used in enzyme-linked immunosorbent assays (ELISA) and enzyme-linked immunosorbent assays (EMIT)), ligands capable of binding to a label component, and the like. Suitable enzymes that serve as indirect labels include, by way of example and without limitation, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and urease. The use of these enzymes in ELISA and EMIT immunoassays is described in detail in Engvall, 1980, Methods Enzym. 70: 419-439 and U.S. Patent No. 4,857,453.

[0024] The terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer comprising two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.), or by chemical modification techniques that are well known in the art.

[0025] The "positive predictive value (PPV)" or "precision rate" of a diagnostic test is a summary statistic used to describe the proportion of subjects with a positive test result who are correctly diagnosed. It is a measure of the power of a diagnostic method because it reflects the probability that a positive test reflects the underlying condition being tested for. However, its value depends on the prevalence of the outcome of interest, which may not be known for a particular target population. PPV can be derived using Bayes' theorem. PPV is defined as follows:

number

[0026] "Negative predictive value (NPV)" is defined as the proportion of subjects with a negative test result who are correctly diagnosed. A high NPV means that when a diagnostic test produces a negative result, it is less likely that the result should have been positive. NPV is defined as follows:

number

[0027] If the prevalence, sensitivity, and specificity are known, the positive and negative predictive values ​​(PPV and NPV) can be calculated for any prevalence as follows:

number

[0028] If the prevalence of a disease is very low, the positive predictive value will not be close to 1, even if both sensitivity and specificity are high. It is therefore inevitable that many people with positive test results in screening of the general population will be false positives. The rarer the abnormality, the higher the certainty that a negative test does not indicate an abnormality and the lower the certainty that a positive result truly indicates an abnormality. Prevalence can be interpreted as the probability that a subject has a disease before the test is taken, and is known as the prior probability of the disease. Positive and negative predictive values ​​are modified estimates of the same probability for a given subject to test positive and negative, and are known as the posterior probabilities. The difference between the prior and posterior probabilities is one way of assessing the usefulness of a test. For any test result, the probability of obtaining that result if the patient truly had the condition of interest can be compared with the corresponding probability if he or she was healthy. The ratio of these probabilities is called the likelihood ratio and is calculated as sensitivity / (1-specificity) (Altman DG, Bland JM (1994). BMJ 309 (6947):102).

[0029] As used herein, the terms "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In preferred embodiments, the individual, patient, or subject is a human. The term "reference level" refers to the detection level of a positive or negative control. For example, a positive control reference level may be a known amount of group A streptococcus specific antigen obtained from a sample or culture of known group A streptococcus bacteria, a subject known to be infected with group A streptococcus, or may refer to a value derived from a known source of group A streptococcus specific antigen. As used herein, the term "sample" refers to a clinical sample obtained from a patient or an isolated microorganism. In a preferred embodiment, the sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, body fluid, or a microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or raw), bronchoalveolar lavage fluid (BAL), bronchial washing fluid (BW), blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). Preferred sample sources include nasopharyngeal and / or throat swabs or nasal washes. In the context of this disclosure, biological fluids can be solid or semi-solid samples, such as feces, biopsy specimens, skin, nails, and hair, or liquid samples, such as urine, saliva, sputum, mucus, throat swab samples, blood, blood components, such as plasma or serum, amniotic fluid, semen, vaginal fluid, tears, cerebrospinal fluid, lavage fluid, and other body fluids. Samples include, for example, swabs from the cervix, urethra, nasal passages, and throat. Any of the samples may be from living, dead, or dying animals or plants.

[0030] In statistics and diagnostic testing, sensitivity and specificity are statistical measures of the ability of a binary classification test. "Sensitivity" (also called "recall") assesses the proportion of real positives that are correctly identified as such (e.g., the percentage of subjects correctly identified as having a condition). Sensitivity is related to the ability of a diagnostic test to identify positive results and is calculated as the number of true positives divided by the sum of the number of true positives and the number of false negatives. "Specificity" assesses the proportion of negatives that are correctly identified (e.g., the percentage of healthy subjects correctly identified as not having the condition). Specificity is related to the ability of a diagnostic test to identify negative results and is calculated as the number of true negatives divided by the sum of the number of true negatives and the number of false positives. Sensitivity and specificity are closely related to the concepts of type I error and type II error. The theoretical optimal prediction aims to achieve 100% sensitivity and 100% specificity, but in theory any predictor will have a minimum error bound known as the Bayes error rate. In any test, there is a trade-off between sensitivity and specificity, which can be represented graphically using a receiver operating characteristic (ROC) curve. In some embodiments, the ROC is used to generate summary statistics. There are several common versions: the intercept of the ROC curve with a line at 90 degrees to the indifference line (also called Youden's J statistic); the area between the ROC curve and the indifference line; the area under the ROC curve, or "AUC" ("Area Under Curve"), or A' (an obvious "a prime"); d' (an obvious "d prime"), the distance between the mean of the system's activity distribution under noise-only conditions and the mean of that distribution under signal-only conditions, divided by their standard deviations (under the assumption that both distributions are normal with the same standard deviation). Under these assumptions, it can be shown that the shape of the ROC depends only on d'.

