BRPI0805220A2 - methods for producing a protein extract from fixed cells and for detecting a protein, system and kit for producing a protein extract, and method for extracting a target viral protein from a cell sample - Google Patents

Abstract

Methods for producing a protein extract from cells, such as cells containing viral proteins, are provided. Generally speaking, the methods involve: raising the pH of cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a nonionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract. Kits and compositions for practicing object methods are also provided.

Description

"METHODS FOR PRODUCING A FIXED CELL PROTEIN EXTRACT AND DETECTING A PROTEIN, SYSTEM AND KIT TO PRODUCE A PROTEIN EXTRACT, AND A METHOD FOR EXTRACTING A TARGET CIRCLE VIRAL PROTEIN"

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 60 / 747,076, filed May 11, 2006, which application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

In many diagnostic methods, cells are taken from a patient and deposited in a liquid medium containing a fixative. Cells are fixed in the middle and examined cytologically to provide a diagnosis. For example, the detection of precancerous or cancerous cells in cervical tissue is routinely performed by microscopic evaluation of exfoliated cervical cells. This method, developed by George N. Papanicolaou and known as the "Pap" test, involves exfoliating a woman's cervical cells using a sampling device, depositing the exfoliated cells in a transport medium containing a fixative and then depositing the cells. on a blade. The cells are then stained and examined by light microscopy for cellular abnormalities by a trained medical professional. More than 55 million Pap tests are performed each year in the United States alone.

Despite the success of such cytological tests, the tests are prone to error. For example, it has been estimated that up to 40% of conventional Pap tests are compromised by the presence of contaminants such as mucus, blood cells and obscured inflammatory cells. These contaminants lead to false negative results, false positive results and a significant amount of follow-up work. See, for example, Koss, L. G. (1989), The Papanicolaou Test for Cervical Cancer Detection: ATriumph and a Tragedy, JAMA 261: 737-743; see also DeMay, "Problems in Pap Smear Interpretation", Arch. Pathol. Lab. Med. 121: 229-23 (1997).

In view of the above, there is a need for complementary molecular diagnostic methods for the analysis of cells that are present in a liquid medium containing a fixative. Such methods are not direct, however, because it is not always possible to perform such methods on fixed cells. For example, certain binders (e.g., those transport media used in the THINPREP or SUREPATH® test systems) may cause particular cellular proteins to precipitate or aggregate, thereby making these proteins insoluble and difficult or reliably detectable using conventional means, for example. using an enzyme-linked immunosorbent assay (ELISA) or other immunoassay.

There is therefore a huge need for methods and compositions to extract proteins from fixed cells in a manner that allows them to be suitable for use in molecular, e.g., immunological, detection assays. The invention described herein achieves a necessity and the like.

Literature

The literature of interest includes: U.S. Patents 6,890,729,6,337,189 and U.S. Patent Application published 20050032105.

SUMMARY OF THE INVENTION

Methods for producing a cell protein extract, preferably fixed cells, are provided. Generally speaking, the methods involve raising the cell pH to a pH at least around pH10.0 to produce an intermediate composition and then, in the presence of a nonionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract. The method may include: a) contacting the cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and b) contacting the intermediate composition with a neutralizing reagent to neutralize the pH of the intermediate composition and produce the protein extract. One or both of the extraction agent and neutralization reagent contains the nonionic detergent. In certain embodiments, fixed cells may be fixed exfoliated cervical cells.

DEFINITIONS

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 invention belongs. Still, certain elements are defined below because of clarity and ease of reference.

The term "cell sample" as used herein refers to a liquid composition containing one or more cells of interest. A cellular sample may be a clinical sample containing cells removed from (for example, dissected or exfoliated) an individual, including but not limited to, for example, plasma cells, serum, spinal fluid, semen, lymphoid fluid, the outer sections of the skin, respiratory tract, egenitourinary bowel, tears, saliva, milk, blood cells, tumors or organs.

In other embodiments, the cell sample may contain in vitro cultured cells (including but not limited to cells in cell culture medium, virally infected cells, recombinant cells, etc.). In certain embodiments, the cell sample may contain cells that are most at risk for HPV infection. In these embodiments, ascells can be obtained from a cervix, vulva, vagina, anus, penis, mouth or throat. In certain embodiments, the cells are mucous membrane and may be epithelial in origin. A cell sample may or may not contain contaminants other than exfoliated or dissected cells. For example, mucus or bacterial, yeast or blood cells may be present in a cell sample.

"HPV" is human papillomavirus, including but not limited to HPV strains 4, 11, 20, 24, 28, 36, 48, 50, 16, 18, 31, 35, 30, 39, 45, 51,52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 73 and 82.

An "oncogenic HPV strain" is a strain known to cause cervical cancer as determined by the National Cancer Institute (NCI, 2001).

