WO2008057605A2 - Nuclear protein extraction medium and method of use - Google Patents

Abstract

An extraction medium for use in detection of a protein in a biological sample is described. The extraction medium contains a combination of reagents, including a nuclease, that aid in nuclear protein extraction from the biological sample. The medium is particularly suited for use in commercially-available rapid diagnostic assays, where the medium improves sensitivity of the assay to the presence of viral nuclear proteins. Kits comprising the extraction medium and methods of using the extraction medium are also described.

Description

NUCLEAR PROTEIN EXTRACTION MEDIUM AND METHOD OF USE

[0001] The application claims the benefit of U.S. application serial no. 60/857,894, filed November 10, 2006, which is incorporated by reference herein.

TECHNICAL FIELD

[0002] An extraction medium for use in the detection, quantification, and/or identification of a protein, particularly a nuclear protein in a sample, is described. The medium is suitable for use with commercially available rapid diagnostic systems for detecting the presence, or absence, of infectious agents in a biological sample. Kits comprising the extraction medium and methods of using the extraction medium are also described.

BACKGROUND

[0003] Various methods of detecting or identifying infectious agents, such as viruses in a sample are known, and include radio or enzyme immunoassay, in vitro culture of the virus, and extraction of the viral DNA or RNA for genomic identification. These methods are suitable in cases where a rapid medical decision at the point-of-care is not necessary. For point-of-care decisions, rapid diagnostic assays are a preferred approach, where detection of viral antigen in a patient sample can be made at the point-of-care of the patient.

[0004] Rapid diagnostic assays generally rely upon immunological detection of viral antigens, frequently employing a sandwich-type immunoassay method. A biological sample, such as blood, urine, or a nasopharyngeal or nasal discharge acquired via wash, aspirate or swab, is often employed in such devices. Detection of pathogenic organisms, such as a virus, and/or macromolecular entities associated with the organism, in the biological sample can be limited by the sensitivity and/or rapidity of the detection system. Moreover, the amount of biological sample available may be small and/or the concentration of the pathogenic organism in the sample may be very low, further complicating identification and/or detection of pathogenic organisms in the sample.

[0005] Nucleases are enzymes that degrade DNA or RNA molecules by catalysing the cleavage of the phosphodiester bonds that link adjacent nucleotides. Deoxyribonuclease (DNAse) utilizes DNA as its substrate and ribonuclease (RNAse) utilizes RNA as its substrate. Endonucleases cleave at internal sites in the substrate molecule, while exonucleases progressively cleave from the end of the substrate molecule. Nucleases have varying degrees of base-sequence specificity, the most specific being the restriction endonucleases. In assays and methods where preserving the integrity of RNA or DNA in a sample is required, nucleases are detrimental and much effort is spent to ensure the media involved in the assay or method are free of nucleases.

[0006] For detection and/or identification of viral antigens, the sensitivity of rapid assays is dependent, in part, upon the presence of sufficient viral antigen to provide a visual signal within the assay. Such assays can be adversely affected when the biological sample lacks ample viral antigen, whether due to dilution of the sample, for example as may happen with a nasal wash, or due to insufficient viral shedding, for example during the early stages of infection in an adult. In situations where a sample comprises a low concentration of viral particles, efficient extraction of viral antigen from the sample is desired. Extraction of sufficient antigen protein when the antigen protein is part of a nucleic acid/protein complex, that is, where the antigen protein is associated with a nucleic acid, is particularly desirable to generate a sufficient assay signal for detection or identification of the antigen protein. At the same time, it is desirable that a user of a rapid diagnostic assay be able to readily distinguish between background noise and signal, so that positive versus negative results may be easily read. Too often, approaches to increase signal to improve detection of smaller analyte quantities are accompanied by an unacceptable increase in background noise. Thus, there remains a need for reagents used in the detection of infectious agents, particularly viral nuclear proteins that improve the sensitivity of the assay. BRIEF SUMMARY

[0007] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.

[0008] In one aspect, a method for detecting protein in a biological sample is provided, the method comprising contacting the sample with an extraction medium comprising a nuclease, incubating the sample and extraction medium to achieve extraction of protein from the sample, and detecting protein in the sample.

[0009] In another aspect, a kit for use extraction, analysis, and/or detection of a protein in a biological sample is provided, the kit comprising a rapid diagnostic assay, an extraction medium comprising a nuclease, and instructions for use.

[0010] In one embodiment of both aspects, the nuclease is a ribonuclease (RNase) or a deoxyribonuclease (DNase).

[0011] In some embodiments, the nuclease is an RNase selected from the group consisting of RNase A, RNAse II, RNase T1 , RNase T2, RNase III, RNase IV. In some embodiments, the RNase is a biologically active fragment or derivative of such RNases.

[0012] In some embodiments, the nuclease is a DNase, such as DNase I. In some embodiments, the DNase is a biologically active fragment or derivative of a DNase.

[0013] In some embodiments, the protein is nuclear protein. In other embodiments, the protein is a viral protein or a viral nuclear protein.

[0014] In some embodiments, the rapid diagnostic assay is selected from the group consisting of an immunochromatographic lateral flow assay, an enzyme-linked immunosorbent assay, a time-resolved fluorescence assay, and a chemiluminescence assay.

[0015] In another embodiment, the extraction medium further comprises a zwitterionic detergent. In particular embodiments, the zwitterionic detergent is Λ/-dodecyl-Λ/,/V- dimethylglycine;

[0016] In some embodiments, the extraction medium further comprises Tris (2- carboxyethyl)phosphine hydrochloride (TCEP).

[0017] In some embodiments, the extraction medium further comprises a zwitterionic detergent. In particular embodiments, the zwitterionic detergent is Λ/-dodecyl-Λ/,Λ/- dimethylglycine. [0018] In some embodiments, the extraction medium further comprises Tris (2- carboxyethyl)phosphine hydrochloride (TCEP).