[0031] "Rule-out criteria", "Rule-Out", or "RO" is a term used in the medical differential diagnosis of a disease or condition, where certain criteria are evaluated in the process of making clinical decisions to exclude or include. With consideration of the criteria, a subject is "excluded" when it is determined that the subject does not meet all or a substantial number of the criteria for having the disease. As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize and bind to other molecules. The terms "specific binding to," "specifically binds to," or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide) are used herein to refer to, for example, the K D About 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12M. The term "specifically binds" can also refer to binding where a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a particular polypeptide (e.g., a target polypeptide), or an epitope on a particular polypeptide, but does not bind substantially to any other polypeptides or polypeptide epitopes.

[0032] "Substrate", "support", "solid support", "solid carrier", or "resin" are interchangeable terms and refer to any solid phase material. Substrate also encompasses terms such as "solid phase", "surface", and / or "membrane". A "solid support" can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. A solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica, or metal, such as gold or platinum. "Solid supports" include membranes (e.g., nitrocellulose), microtiter plates (e.g., PVC, polypropylene, polystyrene), dipsticks, test tubes, and glass or plastic beads. The shape of the substrate can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. The surface can be planar, substantially planar, or non-planar. A solid support may be porous or non-porous and may have swelling or non-swelling properties. A solid support may be formed in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports may be formed in an array at various locations addressable for robotic delivery of reagents or by detection methods and / or instruments. Methods for immobilizing biomolecules are well known in the art, and antibodies may be covalently or non-covalently attached to a substrate. In a particular embodiment, the solid support is a streptavidin-coated plate to which biotinylated antibodies are non-covalently attached.

[0033] Diagnostic method of the present technology In one aspect, the disclosure provides a method for detecting the presence of at least one Group A Streptococcus species in a biological sample, comprising: (a) administering to the biological sample an antibody or antigen-binding fragment that specifically targets a Group A streptococcus specific antigen and an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m- and (b) detecting binding of an antibody or antigen-binding fragment to a group A streptococcus specific antigen, if present in the sample, wherein detection of binding of the antibody or antigen-binding fragment to the group A streptococcus specific antigen indicates the presence of at least one group A streptococcus species in the sample.

[0034] Examples of antibodies or antigen-binding fragments for use in the immunoassays of the present disclosure include, but are not limited to, polyclonal antibodies, such as affinity purified rabbit anti-Strep A antibodies. Illustrative publications describing components of precursor compositions, methods and kits for detecting Group A streptococcus, as well as various antibodies, include the following: U.S. Patent Nos. 5,415,994; 5,763,262 and 5,770,460. All of these patents, applications and publications are incorporated herein by reference in their entirety. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment binds to one or more epitopes of Group A streptococcus and also binds to N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, the antibody is a population of polyclonal antibodies, which population includes portions of antibodies that have specific binding to N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, the antibody or antigen-binding fragment does not bind to glucosamine, galactosamine, mannosamine, acetyl-muramic acid, chitin, chitosan, and / or hyaluronic acid (eg, HA-50K).

[0035] In some embodiments, the antibody or antigen-binding fragment has high specificity and low sensitivity for detecting group A streptococcus antigens. In some embodiments, the antibody or antigen-binding fragment has high sensitivity and low specificity for detecting group A streptococcus antigens. In some embodiments, the antibody or antigen-binding fragment has high specificity and high sensitivity for detecting group A streptococcus antigens. Additionally or alternatively, in some embodiments, the group A streptococcus specific antigen may be selected from the group consisting of capsular polysaccharide ("C-substance"), cell wall peptidoglycan, lipoteichoic acid (LTA), M protein, pilus protein, fibronectin binding protein, and cell-associated streptokinase. Examples of group A streptococcus species include Streptococcus pyogenes or Streptococcus dysgalactiae.