An "oncogenic E6 protein" is an E6 protein encoded by an oncogenic HPV strain. Exemplary oncogenic strains are: HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 45, HPV 51, HPV 52, HPV 56 , HPV 59, HPV 58, HPV 33, HPV 66, HPV 68, HPV 69, and HPV 82. The amino acid sequences of oncogenic E6 proteins are deposited in the NCBI GenBank database. While not wishing to be bound by theory, it is generally believed that HPV strains 4, 11, 20, 24, 28, 36, 48, and 50 are not congenital.

The terms "polypeptide" and "protein" are used interchangeably. The term "polypeptide" includes polypeptides in which conventional main chain has been replaced with non-naturally occurring or synthetic main chains and peptides wherein one or more conventional amino acids have been substituted with one or more non-naturally occurring or synthetic amino acids.

The term "fusion protein" or grammatical equivalents allude to a protein compound of a plurality of polypeptide components, which although not bound in their native state, are joined by their amino and carboxyl termini through a depeptide bond to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminus methionine residues; immunologically targeted proteins; fusion proteins with detectable fusion partners, for example fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase and others.

In general, polypeptides may be of any length, for example, greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than four. 100 amino acids, greater than about 300 amino acids, usually up to about 500 or 1000 or more amino acids. "Peptides" are generally greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, usually up to about 50 amino acids. In some derealization forms, the peptides are between 5 and 30 amino acids in length. Polypeptides may be natural in that they may be encoded by the genome of an organism or virus or unnatural in which they are not naturally occurring.

The term "capture agent" refers to an agent that binds a protein through an interaction that is sufficient to allow the agent to bind and concentrate the protein from a homogeneous mixture of different proteins. Accordingly, the term "capture agent" refers to a molecule or a multi-molecular complex that can specifically bind an analyte, for example specifically bind an analyte to the decapturing agent, with a dissociation constant (Kd) of less than about 10-6 M without binding to other targets. The binding interaction may be mediated by a capture agent affinity region. Representative capture agents include antibodies (including fragments and mimetics thereof) and PDZ domain-containing proteins, etc. .

The term "specific binding" refers to the ability of a capture agent to preferentially bind to a particular protein that is present in a homogeneous mixture of different proteins. In certain embodiments, a specific binding interaction will discriminate between a particular protein and other proteins in a sample, in some embodiments more than about 10 to 100 times or more (for example, more than about 1000 or 10,000 times).

The term "capture agent / protein complex" is a complex that results from the specific binding of a capture agent with a protein, that is, a "binding partner pair". A capture agent and a capture agent protein specifically bind to each other under "conditions suitable for specific binding" where such conditions are those conditions (in terms of saline concentration, pH, detergent, protein concentration, temperature, etc.). .) which allows binding to occur between capture agents and proteins to bind in solution. Such conditions, particularly with respect to antibodies and their antigens, are well known in the art (see, for example, Harlow and Lane (Antibodies: Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). The affinity between a capture agent and protein that are specifically bound in a capture agent / protein complex is characterized by a KD (constant de-coupling) of less than 10 "M, less than 10" 'M, less than 10 " 8 M, less than 10 "9 M or less than about 10" 10 M.

"Binding partners" and equivalents refer to demolecular pairs that can be found in a decapture agent / analyte complex, that is, exhibit specific binding to each other.

The terms "antibody" and "immunoglobulin" are used interchangeably herein to refer to a capture agent that has at least one epitope binding domain of an antibody. These terms are well understood by those in the field and refer to a protein containing one or more polypeptides that specifically binds an antigen. An antibody form is the basic structural unit of an antibody. This is a tetramer and consists of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen and the constant regions are responsible for the antibody effector functions.

Recognized immunoglobulin polypeptides include kappa and lambda light chains and the alpha, gamma (IgGi, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Life-size immunoglobulin "light chains" (about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2 terminus and a noterminal COON kappa or lambda constant region. Life-size immunoglobulin "heavy chains" (about 50 kDa or about 446 amino acids) similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g. about 330 amino acids).

The terms "antibodies" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain antigen specific binding, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies and fusion proteins comprising an antigen binding portion of a antibody antibody and a non-antibody protein. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. Antibodies may be further conjugated to other moieties, such as specific binding pair members, for example, biotin (biotin-avidin specific binding pair member) and the like. Antibodies may also be attached to a solid support, including, but not limited to, polystyrene plaques or beads and the like. Also encompassed by the terms are Fab ', Fv, F (ab') 2 and or other antibody fragments that retain antigen specific binding.

Antibodies may exist in a variety of other forms including, for example, Fv, Fab and (Fab) 2, as well as bi-functional (i.e. bispecific) hybrid antibodies (for example, Lanzavecchia et al., Eur. J. Immunol 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, General, Hood et al., "Immunology", Benjamin, N.Y., 2nd ed. (1984) and Hunkapiller and Hood, Nature, 323, 15-16 (1986)). Monoclonal antibodies and "phage demonstration" antibodies are well known in the art and encompassed by the term "antibodies".