[0019] In another aspect, an extraction medium for extracting a nuclear protein from a biological sample is provided, the extraction medium comprising a nuclease and, optionally, a zwitterionic detergent and/or a buffer.

[0020] In some embodiments, the nuclease is an RNase. In some embodiments, the nuclease is a DNase. In some embodiments, the zwitterionic detergent is N-dodecyl-

N,N-dimethylglycine betaine.

[0021] In some embodiments, the extraction medium further comprises a reducing agent, such as tris (2-carboxyethyl)phosphine hydrochloride (TCEP). In some embodiments, the extraction medium further comprises a chelator, such as ethylenediaminetetraacetic acid (EDTA).

[0022] In some embodiments, the extraction medium includes a salt, such as MgCI₂ or NaCI, and/or a serum albumin, such as bovine serum albumin.

[0023] In particular embodiments, the concentration of RNase A in the medium is between about 0.05 mg/mL and about 0.10 mg/mL, more preferably between about

0.10 mg/mL and about 0.20 mg/mL, and still more preferably between about 0.20 mg/mL and about 0.50 mg/mL.

[0024] An exemplary extraction medium comprises RNase A, Tris hydroxymethyl- aminoethane buffer, ethylenediaminetetraacetic acid (EDTA), and a zwitterionic detergent. In one embodiment, the concentrations of each component in the medium are 0.2 mg/mL RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 10 mM MgCI₂; 124 mM NaCI; 1wt% bovine serum albumin (BSA); and 0.025 wt% zwitterionic detergent.

[0025] In another aspect, an improvement in a method for detecting a nuclear protein in a biological sample using a rapid diagnostic assay is provided, the improvement comprising providing a medium comprising a nuclease to achieve a decreased time to signal when compared to a time to signal obtained using the same rapid diagnostic assay with a medium that does not comprise nuclease.

[0026] In some embodiments, the medium further comprises a zwitterionic detergent.

[0027] Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the subject matter described herein will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in a one-step commercially available immunoassay diagnostic device as a function of RNase A concentration, in μg/mL, in the reagent mixture supplied with the device, for detection of inactivated influenza A/Panama/2007/99, subtype H3N2 virus.

[0029] Fig. 2 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in a one-step commercially available immunoassay diagnostic device as a function of protein (inactivated influenza A/Panama/2007/99, subtype H3N2 virus) concentration, in ng/mL, with (diamonds) and without (squares) RNase A added to the regent mixture supplied with the device. [0030] Fig. 3 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in a one-step commercially available immunoassay diagnostic device, as a function of protein (inactivated Flu B/Qindao/102/91 virus) concentration, in ng/nL, with (diamonds) and without (squares) RNase A added to the regent mixture supplied with the device.

[0031] Fig. 4 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in an immunoassay diagnostic device, as a function of RNase A concentration, in μg/mL, in the reagent mixture employed with the device, for detection of inactivated influenza A/Panama/2007/99, subtype H3N2 virus. [0032] Fig. 5 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in an immunoassay diagnostic device, as a function of protein (inactivated influenza A/Panama/2007/99, subtype H3N2 virus) concentration, in μg/mL,. with (diamonds) and without (squares) RNase A added to the reagent mixture employed with the device. [0033] Fig. 6 shows the time to appearance of a discernible signal at the capture line ("Time to Signal", in minutes:seconds) in an immunoassay diagnostic device, as a function of protein (inactivated influenza type B/Qingdao/102/91 virus) concentration, in μg/mL, with (diamonds) and without (squares) RNase A added to the reagent mixture employed with the device.

DETAILED DESCRIPTION I. Definitions

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. For example, while particular reference is made to extraction of viral proteins, it will be understood that the extraction medium can be used with any protein, viral or non-viral. [0035] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. [0036] The term "or" is not meant to be exclusive to one or the terms it designates. For example, as it is used in a phrase of the structure "A or B" may denote A alone, B alone, or both A and B.

[0037] As used herein, the term "nuclear protein" or "nucleoprotein" refers to a protein localized to a cell nucleus, especially one that is associated with, whether covalently, ionically or otherwise, a nucleic acid. A viral nuclear protein is a nuclear protein that is found within a virus.

II. Extraction Medium and Methods of Use

[0038] An extraction medium for use with a diagnostic assay, and preferably with a rapid diagnostic assay, is described. The extraction medium enhances sensitivity of the diagnostic device to an analyte in a biological sample, as will be seen in the examples set forth below. Without being bound by any particular theory for the mechanism of action, it is believed that the extraction medium disclosed herein increases the availability of nuclear protein for detection in the relevant assay. The extraction medium, particularly when employed in conjunction with a diagnostic assay device, permits accurate detection of an analyte in, for example, clinical, experimental, and/or epidemiological samples.

[0039] The extraction medium can be used for detecting, identifying, and/or quantifying at least one protein in a sample. In a preferred embodiment, the medium is used as part of a diagnostic tool for detecting a viral infection in an animal. By increasing the sensitivity of the diagnostic tool to the viral antigen, the extraction medium permits detection of a viral antigen in a sample in situations where the viral antigen may normally go undetected.

A. Extraction Medium Components

[0040] Accordingly, in a first aspect, an extraction medium comprising a nuclease is provided. The nuclease may be, for example, any enzyme that cleaves a nucleic acid. Exemplary nucleases include endonucleases and exonucleases, preferably endoribonucleases and exoribonucleases. Specific examples of nucleases include, but are not limited to, a DNase or an RNase, for example, single stranded RNases such as RNase A, RNAse II, RNase T1 , RNase T2 or double stranded RNases such as RNase III or RNase IV. It will be appreciated that enzymatically active fragments of an RNase or a DNase are suitable for use as the nuclease. Other nucleases can be used without departing from the scope of the reagents and methods.