[0036] Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment comprises a gold nanoparticle label, a chemiluminescent label, a radioactive label, a bioluminescent label, a fluorescent label, a chromogenic label (e.g., gold nanoparticles), a spectroscopic label, a photochemical label, or an electrochemiluminescent label. Examples of fluorescent labels include, but are not limited to, Europium, TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, Monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, and the like. Green), TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOK, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Examples of chemiluminescent labels include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), and alkaline phosphatase. Examples of bioluminescent labels include aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of radioactive labels include, but are not limited to, 3 H,14 C. 32 P, 35 S, 36 Cl, 57 Co, 131 I and 186 Re is an example.

[0037] In some embodiments of the methods disclosed herein, the biological sample is a bodily fluid, such as urine, saliva, sputum, mucus, a throat swab sample, blood, blood components, such as plasma or serum, amniotic fluid, semen, wound fluid, vaginal fluid, tears, spinal fluid, lavage fluid, and other bodily fluids. Biological samples include, for example, swab specimens from the cervix, urethra, nasal passages, and throat. In some embodiments, the biological sample is collected via the use of a throat swab. In some embodiments, the biological sample is collected via a swab of the throat, tongue, cheek, teeth, gums, or nasal passages. Additionally or alternatively, in some embodiments of the methods of the present technology, binding of the antibody or antigen-binding fragment to a group A streptococcus specific antigen is detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunosorbent assay (EMIT). Additionally or alternatively, in some embodiments, the biological sample is obtained from a subject suffering from or suspected of having a bacterial infection. In particular embodiments, the subject is a human.

[0038] Without being bound to a particular theory, the data demonstrate that false positive signals from glycoproteins present within epithelial cells inadvertently collected on buccal swab specimens can be effectively blocked or suppressed by the addition of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine to the immunoassay without compromising the overall sensitivity and / or PPV of the assay. The polyclonal or monoclonal anti-Strep A antibodies used in the immunoassay may, in some embodiments, comprise a population of antibodies that recognize glycoproteins of human epithelial cell walls that mimic group A streptococcal cell wall proteins. By providing the immunoassay with a reagent capable of blocking this population of antibodies, the performance of the immunoassay can be improved in terms of overall accuracy by reducing the false positive rate. The results also demonstrate that, unlike NAG, the addition of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine reduces the overall false negative rate of the immunoassay. In some embodiments, the NPV of the immunoassay is improved compared to that observed in the absence of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine.

[0039] Thus, some embodiments contemplate the addition of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine to an immunoassay, where the presence of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine is indicative of a genotype or phenotype of Strep. A is effective to reduce non-specific binding between antibodies in a polyclonal antibody population and components in a test sample by at least about 50%, more preferably at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of the signal obtained in the absence of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine.

[0040] Immunoassay device of the present technology Immunoassays for the detection of Group A streptococcus (see FIG. 12) comprising N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine are contemplated, where the assay comprises a lateral flow device that allows for pretreatment and detection of Group A streptococcus organisms with enhanced sensitivity. Immunoassay devices are well known in the art and typically have at least a sample receiving zone, a label zone, and a capture zone, and can be made as described in any of U.S. Pat. Nos. 5,415,994; 5,763,262 and 5,770,460, and U.S. Patent Application Publication No. 20090305231, which are incorporated by reference in their entireties. Thus, in one aspect of the present disclosure, a device is provided for detecting the presence of group A streptococcus in a sample. Various embodiments of the device are contemplated, and for purposes of illustration, an exemplary embodiment is described herein. However, those skilled in the art will recognize that the illustrative embodiment is non-limiting on the inventive concepts set forth herein.