The term "evaluate" includes any form of measurement and includes determining whether an element is present or not. The terms "determine", "measure", "evaluate", "evaluate" and "test" are used interchangeably and may include quantitative and / or qualitative determinations. The rating may be relative or absolute. "Assessing the presence of includes determining the amount of something present, and / or determining whether it is present or absent.

By "remote location" is meant a location other than the location at which cells are obtained and deposited in a fixative-containing liquid. For example, a remote location may be a different room in the same building from which cells are obtained (for example, another laboratory), a different building in the same building complex as cells are obtained, or a different location in the same city, state, or country, etc. When a cellular sample is indicated as being "received" from a remote location, the cellular sample may be obtained from a remote location or personally delivered, mailed or a messenger from the remote location, for example.

"Communicating" information refers to any means of obtaining this information from one location to the next, whether by physically transported printed material or computer readable media containing the information (for example, by mail) or by transmitting the information. If the information is transmitted , a digital or analog signal representing information (for example, an electromagnetic signal such as a light or electrical signal) is transmitted on a suitable communication channel (for example, a private, public or wireless network). Any convenient means may be used to transmit the data, for example, facsimile, modem, internet, email, etc.

As used herein, the term "means of transport" is used to describe liquids suitable for cell collection and preservation of cells in a manner that allows them to be suitable for liquid-based studies. The means of transport are commonly used in the Pap test. Cells deposited on the transport medium may or may not be transported from one location to another on this medium. The means of transport contain fastener. Deposition of cells in a transport medium fixes the cells to produce fixed cells. Representative transporter means include the SUREPATH® or PRESERVCYT® transport means.

A "fixed cell" is a cell that has been treated with ecologically preserved by a chemical fixative. Fixed cells are also suitable for dyeing and subsequent morphological and / or cytological analysis by light microscopy.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same degree as each individual publication or patent application is specific and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are hereby encompassed.

Methods for producing a protein extract from fixed cells are provided. Generally speaking, the methods involve: raising the pH of the fixed cells to a pH of at least about pH 10.0 to produce an intermediate composition and then, in the presence of a nonionic detergent, neutralizing the pH of the intermediate composition to produce the extract. protein. The method may include: a) contacting the fixed cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and b) contacting intermediate composition with a neutralizing reagent to neutralize the pH of the intermediate composition and producing the protein extract. Both of the extraction reagent and neutralization reagent contain nonionic detergent. In certain embodiments, fixed cells may be fixed exfoliated cervical cells. Kits and compositions for practicing the object methods are also provided. Object methods find use in a variety of different applications, including diagnostic tests that detect particular proteins in the resulting protein extract.

Before the subject invention is described below, it should be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of particular embodiments may be made and still fall within the scope of the appended claims. It should also be understood that the terminology used is for the purpose of describing particular embodiments and is not intended to be limiting. Rather, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms "one," "one" and "o", "a" include mild plural references that the context clearly dictates otherwise.

Where a range of values is provided, it is understood that each value brought to the tenth of the lower boundary unless the context clearly dictates otherwise between the upper and lower boundary of this range and any other established or interposed value in which the established range, is encompassed within the invention. The upper and lower limits of these minor ranges may be independently included in the minor ranges and are also encompassed within the invention, subject to any limit specifically excluded within the established range. Where the established range includes one or both of the limits, bands excluding each or both of those included limits are also included in the invention.

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 invention belongs. While any methods, devices and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods, devices and materials are now described.

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing components that are described in the publications that would be used in connection with the presently disclosed invention.

As summarized above, the subject invention provides methods and compositions for producing a protein extract from fixed cells. In describing the invention in more detail, the methods are described first followed by a description of the kits and systems for use in object method practice.

PROTEIN EXTRACTION METHODS

As mentioned above, the invention provides a method for producing a protein extract from fixed cells. In general, the methods involve two steps: a) contacting fixed cells with an extraction reagent having a pH that is greater than around pH 10.0 to produce an intermediate composition and b) contacting the intermediate composition with a neutralizing reagent. The extraction reagent and / or neutralization reagent contains a nonionic detergent. The resulting protein extract contains a nonionic detergent and has a pH that is neutral (ie, between about pH 7.0 and about pH 8.0). The methods generally produce a protein extract containing proteins that are easily detectable using capture agents for these proteins. As such, a protein extract produced by the present methods is generally suitable for use in binding assays, for example immunological assays, for the detection of these proteins. In certain embodiments the methods may include: raising the pH of cells fixed to a pH at least around pH 10.0 to produce an intermediate composition and then, in the presence of a nonionic detergent, neutralize the pH of the intermediate composition to produce the protein extract. Since, as mentioned above, nonionic detergent may be present in the extraction reagent or neutralizing agent (or in both the extraction reagent and neutralization agent), certain embodiments of the present methods include: a) contacting fixed cells with a reagent extraction to produce an intermediate composition having a pH of at least about pH10.0; and b) contacting the intermediate composition with a neutralizing reagent comprising a nonionic detergent; to neutralize the pH of the intermediate composition and produce the protein extract. In other embodiments, the method may include: a) contacting the fixed cells with an extraction reagent comprising a nonionic detergent to produce an intermediate composition having a pH of at least about 10.0 pH; and b) contacting the intermediate composition with a neutralizing reagent; to neutralize the pH of the intermediate composition and produce the protein extract.