[0041] In certain embodiments, the extraction medium further comprises a salt buffer and/or a zwitterionic detergent. Suitable zwitterionic detergents include, for example, N- dodecyl-N,N-dimethylglycine betaine (Empigen^® BB, CalBiochem, San Diego, CA), amidosulfobetaine-16, NDSB 201 , and 3-(N, N-Dimethylpalmitylamononic)-propane sulfonate. The amount of detergent included in the extraction medium can vary as will be appreciated by a skilled artisan, but is typically in the range of from about 0.01 to 0.1 wt%, 0.01 to 0.07 wt%, 0.01 to 0.05 wt%, and 0.01 to 0.03 wt%. In a preferred embodiment 0.025 wt % of a zwitterionic detergent is included in the extraction medium. [0042] The salt buffer component in the medium is selected based on the particular protein, virus, biological sample, and detection device involved in the assay. The salt buffer components are also selected, where needed, to maintain nuclease activity. Exemplary buffers and components of the buffer include, for example, Tris, phosphate buffer, HEPES, at the appropriate pH, such as, for example, pH 9.4, or the pH appropriate for the particular nuclease activity. The buffer may further include a reducing agent, such as, for example, TCEP or DTT. The buffer may further include a chelator such as, for example, EDTA, for example from 20 to 50, 25 to 45, 30 to 35, or 32.9 mM EDTA. The buffer may further include an antibody, such as, for example, mouse IgG, and may further include bovine serum albumin (BSA). [0043] In another embodiment, the medium may further include a salt such as MgCI₂, MnCI₂, CoCI₂, NaCI, or CaCI₂. The amount of salts may vary, and may include, for example, from 1 to 25, 5 to 20, 7 to 15, 8-12, or 10 mM MgCI₂, may include, for example, from 50 to 250, from 75 to 200, from 80 to 160, from 90 to 140, from 110 to 130, from 120 to 125, or 124 mM NaCI.

[0044] As known by those of skill in the art, each of the components, the pH of the medium, and the temperature of the assay, may be titrated to obtain optimal activity. The medium may include components as suggested by the nuclease or device manufacturer's instructions.

[0045] In a preferred embodiment, particularly useful for detecting a nuclear protein antigen of a single-stranded RNA virus, the nuclease RNase A is used in the extraction medium. Those of ordinary skill in the art may modify the amount of RNase A within the ranges taught herein and likewise may modify the incubation time as appropriate for the particular virus to be detected and/or specimen being used. By way of example, those of ordinary skill in the art will recognize that a longer incubation time may be appropriate where the virus to be detected may be at a low concentration in the sample and or the sample size is small. Advantageously, the extraction medium of the present invention may used with various commercially available rapid detection devices, such as rapid immunoassays, and/or typical ELISA assays. Reference to the assay's protocol or device's package insert may be relied upon to determine the approximate time for such incubation step.

[0046] The amount of RNase, for example, RNase A, in the extraction medium may be, for example from 0.02 to 1 mg/mL, 0.02 to 0.75 mg/mL, 0.02 to 0.6 mg/mL, 0.02 to 0.1 mg/mL, 0.02 to 0.5 mg/mL, 0.02 to 0.45 mg/mL, 0.02 to 0.4 mg/mL, 0.02 to 0.35 mg/mL, 0.02 to 0.3 mg/mL, 0.02 to 0.25 mg/mL, 0.02 to 0.2 mg/mL, 0.02 to 0.15 mg/mL, 0.02 to 0.1 mg/mL, 0.02 to 0.5 mg/mL, 0.05 to 1 mg/mL, 0.05 to 0.75 mg/mL, 0.05 to 0.6 mg/mL, 0.05 to 0.1 mg/mL, 0.05 to 0.5 mg/mL, 0.05 to 0.45 mg/mL, 0.05 to 0.4 mg/mL, 0.05 to 0.35 mg/mL, 0.05 to 0.3 mg/mL, 0.05 to 0.25 mg/mL, 0.05 to 0.2 mg/mL, 0.05 to 0.15 mg/mL, 0.05 to 0.1 mg/mL, 0.07 to 1 mg/mL, 0.07 to 0.75 mg/mL, 0.07 to 0.6 mg/mL, 0.07 to 0.1 mg/mL, 0.07 to 0.5 mg/mL, 0.07 to 0.45 mg/mL, 0.07 to 0.4 mg/mL, 0.07 to 0.35 mg/mL, 0.07 to 0.3 mg/mL, 0.07 to 0.25 mg/mL,.0.07 to 0.2 mg/mL, 0.07 to 0.15 mg/mL, 0.07 to 0.1 mg/mL, 0.1 to 1 mg/mL, 0.1 to 0.75 mg/mL, 0.1 to 0.6 mg/mL, 0.1 to 0.1 mg/mL, 0.1 to 0.5 mg/mL, 0.1 to 0.45 mg/mL, 0.1 to 0.4 mg/mL, 0.1 to 0.35 mg/mL, 0.1 to 0.3 mg/mL, 0.1 to 0.25 mg/mL, 0.1 to 0.2 mg/mL, 0.1 to 0.15 mg/mL, 0.2 to 1 mg/mL, 0.2 to 0.75 mg/mL, 0.2 to 0.6 mg/mL, 0.2 to 0.1 mg/mL, 0.2 to 0.5 mg/mL, 0.2 to 0.45 mg/mL, 0.2 to 0.4 mg/mL, 0.2 to 0.35 mg/mL, 0.2 to 0.3 mg/mL, 0.2 to 0.25 mg/mL, 0.3 to 1 mg/mL, 0.3 to 0.75 mg/mL, 0.3 to 0.6 mg/mL, 0.3 to 0.1 mg/mL, 0.3 to 0.5 mg/mL, 0.3 to 0.45 mg/mL, 0.3 to 0.4 mg/mL, 0.3 to 0.35 mg/mL, 0.4 to 1 mg/mL, 0.4 to 0.75 mg/mL, 0.4 to 0.6 mg/mL 0.4 to 0.1 mg/mL, 0.4 to 0.5 mg/mL, or 0.4 to 0.45 mg/mL.