[0041] In general embodiments, the device comprises a series of zones in fluid communication. In particular embodiments, the sample-receiving zone is in fluid communication with second and subsequent zones, such as a label zone, a capture zone, and / or an absorption zone. One particular embodiment of the device of the present technology includes an immunoassay test strip for the detection of group A streptococcus. The exemplary test strip is comprised of a support layer, which optionally extends the length of the test strip. The support layer in turn supports a sample pad, a label pad (which may include a conjugate pad where an antibody or antigen-binding fragment is conjugated to a label), a nitrocellulose member, and an optional absorbent pad. On top of the nitrocellulose member are a test line and a control line. For detection of Strep A, the label pad contains an anti-Strep A antibody to aid in the test line. In one embodiment, the antibody or antigen-binding fragment deposited on the label pad is a detection antibody or detection antigen-binding fragment that contains a label that aids or allows detection of the antibody or antigen-binding fragment. The labeled antibody or antigen-binding fragment specifically binds to the Strep A antigen as it passes through the label zone. The capture zone contains a means for specifically binding the labeled antigen thereon. In some embodiments, the sample is contacted with N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine prior to application to the device and / or while it is flowing through the device. The studies described herein are illustrative of the improved performance of devices intended for the detection of Strep A when N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine are incorporated into the assay.N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine can be incorporated into the device, for example, in the sample receiving zone, labeling zone, capture zone, or any combination thereof, and / or the sample can be treated with N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine prior to application of the sample to the sample receiving zone of the device.

[0042] In one embodiment, the lateral flow immunoassay comprises an immunoassay using a detection label that can be read by the naked eye, such as colored beads or particles, and the collection of beads or particles at the test line of the immunoassay can be seen by the user with the naked eye. In another embodiment, the lateral flow immunoassay comprises an immunoassay using a label that is read by the eye by an instrument or with the aid of an instrument. For example, the immunoassay detects fluorescent, chemiluminescent, bioluminescent, gold nanoparticles, or radioactive labels using an instrument such as a reflectance reader or a fluorescence detector. Exemplary instruments and lateral flow immunoassays are described in U.S. Patent Application No. 61 / 666,689 and U.S. Patent No. 9,207,181, the contents of which are incorporated herein by reference. Another exemplary instrument and lateral flow immunoassay is described in U.S. Patent Application No. 12 / 420,574, the contents of which are incorporated herein by reference.

[0043] In another aspect, there is provided a device for detecting the presence of group A streptococcus in a sample, the device comprising a matrix having (i) a sample receiving zone for receiving a sample containing or suspected of containing a group A streptococcus specific antigen, (ii) a labeling zone containing a detection antibody for specifically labeling the antigen as it passes therethrough, and (iii) a capture zone having means thereon for specifically binding the labeled antigen, the sample receiving zone, the labeling zone and the capture zone being arranged in a liquid flow path on the matrix, and the sample receiving zone, the labeling zone, or any combination thereof, contains N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine.

[0044] Another embodiment of the device contemplated for use is described in U.S. Patent No. 5,415,994, the contents of which are incorporated herein by reference. In this embodiment, the device includes a receiving chamber that is positioned or positionable for fluid contact with a lateral flow immunoassay device, preferably for fluid communication with a sample receiving zone or labeling zone of an immunoassay test strip. A biological sample suspected of containing Strep A is received in the receiving chamber, for example, by inserting a sample-containing swab or by dispensing a volume of the sample into the receiving chamber. Additionally, one or more extractants or processing agents can be added to the receiving chamber or swab. In some embodiments, the processing agent includes N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In certain embodiments, the receiving chamber is sized to receive a liquid extraction reagent comprising N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine, and is positioned above a sample receiving zone that optionally includes a cylindrical portion for receiving a swab containing a patient sample. The immunoassay test strip comprises a matrix having a sample receiving zone for receiving an extraction liquid containing a processed sample suspected of containing Strep A antigen, a label zone having a detection antibody or detection antigen binding fragment for specifically labeling the antigen as it passes therethrough, and a capture zone having a means for specifically binding the labeled antigen thereon, where the sample receiving zone, label zone, and capture zone are arranged on the matrix in a liquid flow path. In some embodiments, the detection antibody is polyclonal. In some embodiments, the detection antibody is a monoclonal antibody.

[0045] In some embodiments of the devices described herein, the extraction reagent provided for processing the biological sample comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, the label zone of the immunoassay device comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In certain embodiments, the capture zone of the immunoassay device comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, the sample receiving zone of the immunoassay device comprises N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. In some embodiments, all or some of these specific zones contain N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine in the extraction reagent.