In certain embodiments, the protein extract produced by the present methods may contain more protein that is accessible to capture agents than a protein extract made using other methods, for example methods that do not use: a high pH extraction step (i.e. , a step that raises the pH more than around pH 10.0 or pH 11.0), a neutralization step (that is, a step that raises the pH to around pH 7.0 to around pH 8.0) and a nonionic detergent. Neither high pH alone nor nonionic detergent produces such a protein extract. In particular embodiments, the high pH extraction reagent solubilizes proteins in the fixed cells, whereas the nonionic detergent prevents the solubilized proteins in the intermediate composition from rearranging or precipitating as the pH of the intermediate composition is neutralized.

The reagents used in the present methods and the protein extract produced by the present methods are described in more detail below, as is a description of how the reagents may be used to produce the protein extract. As will be discussed below, the ideal epH concentration of reagents used in the present methods may vary depending on which reagents are used. However, the concentration and pHideal of the reagents are easily determined experimentally or empirically.

The cells from which the protein is extracted using the invention method

The methodology according to the present invention may be used to extract a target protein or protein of interest from a sample cell. The cell sample may be a homogeneous cell population or a heterogeneous mixture of cells of a different type. The cell sample may also contain "contaminants" such as mucus, blood cells, and inflammatory cells that are not of interest for the purpose of target protein extraction or do not contain the target protein.

In some embodiments, the target protein is a viral protein present in cells infected with a virus, preferably a pathological virus, and the cells are preferably those isolated from a mammal, for example, a human.

The pathogenic virus can be any pathogenic virus that causes pathogenic effects or disease in humans or other animals. Pathogenic ovirus can be from various strains of the human immunodeficiency virus (HIV), such as HIV-I and HIV-2. The viral protein may be HTV umaglycoprotein (or surface antigen) such as HIV GP120 eGP41 or a capsid protein (or structural protein) such as HIV protein P24.

The pathogenic virus can be Ebola or Marburg virus. Viral protein may be an Ebola glycoprotein or a surface antigen such as Ebola GP1 or GP2 proteins.

The pathogenic virus may be hepatitis virus such as hepatitis A, B, C, D, or E. Virus For example, the viral protein may be a surface antigen or a hepatitis B virus core protein such as a hepatitis surface antigen. Small B (SHBsAg) (also referred to as the Australian antigen), medium hepatitis B surface antigen (MHBsAg) and large hepatitis B surface antigen (LHBsAg). The viral antigen may be a surface antigen or hepatitis C virus core protein such as NS3, NS4 and NS5 antigens.

The pathogenic virus may be a respiratory syncytial virus (RSV). For example, the RSV viral protein may be glycoprotein (G protein) or RSV fusion protein (F protein).

The pathogenic virus may be a herpes simplex virus (HSV) such as HSV-I and HSV-2. For example, the HSV viral antigen may be HSV-2 glycoprotein D.

The target protein may be a tumor antigen, such as Her2 from breast cancer cells and CD20 in lymphoma cells, an oncogeneviral such as human papilloma virus E6 and E7, or a cellulartal oncogene such as mutated ras.

In some embodiments, the cell sample contains fixed cells in which the target protein is present. The fixed cells used in the present methods are generally obtained by depositing a sample of cells (obtained by removing a patient's cells by dissecting, exfoliating or washing, for example) in a liquid medium. The cell sample may be deposited in a liquid medium that already contains a chemical fixative or a chemical fixative may be added to the liquid medium after the cells have been placed in the medium. A liquid medium containing a fixed and fixed cells is here called a "cell sample".

Representative chemical fixatives which may be used in the present methods include: alcohols (eg methanol or ethanol), aldehydes (eg glutaraldehyde or formaldehyde) and ketones (eg acetone) as well as osmium tethoxide, acetic acid, citric acid and salts heavy metal ionic. Other examples of fasteners that may be used in the present methods include embisulfite based fasteners (which may also include acetic acid), PVP based fixtures (which may also contain propylene glycol and methanol) as described in US Patent 3,546,334, 4,578. 282, 4,857,300, 5,104,640.5,256,571, 5,432,056 and 5,196,182. Examples of fasteners that may be used in the present methods, including the concentrations of these fasteners, can be found in Baker, (Principies of Biological Micro-Technique: A Study of Fixation and Dyeing, 1959) and Williams ("Tissuepreparation for immunocytochemistry." J Clin. Pathol 1997 50: 422).