[0047] Where the nuclease in the extraction medium is DNase, such as, for example, DNase I, the amount of DNase in the extraction medium may be, for example, from 0.02 to 1 mg/mL, 0.02 to 0.75 mg/mL, 0.02 to 0.6 mg/mL, 0.02 to 0.1 mg/mL, 0.02 to 0.5 mg/mL, 0.02 to 0.45 mg/mL, 0.02 to 0.4 mg/mL, 0.02 to 0.35 mg/mL, 0.02 to 0.3 mg/mL, 0.02 to 0.25 mg/mL, 0.02 to 0.2 mg/mL, 0.02 to 0.15 mg/mL, 0.02 to 0.1 mg/mL, 0.02 to 0.5 mg/mL, 0.05 to 1 mg/mL, 0.05 to 0.75 mg/mL, 0.05 to 0.6 mg/mL, 0.05 to 0.1 mg/mL, 0.05 to 0.5 mg/mL, 0.05 to 0.45 mg/mL, 0.05 to 0.4 mg/mL, 0.05 to 0.35 mg/mL, 0.05 to 0.3 mg/mL, 0.05 to 0.25 mg/mL, 0.05 to 0.2 mg/mL, 0.05 to 0.15 mg/mL, 0.05 to 0.1 mg/mL, 0.07 to 1 mg/mL, 0.07 to 0.75 mg/mL, 0.07 to 0.6 mg/mL, 0.07 to 0.1 mg/mL, 0.07 to 0.5 mg/mL, 0.07 to 0.45 mg/mL, 0.07 to 0.4 mg/mL, 0.07 to 0.35 mg/mL, 0.07 to 0.3 mg/mL, 0.07 to 0.25 mg/mL, 0.07 to 0.2 mg/mL, 0.07 to 0.15 mg/mL, 0.07 to 0.1 mg/mL, 0.1 to 1 mg/mL, 0.1 to 0.75 mg/mL, 0.1 to 0.6 mg/mL, 0.1 to 0.1 mg/mL, 0.1 to 0.5 mg/mL, 0.1 to 0.45 mg/mL, 0.1 to 0.4 mg/mL, 0.1 to 0.35 mg/mL, 0.1 to 0.3 mg/mL, 0.1 to 0.25 mg/mL, 0.1 to 0.2 mg/mL, 0.1 to 0.15 mg/mL, 0.2 to 1 mg/mL, 0.2 to 0.75 mg/mL, 0.2 to 0.6 mg/mL, 0.2 to 0.1 mg/mL, 0.2 to 0.5 mg/mL, 0.2 to 0.45 mg/mL, 0.2 to 0.4 mg/mL, 0.2 to 0.35 mg/mL, 0.2 to 0.3 mg/mL, 0.2 to 0.25 mg/mL, 0.3 to 1 mg/mL, 0.3 to 0.75 mg/mL, 0.3 to 0.6 mg/mL, 0.3 to 0.1 mg/mL, 0.3 to 0.5 mg/mL, 0.3 to 0.45 mg/mL, 0.3 to 0.4 mg/mL, 0.3 to 0.35 mg/mL, 0.4 to 1 mg/mL, 0.4 to 0.75 nng/mL, 0.4 to 0.6 mg/mL, 0.4 to 0.1 mg/mL, 0.4 to 0.5 mg/mL, or 0.4 to 0.45 mg/mL. Those of ordinary skill in the art may readily modify nuclease concentrations in the extraction medium based upon the above and their knowledge of the particular nuclease being used, including the expected activity of that nuclease in the particular sample being tested. Simple titrations may be employed, where appropriate, to identify most preferred nuclease concentrations for the particular organism being treated.

B. Methods of Using the Extraction Medium

[0048] The improved extraction medium described herein may be used for a general procedure, such as detection, quantification, and/or identification, of infectious agents with distinct biological and architectural characteristics, as in the diagnosis of viral or bacterial infections, particularly viral infections. Viruses that may be detected, identified, and/or quantified can be any DNA or RNA virus. In a preferred embodiment the extraction medium is used to detect, identify, and/or quantify a single-stranded or double-stranded RNA virus that infects a human. Exemplary DNA and RNA viruses include, but are not limited to, adenoviruses 1-49, astroviruses, B-virus (Cercopithecus herpesvirus), BK virus, Bunyamwera virus, California encephalitis virus, Central European encephalitis virus, Colorado tick fever virus, Cong-Crimean hemorrhagic fever virus, cornoaviruses, cowpox virus, coxsackie A 1-22, A 24 viruses, coxsackie B 1-6 viruses, Creutzfeldt-Jakob virus, cytomegalovirus, delta virus, Dengue 1-4 virus, Duvenhage virus, Eastern equine encephalitis virus, ebola virus, echo 1-9, 11-27, 29-34 viruses, enteroviruses 68-71 , Epstein-Barr virus, ET non-A/non-B hepatitis virus, Hantaan virus, hepatitis A virus, hepatitis B virus, herpes simplex 1 and 2 viruses, human immunodeficiency 1 and 2 viruses, human T-lymphotropic 1 and 2 viruses, Japanese encephalitis virus, JC virus, Junin virus, Kuru virus, Kyasanur Forest virus, La Crosse virus, Lassa virus, Lymphocytic choriomeningitis virus, respiratory syncytial virus, influenza virus, including, but not limited to influenza A virus (including, but not limited to, influenza A/Panama/2007/99 subtype H3N2, influenza B (including, but not limited to, influenza B/Qingdao/102/91) and influenza C. [0049] In a study conducted to illustrate the improved sensitivity of a diagnostic device to detect a viral protein using the extraction medium described herein, an extraction medium comprising RNase A, Tris buffer, a zwitterionic detergent, N-dodecyl- N,N-dimethylglycine, was prepared. As described in Example 1 , the extraction medium, containing various concentrations of RNase A, was used in a one-step commercially available rapid diagnostic device (QuickVue^® Influenza A+B) for extraction of nucleoprotein antigen from inactivated influenza A virus (A/Panama/2007/99). The sample and the extraction medium were incubated at a temperature and for a time sufficient to extract the protein from the sample. Specifically, the Flu A and the extraction medium were incubated at room temperature for about 30 seconds. After incubation, the samples, containing a fixed amount of influenza A virus and the extraction medium, were applied individually to a test strip in a rapid diagnostic device. The time to appearance of a discernible signal at the capture line on the test strip, referred to herein as the "time to signal", was recorded. An extraction medium without RNase A was used as a control. The results are shown in Fig. 1. [0050] As seen in Fig. 1 , increasing concentrations of RNase A decreased the time to appearance of a discernible signal on the diagnostic test strip, to indicate the presence (or absence) of the viral protein. The decreased time to signal is evidence of the extraction medium providing an enhanced sensitivity of the diagnostic device. The optimal concentration of RNase A using this diagnostic device with this flu strain was approximately 0.2 mg/mL, as further increases in RNase A concentration, e.g, from 0.2 mg/mL to 0.4 mg/mL, did not result in any further significant decrease in the time to signal. This study was carried out at room temperature since the diagnostic device is designed to perform at room temperature. It will be appreciated that temperature affects enzymatic activity and conducting the study at different temperatures will change the observed time to signal.