[0046] In another aspect, the present disclosure provides a method for detecting Strep in a biological sample. A method for detecting the presence of Group A comprising the steps of: (a) providing a matrix having (i) a sample receiving zone for receiving a sample containing or suspected of containing a group A streptococcus specific antigen, (ii) a labeling zone containing a detection antibody or detection antigen binding fragment for specifically labeling the antigen as it passes therethrough, and (iii) a capture zone having means thereon for specifically binding the labeled antigen, wherein the sample receiving zone, labeling zone and capture zone are arranged on the matrix in a liquid flow path; (b) contacting the sample receiving zone with the sample, the sample being pretreated with a liquid reagent comprising N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine; and (c) detecting the presence of the antigen in the capture zone. In some embodiments, the matrix further comprises an absorption zone downstream of the capture zone.

[0047] In another aspect, the disclosure provides a method for reducing the false positive rate of a lateral flow assay for the detection of Group A streptococcus in a liquid sample, the method comprising preferentially binding an N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine binding moiety of a polyclonal antibody (or antigen-binding fragment) used in the assay, the method comprising: The method includes treating the bibulous matrix with an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine as a blocking agent to enhance specific binding to the A antigen and reduce the false positive rate of the assay.

[0048] In another aspect, the disclosure provides a method for reducing the false positive rate of a lateral flow assay for the detection of Group A streptococcus in a liquid sample, the method comprising: preferentially binding an N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine binding moiety of a polyclonal antibody (or antigen-binding fragment) used in the assay; the method comprising: The method includes adding to the extraction reagent an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine as a blocking agent to enhance specific binding to the A antigen and reduce the false positive rate of the assay.

[0049] Known immunoassay devices for detecting group A streptococcus can be improved by adding to the sample receiving zone N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine in an amount effective as a blocking agent to enhance the specific binding of at least a portion of the polyclonal antibodies to the Strep A antigen, thereby reducing the false positive rate of the assay. The improvement may also include adding N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine to the labeling zone in an amount effective as a blocking agent to enhance specific binding of the polyclonal antibody to the Strep A antigen and reduce the false positive rate of the assay. The improvement may also include adding to the extraction reagent N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine in an amount effective as a blocking agent to enhance specific binding of the polyclonal antibody to the Strep A antigen and reduce the false positive rate of the assay. The improvement may also include the addition to the capture line of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine in an amount effective as a blocking agent to enhance specific binding of the polyclonal antibody to the Strep A antigen and reduce the false positive rate of the assay.

[0050] Treatment selection method using this technology In another aspect, the disclosure provides a method for selecting a patient suitable for antibiotic therapy, in some embodiments, the method includes detecting the presence of at least one Group A Streptococcus species in a biological sample obtained from the patient using any of the devices disclosed herein, and administering antibiotic therapy to the patient. In another embodiment, the method comprises the steps of (a) contacting a biological sample obtained from a patient with an antibody or antigen-binding fragment that specifically targets a group A streptococcus specific antigen and an effective amount of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine, and (b) administering antibiotic therapy to the patient when binding between the antibody or antigen-binding fragment and the group A streptococcus specific antigen is detected in the sample. In some embodiments of the methods disclosed herein, the antibiotic therapy comprises one or more of penicillin, ampicillin, sulbactam, amoxicillin, clavulanic acid, clindamycin, erythromycin, macrolides, and cephalosporins. In some embodiments, the macrolide antibiotic is selected from the group consisting of azithromycin (Zithromax), clarithromycin (Biaxin), erythromycin (E-Mycin, Eryc, Ery-Tab, PCE, Pediazole, Ilosone), and roxithromycin. Examples of cephalosporins include Cefacetrile (e.g., Cephacetrile), Cefadroxil (e.g., Cefadroxyl; Duricef), Cefalexin (e.g., Cephalexin; Keflex), Cefaloglycin (e.g., Cephaloglycin),Cephalonium (e.g., cephalonium), Cephaloridine (e.g., cephaloradine), Cefalotin (e.g., cephalothin; Keflin), Cefapirin (e.g., cephapirin; Cefadryl), Cefatrizine, Cefazafl ur), Cefazedone, Cefazolin (e.g., cephazolin; Ancef, Kefzol), Cephradine (e.g., cephradine; Velosef), Cefroxadine, Ceftezole, Cefaclor (e.g., Ceclor, Distaclor, Keflor) ), Raniclor), Cefonicid (e.g., Monocid), Cefprozil (e.g., cefproxil; Cefzil), Cefuroxime (e.g., Zefu, Zinnat, Zinacef, Ceftin, Biofuroxsym, Xorimax), Cefuzonam, Cefmetazole (e.g., efmetazole, cefotetan, cefoxitin, loracarbef (e.g., Lorabid), cefbuperazone, cefmetazole (e.g., Zefazone), cefminox, cefotetan (e.g., Cefotan), cefoxitin (e.g., Mefoxin),Cefotiam (e.g., Pansporin), Cefcapene, Cefdaloxime, Cefdinir (e.g., Sefdin, Zinir, Omnicef, Kefnir), Cefditoren, Cefetamet, Cefixime (e.g., Fixx, Zifi, Suprax), Cefmenoxime ( Cefmenoxime, Cefodizime, Cefotaxime (e.g., Claforan), Cefovecin (e.g., Convenia), Cefpimizole, Cefpodoxime (e.g., Vantin, PECEF, Simplicef), Cefteram, Ceftamere (e.g., Enshor t), Ceftibuten (e.g., Cedax), Ceftiofur (e.g., Naxcel, Excenel), Ceftiolene, Ceftizoxime (e.g., Cefizox), Ceftriaxone (e.g., Rocephin), Cefoperazone (e.g., Cefobid), Ceftazidime (e.g., Ceftazidime), tazidime (e.g. Meezat, Fortum, Fortaz, Latamoxef (e.g. moxalactam), Cefclidine, Cefepime (e.g. Maxipime), Cefluprenam, Cefoselis, Cefozopran, Cefpirome (e.g. Cefrom),Cefquinome, Flomoxef, Ceftobiprole, Ceftaroline, Ceftolozane, Cefaloram, Cefaparole, Cefcanel, Cefedrolol, Cefempidone, Cefetri Cefetrizole, Cefivitril, Cefmatilen, Cefmepidium, Cefoxazole, Cefrotil, Cefsumide, Ceftioxide, Cefuracetim, and Nitrocefin.