Of particular interest in the present methods are liquid media which are called "means of transport" and are routinely used for the collection, preservation (i.e. fixation) and transport of cervicovaginal cells (e.g., exfoliated cervical cells) as part of a gynecological examination. FDA-approved means of transport are of particular interest.

Examples of commercially available transport media that may be used include: methanol-based PRESERVCYT® transport media (which is sold as part of the Cytyc, Inc., Marlborough, Mass. THINPREP® Gynecological Sampling Kit), SUREPΑΤΗ® transport media ethanol-based formally known as CYTORICH® (TriPath, Inc. Burlington, NC) and methanol-based CYTOLYT® transport medium (Cytyc, Inc., Marlborough, Mass.) for example.

Cells may be obtained by any convenient method, including but not limited to exfoliation (e.g. scraping), dissection and washing. Of particular interest are epithelial cells of cervical origin, which cells are typically obtained by exfoliation methods using an adapted brush, spatula or scraper and disposed in a fixative-containing liquid medium.

Extraction Reagent

The extraction reagent used in the present method contains components that are present in sufficient amounts in concentration to produce a protein extract having a pH that is at least pH 10.0 upon addition of the extraction reagent to fixed cells. Consequently, the extraction reagent generally has a pH of at least about pH 10.0.

The extraction reagent is contacted with fixed cells to produce the intermediate composition. The pH of the resulting intermediate extraction and extraction reagent is generally at least around pH10.0, for example, in the range from pH 10.0 to around pH 13.0 or around pH 11.0 to around pH 12.0. In certain embodiments, the extraction reagent may have a pH around pH 10.0 to around pH 10.5, pH 10.5 to around pH 11.0, pH 11.0 to around pH 11 0.5 pH 11.5 to around pH 12.0, pH 12.0 to around pH12.5 or pH 12.5 to around pH 13.0. The extraction reagent may be manufactured using any suitable source of hydroxide ions, for example sodium or potassium hydroxide or calcium carbonate, for example.

In certain embodiments, the extraction reagent may not contain any significant amount of denaturant. However, in other embodiments, in addition to having a pH of at least 10.0, the extraction agent may also contain a denaturant, for example, an ionic detergent such as sodium dodecyl sulfate (SDS) or sarkosyl or chaotropic umagent such as urea. . In these embodiments, denaturant, if present, may be present at a concentration that does not significantly decrease the sensitivity of future trials. The denaturant concentration in certain embodiments may be decreased during sample processing, for example by diluting the denaturant using neutralization buffer or by the addition of a diluent, for example, buffering water to the protein extract prior to use.

Depending on the concentration of the denaturant used and the pH of the extraction buffer, the denaturant may be present in the extraction buffer at a concentration of about 0.01 M to about 0.05 M, from about 0.05 M to about 0.1. M, from 0.1 M to about 0.2 M, from about 0.2 M to about 0.5 M, from about 0.5 M to about 1.0 M, from about 1.0 M is about 2.0 M, from about 2.0 M to about 4.0 M or from about 4.0 M to about 8.0 Μ. Denaturant, if present in the extraction reagent, may be present at a concentration well below the denaturant concentration typically used for denaturing protein. In other words, the extraction reagent may contain denaturant at a concentration that allows detection of a protein using a protein capture agent, then producing a protein extract according to the subject methods. The concentration of denaturant used is generally sufficient to produce a protein extract containing proteins that are readily detectable in a binding assay using a decapturing agent, for example, in an antibody detection assay.

Exemplary denaturants and their concentrations in an object extraction reagent: sodium dodecyl sulfate (SDS): from about 0.01% to about 2%, eg 0.05%, sarkosyl: from about 0.01 % to about 5%, eg 0.5%, guanidine: from about 0.1 Ma to about 6 M, for example about 0.5 M and urea: from about 0.1 Ma to about 8 M for example about 0.5 M wt / vol>

SDS is typically used to denature proteins at a concentration of 0.1% to 0.5%, sarkosyl is typically used to denature proteins at a concentration of 2% w / v, urea is typically used to denature proteins at a concentration of 2 M to 8 Μ, guanidine hydrochloride is typically used to denature proteins at a concentration of 3 M to 8 Μ, N-cetyl trimethylammonium chloride typically used to denature proteins at a concentration of 5% w / v and N-octyl glycoside is typically used to denature proteins. proteins at a concentration of 2% w / v (See Protein purification Handbook, Amersham Pharmacia Biotech, p. 71 (1999)).

If no denaturant is present in an extraction reagent, the reagent may have a pH of at least around pH 11.0. If the extraction agent contains detergent, then the pH of the extraction reagent may have a pH of at least around pH 10.0.

As will be described in more detail below, the extraction reagent, in certain embodiments, may also contain a nonionic detergent.