[0051] In another study, described in Example 2, the same immunoassay diagnostic device was used in the presence of an extraction medium containing a fixed amount of RNase A to detect various concentrations of influenza A/Panama/2007/99 and influenza B/Qingdao/102/91. The results are shown in Figs. 2 and 3. The data shows that the presence of RNase A (diamond symbols in Figs. 2-3) in the extraction medium improved the sensitivity of the diagnostic device to both viral proteins, particularly at viral protein concentrations of less than about 300 ng/mL, less than about 200 ng/ml, less than about 150 ng/mL, less than about 100 ng/mL, less than about 75 ng/mL, less than about 50 ng/mL, or less than about 25 ng/mL.

[0052] In other studies, described in Examples 3-4, an immunoassay diagnostic device described in U.S. Patent Publication No. 2006/0078986 was used to ascertain the time to signal at varying concentrations of RNase A and of the viral proteins influenza A/Panama/2007/99 and influenza B/Qingdao/102/91. The results are seen in Figs. 4-6. Fig. 4 shows that addition of RNase A to the extraction medium decreased the time to signal, where 50 μg/mL of RNase A in the medium achieved a 42% decrease in time to signal. Thus, in one embodiment, the extraction medium improves sensitivity of a diagnostic device by decreasing the time to signal by at least about 30%, more preferably by at least about 40%.

[0053] Figs. 5-6 show the time to signal for detection of various concentrations of influenza A/Panama/2007/99 (fig. 5) and influenza B/Qingdao/102/91 (Fig. 6) in the presence of extraction media with RNase A (diamonds) and without RNase A (squares), using an immunoassay diagnostic device described in U.S. Patent Publication No. 2006/0078986. At viral protein concentrations of less than about 400 ng/mL, more particularly of less than about 100 ng/mL, an extraction medium having RNase A improved the time to signal by at least about 25%, more preferably by at least about 50%. The greatest improvement in time to signal was observed at low protein concentrations in the range of 1-50 ng/mL, more preferably in the range of 1-35 ng/mL. Thus, the extraction medium permits early detection of host infection by viral pathogens since lower concentrations of the pathogen is required for detection. [0054] In summary, the studies described herein show that addition of a nuclease to the extraction medium used in a diagnostic device improves the analytical sensitivity of the device. In particular, the nuclease RNase A.which is known to cleave single stranded RNA on the 3' side of pyrimidine residues, improved detection of viral nucleoprotein antigens. Nucleoproteins are known to be associated with, and possibly occluded by, viral RNA. Therefore, without limitation as to the scope of the subject matter described herein, cleaving the viral RNA with RNase A may act in releasing the nucleoprotein antigen from the RNA, making it more available to the antibodies within the device and thereby increasing sensitivity to the presence of the virus(es) being tested.

[0055] In one embodiment, the extraction medium is used for detection of an intact virus. In another embodiment, the extraction medium is used for detection of a non- intact virus. Intact verses non-intact viruses are strain dependent. In general, a non- intact virus may be less stable and more easily degradable than an intact virus. This may especially be in the case where the antigen being assayed for is an internal component of the virus such as a nuclear protein. A non-intact virus also may not require extraction if the antigen being assayed for is already exposed and readily available for an antibody to bind to it. However, the antigen being assayed for in a non- intact virus may be more susceptible to degradation by the surrounding environment because the virus is not a complete unit and cannot protect the antigen from being exposed. For example, in the case of a nucleoprotein antigen, of an RNA virus, this antigen can form a complex with the RNA and polymerase within the virus, which has a protective coating typically consisting of proteins, lipids, glycoproteins or a combination thereof. Therefore, extraction with an extraction medium with, in this example, RNase A enhances the ability to detect the nucleoprotein antigen by cleaving the RNA and exposing multiple potential binding sites for an antibody to bind. An example of an exemplary intact virus is influenza A virus (A/Panama/2007/99). Hence assaying for the presence of a nucleoprotein antigen in an intact virus such as influenza A virus (A/Panama/2007/99) with and without using an RNase A extraction medium in accordance herewith displays a greater sensitivity difference than with a non-intact virus.

[0056] In one embodiment, the extraction medium is used in the detection, quantification, and/or identification of viral pathogens for which no antibodies are available against the capsid proteins.