[0051] kit The present disclosure also provides a kit for detecting a target epitope corresponding to a group A streptococcus species, comprising a detection antibody (or a detection antigen-binding fragment thereof) targeted to a group A streptococcus specific antigen, at least one of N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine, and instructions for use. The detection antibody may be a polyclonal or monoclonal antibody. In certain embodiments, the detection antibody is a polyclonal antibody. Additionally or alternatively, in some embodiments, the kit comprises an immunoassay device as described herein. In addition to the immunoassay device, the kit may further include one or more of the following: written instructions for using the device and collecting a biological sample; an instrument or tool for collecting the biological sample, a label for characterizing the device, a container or vial containing an extraction reagent for processing the collected sample, and a container or vial containing a reagent for preparing the processed sample. The kit may optionally include instructions for reading and interpreting the assay results. The kit may further include a reference sample that may be used to compare the test results to the specimen sample. In certain embodiments, the kit includes a swab for collecting the biological sample, a container or vial containing an extraction reagent for processing the collected biological sample, and instructions for using the assay and collecting a sample, the instructions not including, for example, a warning against contacting one or more of the throat, tonsils, cheek, or back of the tongue.

[0052] In another aspect, the disclosure provides a method for detecting streptococcus specific antigens comprising the steps of: (a) a matrix having (i) a sample receiving zone for receiving a sample containing or suspected of containing a group A streptococcus specific antigen; (ii) a labeling zone containing a detection antibody (or a detection antigen binding fragment thereof) for specifically labeling the antigen as it passes therethrough; (iii) a capture zone having means thereon for specifically binding the labeled antigen; and optionally (iv) an absorption zone, wherein the sample receiving zone, the labeling zone, and the capture zone (and any present and (b) a container containing an extraction reagent, wherein at least one of the extraction reagent, the sample-receiving zone, the capture zone, and the label zone contains N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamine, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine.