In certain embodiments, the extraction reagent may contain a buffer to maintain the reagent at a desired pH. If a buffer is present in an object extraction reagent, the buffer may have a pKana range of from about 9.0 to about 12.5 to 25 ° C. Exemplary buffers that may be used in an object protein extraction reagent includeCABS , piperidine, phosphate, CAPS, glycine or ethanolamine, for example. Buffers that have little or no buffering capacity at umpH above pH of about 10.0 (eg tris, tricine, hepes, etc.) are generally not used. to buffer the pH of the extraction reagent but not enough may be present in an extraction reagent. The object protein extract reagent may contain other components eg saline ion chelators, protease inhibitors, etc., in addition to the components stated above. .

The protein extraction reagent may be a liquid or solid composition and, in certain embodiments, may contain a combination of different denaturants.

Denaturants which may be used in the present extraction buffer are generally strong denaturants and include but are not limited to: chaotropic agents (e.g. urea, guanidine hydrochloride or a thiocyanate salt such as sodium thiocyanate or guanidinium thiocyanate, sodium iodide, sodium perchlorate and others, see K. Hamaguchi et al., Proc. Natl.Acad. Sci. 62: 1129-1136, 1962) and ionic detergents (e.g., sodium dodecyl sulfate (SDS), sarkosyl or N- trimethylammonium), including cationic, anionic and zwitterionic detergents (such as CHAPS or CHAPSO). Other denaturants that may be used in the present methods are listed in columns 7 and 8 of U.S. Patent 6,488,671, which patent is incorporated herein by reference in its entirety.

In certain embodiments, a weak denaturant such as LiCl, L1CIO4, LiBr, CaCl3 or NaCl is not used as a denaturant in the extraction buffer, although such a compound may be present in an extraction buffer or protein extract in addition to a denaturant listed in previous paragraph.

As mentioned above, the extraction reagent is contacted with (e.g., combined or mixed with) fixed cells. In certain embodiments, a cell sample containing the fixed cells (e.g., a transport medium containing fixed cells) may be directly added to the extraction reagent. In other embodiments, fixed cells may be isolated from the cell sample (e.g., by sedimentation, centrifugation, filtration or affinity methods) prior to their addition to the protein extraction reagent. Cells may be washed or contacted with other reagents prior to their addition to the extraction reagent.

All or a portion of the available fixed cells may be combined with the extraction reagent. For example, in certain derealization forms, a portion of the fixed cells may be used in decitology testing and a portion of the fixed cells may be contacted with the extraction reagent to produce the intermediate composition. The fixed cells and extraction agent may be combined and maintained at a suitable temperature (eg on ice, around room temperature or about 37 ° C) and for a suitable time (eg 10 seconds to 24 hours) to produce the intermediate composition. In certain embodiments, the neutralizing reagent is contacted with the intermediate composition immediately after the fixed cells have been contacted with the extraction reagent.

Neutralization Reagent

The neutralizing reagent used in the present methods has a pH which is sufficient to neutralize the pH of the intermediate composition discussed above upon contact with the intermediate composition.

In other words, the neutralizing reagent contains a nonionic detergent and has a pH that is sufficient to neutralize the pH of the intermediate composition discussed above when the neutralizing reagent is mixed with the intermediate composition. As will be described in more detail below, the neutralizing reagent, in certain embodiments, may contain a nonionic detergent.

The pH of the neutralization reagent is sufficient to neutralize the manufactured intermediate composition by contacting fixed cells with an object extraction reagent. Depending on the pH of the extraction reagent and buffers are used, the pH of the neutralization reagent may be between 4.0 pH to 8.0. In certain embodiments, the neutralizing reagent may have a pH around pH 4.0 to around pH 4.5, pH 4.5 to around pH 5.0, pH 5.0 to around pH 5.5, pH 5.5 to around pH 6.0, pH 6.0 to around pH 6.5, pH 6.5 to around pH 7.0, or pH 7.0 to around pH 7.5. The neutralizing reagent may be manufactured using any suitable source of hydrogen ions, for example hydrochloric acid or acetic acid, for example. In certain embodiments, the neutralizing agent may have a pH lower than pH 4.0.

The neutralizing reagent may be buffered or non-buffered. If the neutralization reagent is buffered, then the neutralization reagent may be buffered using any buffer having a pKa of from about 6 to about 8, for example tris, hepes or tricin, for example.

As mentioned above, the extraction reagent and / or neutralizing reagent may contain a nonionic detergent.

In certain embodiments, the nonionic detergent used may be nonidet P-40, n-octyl glycoside, a TRITON® detergent such as TRITON® X-100, octyl beta-glycopyranoside, a TWEEN® detergent such as TWEEN-20 or NP-40. ). Depending on the concentration of detergent used, the detergent may be present in the extraction buffer or neutralization buffer at a concentration of about 0.01 M to about 0.05 M, from about 0.05 M to about 0.1 M, from 0.1 Ma to about 0.2 M, from about 0.2 M to about 0.5 M, from about 0.5 M to about 1.0 M, from about 1.0 M to about 2.0 M, from about 2.0 M to about 4.0 M or from about 4.0 M to about 8.0 M. Other detergents that can be used in these methods are listed in columns 7 and 8 of U.S. Patent 6,488,671, which patent is incorporated herein by reference in its entirety. In certain embodiments, detergent may be present in both the extraction and neutralization buffers.