[0057] The extraction medium may be used at room temperature 20-25 ⁰C, or more generally in the temperature range of 15-3O⁰C (59-86⁰F). The medium is preferably suitable for storage at room temperature, but may be stored at 4⁰C or -2O⁰C for long- term storage. Those of skill in the art will appreciate that a storage temperature can be selected according to the requirements of the components in the extraction medium, and storage at other than room temperature is contemplated for components that benefit, for example by longer retention of optimal activity, from storage at particular temperatures. In particularly preferred embodiments, the extraction medium is provided in dried form and reconstituted by the user at the time of conducting the diagnostic assay. Placement out of direct sunlight is recommended.

[0058] In another embodiment, a method for determining the optimal amount of the extraction medium for use with a selected diagnostic device and a particular viral protein is provided. To the extent needed, one of skill in the art can identify with routine studies using extraction media with different concentrations of the nuclease and/or other medium components to ascertain the optimal amounts of the medium, and any particular medium component, for any assay or device.

[0059] As briefly mentioned above, the extraction medium can be used with any diagnostic product on the market or with a homemade or experimental assay or device. In a preferred embodiment, the extraction medium is used in a rapid diagnostic device, which refers to a diagnostic assay capable of signaling to a user the presence or absence of an analyte in a sample in about 30 minutes or less, preferably in about 20 minutes or less, and more preferably in about 15 minutes or less. By way of example, rapid diagnostic devices used to detect the presence or amount of an analyte that can be used in conjunction with the extraction medium described herein include commercially-available lateral flow immunoassays for detection of influenza A, influenza B, or both. Rapid diagnostic tests provide for detection of a pathogen using one of several techniques, such as immunochromatographic lateral flow assay, enzyme-linked immunosorbent assay, time-resolved fluorescence assay, and chemiluminescence assay.

[0060] It is preferred that the overall time for detection and/or discrimination of the presence or amount of at least one virus in a sample is convenient to a researcher, diagnostician, or laboratory technologist. In certain embodiments, the time to detection is, for example, about 20 minutes or less, about 10 minutes or less, about 9 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less, from the time the extraction medium-treated sample is added to the testing device. In other examples, the time for detection and/or discrimination of the presence or amount of at least one virus in a sample is, for example, about 10 minutes or less, about 9 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, from the time the extraction medium is added to the sample.

[0061] In another aspect, a kit that utilizes the extraction medium is provided. A basic kit for use in extraction of proteins from a biological sample may comprise, for example, RNase A, a salt buffer, and a zwitteronic detergent. The kit may comprise the components of the extraction medium in one, or in multiple containers. For example, the buffer and detergent may be in one container, and the RNase A in a separate container. Or, each of the components may be in separate containers. Or, the buffer, detergent, and RNase A may be present in the same container, under suitable conditions to maintain RNase activity. The kit may further comprise a device used to detect, identify, or quantify proteins, such as, for example, viral proteins, such as, for example, viral nuclear proteins. The kit may comprise diagnostic devices used to diagnose a disease, condition, or infection, such as, for example, a viral infection. Further, the kit may comprise a diagnostic device wherein the extraction medium is contained within a tube or chamber or similar holder that is integral to the diagnostic device such that the assay may be performed as essentially a single step assay with the sample simply being introduced into the extraction medium portion of the device and, after an appropriate incubation time released into the diagnostic portion of the device. Such release may be actuated by, for example, the person administering the test. [0062] The kit may also comprise instructions for use. Such "instructions for use" may describe a reagent concentration, or may describe at least one assay method parameter such as the relative amount of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions, and the like. The instructions for use are suitable to enable a user to carry out the desired assay.

[0063] Other materials useful in the performance of assays, or in preparing the extraction medium can also be included in the kit, including test tubes, transfer pipettes, and the like.

[0064] The amounts of the various reagents in the kits can be varied depending on various factors, such as the optimum sensitivity of the assay, the number of assays to be performed, etc.

[0065] It is contemplated to provide kits for use in manual diagnostic devices or in automated diagnostic analyzers.

III. EXAMPLES

[0066] The following examples are illustrative in nature and are in no way intended to be limiting.

EXAMPLE 1

Use of Extraction Medium for Detection of Inactivated Flu A (A/Panama/2007/99. H3N2) [0067] An extraction reagent comprised of 12.5 mM Tris hydroxymethyl-aminoethane buffer (Tris) at pH 9.4, 32.9 mM ethylenediaminetetraacetic acid (EDTA), 1.5mM Tris(2- carboxyethyl)phosphine hydrochloride (TCEP), 0.025 mg/mL mouse immunoglobulin G (IgG), and 0.025 wt% N-dodecyl- N,N-dimethylglycine (Empigen^® BB) ("reagent mix A") was prepared.

[0068] RNase A was serially diluted with reagent mix A in the ratios shown in Table 1. Inactivated Flu A (A/Panama/2007/99, H3N2) was obtained from Hytest (Cat. 81 N74-1) at 1.5 mg/mL and was repurified to a final total protein concentration estimated at 102 μg/mL. The repurified Flu A was diluted with saline solution in a Flu A:saline ratio of 1 :100 to make a "1 :100 Flu A" dilution. Ten (10) μL of the 1 :100 Flu A dilution was added to 190 μL of the reagent mix A, with and without the RNase A dilutions, at a final ratio of 1:2000 Flu A.

[0069] A rapid diagnostic device for detection of influenza A and B available under the trade designation QuickVue^® Influenza A + B (Quidel Corporation, San Diego, CA) was obtained. Instructions for using and reading the detection assay were carried out as followed according to the package insert. In brief, the test strip from the device was contacted with the various dilutions and the time, in minutes and seconds, for appearance of a discernible signal at the capture line on the test strip was recorded. The results are shown in Table 1 and in Fig. 1.