[0053] Additionally or alternatively, in some embodiments, the kit includes a lateral flow immunoassay test strip contained in a cassette with a sample receiving chamber, the test strip including a sample pad including N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine. The N-propionyl-D-glucosamine, 2-N-butanoyl-D-glucosamide, bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, m-PEG4-glucosamine, m-PEG6-glucosamine, or m-PEG10-glucosamine may be in a dried form. Additionally or alternatively, in some embodiments, the kit further comprises one or more of a vial containing a reagent solution, a dropper tip that fits securely over the open end of the vial, a sterile rayon swab, a positive control swab (e.g., coated with heat inactivated non-infectious Group A Streptococcus), a negative control swab (e.g., heat inactivated non-infectious Group C Streptococcus), and instructions for use. Test strips can be designed to function in conjunction with an instrument capable of reading reflectance or fluorescent or luminescent signals emitted from test and control lines on the test strip. The kit may further comprise one or more of the following: washing buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, and detection means. These buffers and / or reagents are usually optimized for the particular amplification / detection technique for which the kit is intended. Protocols for using these buffers and reagents to carry out the various steps of the procedure may also be included in the kit. EXAMPLES

[0054] example Example 1: Inclusion of N-propionyl-D-glucosamine reduces false positive and false negative signals in Strep A lateral flow immunoassays Saliva samples were collected from four asymptomatic patients and examined for specificity with immunoassay test strips. Previous studies have reported that samples obtained from oral tissue, saliva, cheek, and tongue of healthy control patients showed high false positive rates for Strep A when assayed by lateral flow immunoassay. See US20130196337A1. N-propionyl-D-glucosamine was examined for its ability to inhibit or block the binding of anti-Strep A polyclonal antibodies in a Strep A immunoassay. Saliva samples from four asymptomatic patients were obtained for testing. Using an immunoassay device for detection of Strep A, each of the four saliva samples was treated with an extraction reagent (e.g., the extraction reagent of the commercially available BD Veritor™ Strep A kit) in the presence of N-propionyl-D-glucosamine, which had dried onto the conjugate pad during spraying of the liquid conjugate onto the conjugate pad (label zone). In the immunoassay, different concentrations of N-propionyl-D-glucosamine were evaluated for blocking Strep A false positive signals.

[0055] As shown in Figures 1-4, false positive signals from saliva samples were effectively suppressed by the addition of N-propionyl-D-glucosamine, demonstrating that cross-reactivity between Strep A polyclonal antibodies and human tissues or cells from the oral cavity was effectively suppressed. Furthermore, the addition of N-propionyl-D-glucosamine did not decrease the overall sensitivity of the Veritor™ Strep A assay. See Figures 1 and 3. These results demonstrate that the addition of N-propionyl-D-glucosamine to the immunoassay for Strep A improves the specificity of the test for group A streptococcus and reduces the false negative rate, unlike NAG. Thus, N-propionyl-D-glucosamine can be used to effectively reduce false positives and false negatives in the test. Those skilled in the art will appreciate that the amounts of antibody, N-propionyl-D-glucosamine, and sample can be adjusted to optimize the observation of a positive signal from S. pyogenes while blocking false positives from N-propionyl-D-glucosamine. These results demonstrate that the compositions and devices disclosed herein are useful in methods for detecting the presence of at least one group A streptococcus species in a biological sample.

[0056] Example 2: Inclusion of 2-N-butanoyl-D-glucosamide or Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide reduces false positive and false negative signals in Strep A lateral flow immunoassays 2-N-butanoyl-D-glucosamide, or Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, were examined for their ability to inhibit or block the binding of anti-Strep A polyclonal antibodies in a Strep A immunoassay (Veritor™ Strep A Assay). Specificity was examined with immunoassay test strips using porcine mucin samples as surrogates for human saliva samples. The effect of 2-N-butanoyl-D-glucosamide, or Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide on the performance of the Strep A assay was evaluated using a 4-fold concentration of mucin standard (approximately 2.5 mg / mL) and Test Standard 1 using an immunoassay device for the detection of Strep A. Either 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide was added directly to the samples. Different concentrations of 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide were evaluated for inhibition of Strep A false positive signals in the immunoassay. As shown in Figures 5-6, false positive signals from porcine mucin samples were effectively suppressed by the addition of 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, demonstrating that cross-reactivity between Strep A polyclonal antibodies and mucin was effectively suppressed. Furthermore, the addition of 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide did not significantly reduce the overall sensitivity of the Veritor™ Strep A assay.