Exemplary detergents and their concentrations in a neutralizing and / or extraction reagent include: Triton X-100: from about 0.1% to about 10%, for example, about 1%, NP40: from about 0, 1% about 10%, for example about 1% and Tween-20: about 0.1% about 10%, for example about 1%, weight / vol.

As mentioned above, the neutralizing reagent is contacted with (e.g., combined or mixed with) the intermediate composition to produce a protein extract having a neutral pH (i.e., a pH in the range from about pH 6.5 to around pH 8.0, for example in the range around pH 7.0 and around pH 7.8). The protein extract further contains fixed cell protein, a nonionic detergent at a concentration listed above and in certain embodiments, a buffer to maintain the protein extract at a particular pH range. If a denaturant is added to the fixed cells, the protein extract may still contain this denaturant. The pH, detergent choice and detergent concentration used (and, if a denaturant is used, denaturant identity and concentration) are sufficient to allow the deprotein extract to be directly used in a binding assay to detect proteins present in the extract. protein.

Neutralization of the cell extract can also be accomplished by passing the extract through a filter or filter tip that is impregnated with neutralization reagent. As the extract passes through the filter material, the neutralization reagent is solubilized and the pH of the extract approaches neutrality.

An alternative method for neutralizing the cell extract is to pass the extract through a pre-equilibrated BioSpin column (BioRad) with a solution at neutral pH. The extract may also be placed in a syringe or similar apparatus containing neutralizing gel (or filtration material) and released from the syringe by positive pressure.

In certain embodiments, the object protein extract contains solubilized HPV E6 protein (particularly oncogenic HPV E6 protein knockouts) that is readily detectable by a capture agent without further treatment of the protein extract (for example, without further denaturant addition). , pH changes or warming up). The protein extract may also contain solubilized or insoluble membranes, proteins other than HPV E6 protein, and other cellular contents such as DNA, RNA, carbohydrates, etc. Other contaminants such as those derived from mucal contamination of the original cell sample may also be present. The protein extract components generally do not contain whole cells (ie cytologically intact).

The protein extract may be used immediately or stored, for example, in frozen form before use.

In particular embodiments, protein extracts produced by the methods set forth above may be used in protein detection methods, which methods are described in greater detail below.

As would be apparent from the above, a variety of denaturant, detergent, buffer, pH and different component concentrations can be used in the reagents described above. The concentration of denaturing agent, detergent, buffer or pH or component in any reagent is readily determined using routine methods.

After neutralization of the cell extract, the E6 protein can be concentrated from the cell extract by incubating the extract with the E6 binder-containing particles. The binder may comprise PDZ, E6 Associated Protein (E6AP) or fragments thereof or E6 Binding Protein (E6BP) or fragments thereof. After E6 is captured by the particles, the particles are washed off and E6 is then released from the particles by incubation with buffer at pHs greater than 10. The particles are separated from the elution solution and the remaining solution is then neutralized by the procedures described above. Alternatively, the protein E6 can be detected without release of capture particles.

PROTEIN DETECTION METHODS

Protein extract manufactured by the methods discussed above may be used directly or indirectly (i.e. after addition of additional reagents) in a method wherein the presence of one or more proteins in the protein extract is evaluated. Protein detection methods generally involve a capture agent that specifically binds a protein. The identity of the proteins to be detected may be of known (i.e. predetermined) identity or unknown at the time of performing the method.

Proteins that can be detected using the object protein detection methods include proteins that are diagnostic markers for a disease or condition, for example, cancer, inflammatory disease or infection with viruses, bacteria or fungi, for example. In certain embodiments, a protein detected using object methods is not routinely detectable unless protein object extraction methods are used.

Exemplary proteins that can be detected using the present methods include proteins that are encoded by an infectious agent, such as human papillomavirus (HPV). In particular embodiments, the present methods can be used to detect HPV E6 protein, a protein that has been difficult or impossible to detect in protein extracts made from cells fixed by other methods.

Generally speaking, protein detection methods are very well known in the art and include binding assays, that is, assays that the binding between a protein and a capture agent for the protein is detected. Such assays include immunoassays, that is, binding assays that use an antibody that specifically binds to a protein, including, but not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay). ), sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name a few. Such assays are routine and well known in the art (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below.