Table 1 : Time to the appearance of a signal of detection of Flu A in the presence of various concentrations of RNase A

^*data is an average of two tests

EXAMPLE 2

Use of Extraction Medium for Detection of Inactivated Flu A and Flu B [0070] The analytical sensitivity of the one-step assay device used in Example 1 was assessed in the presence of RNase A with inactivated samples of influenza A/Panama/2007/99 and influenza B/Qingdao/102/91. The total protein concentration of the virus in each sample solution was known, and is indicated in Tables 2 and 3. An extraction reagent containing 12.5 mM Tris at pH 9.4, 32.9 mM EDTA, 1.5mM TCEP, 0.025 mg/mL mouse IgG, and 0.025 wt% Empigen^® BB₁ with and without 0.2 mg/mL RNase A, was prepared. The time to signal for the various flu A or flu B concentrations in the extraction media with and without RNase A was determined. The data is shown in Tables 2-3 and in Figs. 2-3.

Table 2: Time to signal for detection of Flu A/Panama/2007/99, subtype H3N2 in the presence and absence of RNase

*data is an average of two tests

Table 3: Time to signal for detection of Flu B/ Qingdao/102/91 in the presence and absence of RNase

^*data is an average of two tests

EXAMPLE 3

Use of Extraction Medium for Detection of Inactivated Flu A

[0071] An extraction reagent comprised of 12.5 mM Tris hydroxymethyl-aminoethane buffer (Tris) at pH 9.4, 0.025 mg/mL mouse immunoglobulin G (IgG), 0.025 wt% N- dodecyl- N,N-dimethylglycine (Empigen^® BB) 10 mM MgCI₂, 124 mM NaCI, and 1 % bovine serum albumin (BSA) ("reagent mix B") was prepared. [0072] A mixture of 399 μL of reagent mix B, 1 μL of inactivated influenza A virus (A/Panama/2007/99; "Flu A",102 ug/mL total protein concentration), and 500 μL of an RNase A dilution as listed in Table 4 was prepared, resulting in a final dilution of 1 :1000 of Flu A. The mixtures were incubated 30 seconds, which was a time sufficient to extract protein from the sample, at room temperature (approximately 20°C-25°C) and then 10% BSA was added. [0073] The solutions were applied to an immunoassay device as described in U.S. Patent Publication No. 2006/0078986, herein incorporated by reference. The time to appearance of a discernible signal at the capture line ("time to signal") was recorded. The results are shown in Table 4 and Fig. 4.

Table 4: Time to signal for detection of Flu A using an immunoassay device as described in U.S. Patent Publication No. 2006/0078986

^*data is average of two tests

EXAMPLE 4

Use of Extraction Medium for Detection of Inactivated Flu A and Inactivated Flu B [0074] The analytical sensitivity of the immunoassay device used in Example 3 was assessed in the presence of RNase A with inactivated samples of Influenza A/Panama/2007/99 and Influenza B/Qingdao/102/91. The total protein concentrations of the virus samples were known, as indicated in Tables 5-6. The viruses were subjected to an extraction reagent containing 12.5 mM Tris at pH 9.4, 0.025 mg/mL mouse IgG₁ 0.025 wt% Empigen^® BB, 10 mM MgCI₂, 124 mM NaCI, 1 % BSA, with and without 0.1 mg/mL RNase A. The inactivated virus was diluted in the extraction reagent and 80 μL of the sample was then loaded to the sample port of the immunoassay device. The time required for the user to discern a visible signal at the capture line of immunoassay device ("time to signal") was recorded in minutes and seconds for each test sample. Results are shown in Figs. 5-6 and Tables 5-6.

Table 5: Time to signal for detection of Flu A using an immunoassay device as described in U.S. Patent Publication No. 2006/0078986

'data is average of two tests

Table 6: Time to signal for detection of Flu B using an immunoassay device as described in U.S. Patent Publication No. 2006/0078986

*data is average of two tests

[0075] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Claims

CLAIMS What is claimed is:

1. A method for detecting protein in a biological sample, comprising: contacting the sample with an extraction medium comprising a ribonuclease

(RNase) or a deoxyribonuclease (DNase), incubating the sample and extraction medium to achieve extraction of protein from the sample, and detecting protein in the sample.

2. The method of claim 1 , wherein at least said detecting is performed in a rapid diagnostic assay.

3. The method of claim 2, wherein the rapid diagnostic assay is selected from the group consisting of an immunochromatographic lateral flow assay, an enzyme-linked immunosorbent assay, a time-resolved fluorescence assay, and a chemiluminescence assay.

4. The method of one of claims 1-3, wherein the RNase is selected from the group consisting of RNase A, RNAse II, RNase T1 , RNase T2, RNase III, and RNase IV.

5. The method of one of claims 1-3, wherein the DNase is DNase I.

6. The method of any preceding claim, wherein the extraction medium further comprises a zwitterionic detergent, a salt buffer, or both.

7. The method of claim 6, wherein the zwitterionic detergent is Λ/-dodecyl-Λ/,Λ/- dimethylglycine.

8. The method of claim 6, wherein the salt buffer is Tris hydroxymethyl- aminoethane buffer.

9. The method of any one of claims 2-5, wherein the rapid diagnostic assay comprises contacting the protein with at least one antibody and detecting whether at least one antibody binds to the protein.

10. The method of claim 9, wherein the protein is a nuclear protein or a viral protein.

11. The method of claim 10, wherein said rapid diagnostic assay comprises an antibody for a viral protein, for detection of a viral infection.

12. The method of claim 11 , wherein the viral infection is influenza.

13. The method of claim 12, wherein the influenza is influenza type A or influenza type B.

14. The method of claim 10, wherein said detecting comprises detecting a quantity of said protein.

15. The method of any preceding claim, wherein said contacting comprises contacting a sample selected from the group consisting of a nasal swab sample, a nasal wash sample, nasal aspiration sample, and a nasal secretion sample.