[0057] Figure 7 shows the effect of bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide added to the samples on the performance of two lots of Strep A devices that previously showed mucin cross-reactivity. The dimer slightly suppressed the true sensitivity while significantly reducing the mucin-induced false positive signal. FIG. 8 shows the effect of adding Bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide to the conjugate pad and sample pad soaking solutions on the performance of the Strep A device. These effects demonstrate that false positive signals are significantly reduced by adding the dimer to either the conjugate pad soaking solution and / or the sample pad soaking solution. FIG. 9 demonstrates that lower concentrations of Bis(2-(D-2-deoxy-glucosaminyl))-PEG3-amide dimer can effectively eliminate mucin cross-reactivity without significantly affecting true sensitivity when added to the conjugate pad soaking solution. These results demonstrate that the addition of 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide to the immunoassay for Strep A improves the sensitivity of the test for group A streptococcus and reduces the false negative rate, unlike NAG. Thus, 2-N-butanoyl-D-glucosamide or bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide can be used to effectively reduce false positives and false negatives in the test. One skilled in the art will appreciate that the amounts of antibody, 2-N-butanoyl-D-glucosamide or Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide, and sample can be adjusted to optimize observation of a positive signal from S. pyogenes while blocking false positives with 2-N-butanoyl-D-glucosamide or Bis-(2-(D-2-deoxy-glucosaminyl))-PEG3-amide. These results demonstrate that the compositions and devices disclosed herein are useful in methods for detecting the presence of at least one group A streptococcus species in a biological sample.

[0058] Example 3: Inclusion of m-PEG4-Glucosamine, m-PEG6-Glucosamine, and m-PEG10-Glucosamine reduces false positive and false negative signals in a Strep A lateral flow immunoassay. m-PEG4-Glucosamine, m-PEG6-Glucosamine, m-PEG10-Glucosamine and m-PEG5000-Glucosamine were examined for their ability to inhibit or block the binding of anti-Strep A polyclonal antibodies in a Strep A immunoassay. Porcine mucin was dissolved in water and used as a surrogate for human saliva samples. m-PEG4-Glucosamine, m-PEG6-Glucosamine, m-PEG10-Glucosamine or m-PEG5000-Glucosamine was added directly to the samples. Different concentrations of m-PEG4-Glucosamine, m-PEG6-Glucosamine, m-PEG10-Glucosamine and m-PEG5000-Glucosamine were evaluated for inhibition of Strep A false positive signals in the immunoassay. As shown in Figure 10, different concentrations of m-PEGn(4,6,10)-glucosamine were mixed with either a Strep A antigen-containing sample or a porcine mucin sample before adding them to the sample wells of the lateral flow test device. The results imply that m-PEG4-glucosamine, m-PEG6-glucosamine, and m-PEG10-glucosamine are effective in eliminating mucin cross-reactivity without significantly affecting the sensitivity of the Strep A assay. In contrast, m-PEG5000-glucosamine did not reduce the false positive signal caused by mucin, but instead increased the false positive signal in the presence of mucin. See Figure 11. However, in the absence of mucin, no false positive signal was observed when added to the extraction solution (GAS1+2). These results demonstrate that the addition of m-PEG4-Glucosamine, m-PEG6-Glucosamine, and m-PEG10-Glucosamine to the immunoassay for Strep A improves the sensitivity of the test for group A streptococcus and, unlike NAG, reduces the false negative rate. These results demonstrate that the compositions and devices disclosed herein are useful in methods for detecting the presence of at least one group A streptococcus species in a biological sample.

[0059] Equivalent The present technology is not limited in terms of the specific embodiments described in this application, which are intended to be merely illustrative of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from the spirit and scope of the present technology. From the foregoing description, those skilled in the art will recognize functionally equivalent methods and devices within the scope of the present technology, in addition to those listed herein. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that the present technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, which may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting. The terms "comprising," "including," "containing," and the like, are to be read expansively and open-ended. Moreover, the terms and expressions used herein are used as descriptive and open-ended terms, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described, but rather to recognize that various modifications are possible within the scope of the claims of the present disclosure. Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the disclosure is consequently described in terms of any individual member or subgroup of members of the Markush group.

[0060] As one of ordinary skill in the art will appreciate, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass any and all possible subranges and combinations thereof. Any stated range can be readily recognized as fully describing and enabling the same range broken down into at least an equivalent half, third, quarter, fifth, tenth, etc. As a non-limiting example, each range described herein can be readily broken down into a lower third, middle third, upper third, etc. As one of ordinary skill in the art will also appreciate, all words such as "up to," "at least," "more than," "less than," etc. refer to ranges that include the recited numbers and that can be subsequently broken down into the subranges described above. Finally, as one of ordinary skill in the art will appreciate, ranges include each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc. All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the explicit teachings of this specification.

Claims

[Claim 1] The invention described in the specification.

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