16. The method of any one of claims 1- 5, wherein the extraction medium further comprises Tris (2-carboxyethyl)phosphine hydrochloride (TCEP).

17. The method of any one of claims 1- 5, wherein the extraction medium further comprises a reducing agent.

18. The method of claim 17, wherein the reducing agent is Tris (2- carboxyethyl)phosphine hydrochloride (TCEP).

19. The method of any one of claims 1- 5, wherein the extraction medium further comprises a chelator.

20. The method of claim 19, wherein the chelator is ethylenediaminetetraacetic acid (EDTA).

21. The method of any one of claims 1- 5, wherein the extraction medium further comprises MgCI_∑, NaCI, or a serum albumin.

22. The method of any one of claims 1- 4, wherein the extraction medium comprises between about 0.05 mg/mL and about 0.50 mg/mL RNase A.

23. The method of any one of claims 1- 4, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 32.9 mM ethylenediaminetetraacetic acid (EDTA); and 0.025 wt% zwitterionic detergent.

24. The method of any one of claims 1- 4, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 10 mM MgCI₂; 124 mM NaCI; 1 wt% bovine serum albumin (BSA); and 0.025 wt% zwitterionic detergent.

25. A kit for analysis of a biological sample, comprising: a rapid diagnostic assay, an extraction medium comprising a nuclease, and instructions for use.

26. The kit of claim 25, wherein the rapid diagnostic assay is selected from the group consisting of an immunochromatographic lateral flow assay, an enzyme-linked immunosorbent assay, a time-resolved fluorescence assay, and a chemiluminescence assay.

27. The kit of claim 25 or claim 26, wherein the nuclease is an RNase selected from the group consisting of RNase A, RNAse II, RNase T1 , RNase T2, RNase III, and RNase IV.

28. The kit of claim 25 or claim 26, where wherein the nuclease is a DNase.

29. The kit of claim 28, wherein the DNase is DNase I.

30. The kit of any one of claims 25-29, wherein the extraction medium further comprises a zwitterionic detergent, a salt buffer, or both.

31. The kit of claim 30, wherein the zwitterionic detergent is Λ/-dodecyl-Λ/,Λ/- dimethylglycine.

32. The kit of claim 30, wherein the salt buffer is Tris hydroxymethyl-aminoethane buffer.

33. The kit of any one of claims 25-29, wherein the rapid diagnostic assay is an immunochromatographic lateral flow assay comprising at least one antibody.

34. The kit of claim 33, wherein said antibody binds to a viral protein in the sample.

35. The kit of claim 34, wherein the viral protein is influenza type A or influenza type B protein.

36. The kit of any one of claims 25-35, further comprising a swab.

37. The kit of any one of claims 25-29, wherein the extraction medium further comprises Tris (2-carboxyethyl)phosphine hydrochloride (TCEP).

38. The kit of any one of claims 25-29, wherein the extraction medium further comprises a reducing agent.

39. The kit of claim 38, wherein the reducing agent is Tris (2-carboxyethyl)phosphine hydrochloride (TCEP).

40. The kit of any one of claims 25-29, wherein the extraction medium further comprises a chelator.

41. The kit of claim 40, wherein the chelator is ethylenediaminetetraacetic acid (EDTA).

42. The kit of any one of claims 25-29, wherein the extraction medium further comprises MgCI₂, NaCI, or a serum albumin.

43. The kit of any one of claims 25-29, wherein the extraction medium comprises between about 0.05 mg/mL and about 0.50 mg/mL RNase A.

44. The kit of any one of claims 25-29, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 32.9 mM ethylenediamirietetraacetic acid (EDTA); and 0.025 wt% zwitterionic detergent.

45. The kit of any one of claims 25-29, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 1OmM Mg Cl₂; 124mM NaCI; 1 wt% bovine serum albumin (BSA); and 0.025 wt% zwitterionic detergent.

46. An extraction medium for extracting a nuclear protein from a biological sample, the extraction medium comprising a nuclease.

47. The medium of claim 46, wherein the nuclease is an RNase selected from the group consisting of RNase A, RNAse II, RNase T1 , RNase T2, RNase III, and RNase IV.

48. The medium of claim 46, wherein the nuclease is a DNase.

49. The medium of claim 48, wherein the DNase is DNase I.

50. The medium of any one of claims 46-49, wherein the extraction medium further comprises a zwitterionic detergent, a salt buffer, or both.

51. The medium of claim 50, wherein the zwitterionic detergent is Λ/-dodecyl-Λ/,Λ/- dimethylglycine.

52. The medium of claim 50, wherein the salt buffer is Tris hydroxymethyl- aminoethane buffer.

53. The medium of any one of claims 46-49, wherein the extraction medium further comprises Tris (2-carboxyethyl)phosphine hydrochloride (TCEP).

54. The medium of any one of claims 46-49, wherein the extraction medium further comprises a reducing agent.

55. The medium of claim 54, wherein the reducing agent is Tris (2- carboxyethyl)phosphine hydrochloride (TCEP).

56. The medium of any one of claims 46-49, wherein the extraction medium further comprises a chelator.

57. The medium of claim 56, wherein the chelator is ethylenediaminetetraacetic acid (EDTA).

58. The medium of any one of claims 46-49, wherein the extraction medium further comprises MgCI₂, NaCI, or a serum albumin.

59. The medium of claim 46, wherein the extraction medium comprises between about 0.05 mg/mL and about 0.50 mg/mL RNase A.

60. The medium of claim 46, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 32.9 mM ethylenediaminetetraacetic acid (EDTA); and 0.025 wt% zwitterionic detergent.

61. The medium of claim 46, wherein the extraction medium comprises: 0.2 mg/ml RNase A; 12.5 mM Tris hydroxymethyl-aminoethane buffer, pH 9.4; 10 mM MgCI₂; 124 mM NaCI; 1 wt% bovine serum albumin (BSA); and 0.025 wt% zwitterionic detergent.