Immunoprecipitation protocols generally involve producing a protein extract, adding a capture agent, such as an antibody, to the protein extract and incubating the protein extract and capture agent for an appropriate period of time and temperature. The capture agent is then bound to a solid support, for example, an affinity substrate such as protein A and / or protein G bound beads, and the mixture is incubated and washed. The solid support is resuspended in sample buffer and the protein of interest can be detected by western blotting, for example.

ELISAs may involve preparing a protein extract, binding the protein extract to a solid support (e.g., a reservoir of a multi-reservoir microtiter plate), contacting the support-bound protein extract with a capture agent, for example, an antibody, and detecting the binding between the capture agent and the protein. In certain ELISA methods, the capture agent may be detectably labeled with a detectable moiety such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) prior to contacting the capture agent with the protein extract attached to the support. In other embodiments, however, binding of the capture agent to the protein extract may be detected by a second detectably capture agent (e.g., a second antibody) that binds to the capture agent contacted with the protein extract.

In other ELISA assays, the capture agent may be bound to a solid support and the protein extract is contacted with the capture agent attached to the solid support. Binding of a protein in the protein extract to the solid support antibody can be detected using a second capture agent for the protein. Such "sandwich assays" are well known in the art.

In other assays, binding between a capture agent and aprotein may occur in solution prior to immobilization of the capture agent surface.

In particular, the present methods may be used to detect the E6 protein from oncogenic HPV strains. In such embodiments, the capture agent used in the detection method may be, for example, an antibody or polypeptide comprising a PDZ domain that binds to a PDZ ligand (i.e. a binding site for a PDZ domain) contained in the E6 protein. . For example, the present E6-sensing binding method may use a protein containing the PDZ domain that contains the second MAGI-I PDZ or the DLG or TIP1 PDZ domain, etc., as described in published application US 20040018487 (published 29 January 2004) and incorporated herein by reference in its entirety. Exemplary proteins containing the PDZ domain and PDD domain sequences are shown in TABLE 2 and EXAMPLE 4 of US20040018487. The term "PDZ domain" also encompasses variants (e.g., naturally occurring variants) of sequences (e.g., polymorphic variants, conservatively substituted variants, and others) and alternative species domains (e.g., mouse, rat).

Typically, PDZ domains are substantially identical to those shown in US Ser. Patent Application No. 09 / 724,553 which is incorporated herein by reference, for example at least about 70%, at least about 80% or at least about 90% identity. of amino acid residue when compared and aligned for maximal correspondence, it is evaluated in the art that PDZ domains can be mutated to amino acid changes that can strengthen or weaken binding and change specificity, although PDZ domains remain (Schneider et al., 1998, Nat. Biotech 17: 170-5). Unless otherwise indicated, a reference to a particular PDZ domain (e.g., a MAGI-I2 domain) is intended to encompass the particular PDZ domain and its HPV E6 deletion variants thereof. In other words, if a reference is made to a particular PDZ domain, a reference is also made to PDZ domain-domain variants that bind to the oncogenic HPV E6 protein as described below. In this regard it is mentioned that the numbering of PDZ domains on a protein may change. For example, the MAGI-12 domain (from amino acid sequence PSELKGICFIHTKLRKSSRGEGFTWGGDEPDEFLQIKSLVLDGPAALDGKMETGDVIVS VNDTCVLGHTHAQWKIFQSIPIGAS'VDLELCRGYPLPFDPDtraN), as given here by reference. As such, when a particular PDZ domain of a protein is given as a reference in this application, this reference must be understood in view of the sequence of this domain, as described herein, particularly in the sequence listing in Table 2 of US20040018487, showing the relationship between the sequences. sequence listing and Genbank access names and access numbers for multiple domains, as appropriate. As used herein, the term "PDZ protein" refers to a naturally occurring protein containing a PDZ domain. Exemplary PDZ proteins include CASK5 MPP1, DLG1, DLG2, PSD95, NeDLG5ΤΊΡ-33, SYNla5 43-43, LDP5 LIM5 LIMK1 LIMK2, MPP2, NOS15 AF6, PTN-4, 41.8kD, KIAA0559, RGS12, RGS12 -40, TIAM15 MINT15 MAGI-15 MAGI-2, MAGI-3, KIAA0303, CBP5 MINT3, TIP-2, KIAA0561, and TIP-1. As used herein, the term "PDZ domain polypeptide" refers to a polypeptide containing a PDZ5 domain such as a fusion protein including a PDZ5 domain sequence, a naturally occurring PDZ protein, or a PDZisolated domain peptide. A PDZ domain polypeptide can therefore be about 60 amino acids or more in length, about 70 amino acids or more in length, about 80 amino acids or more in length, about 90 amino acids or more in length, about 100 amino acids or more in length. 200 amino acids or more in length, about 300 amino acids or more in length, about 500 amino acids or more in length5 About 800 amino acids or more in length, about 1000 amino acids or more in length, usually up to about 2000 amino acids or more in length, about from 50 to 2000 amino acids in length, about 50 to 1500 amino acids in length, about 50 to 1000 amino acids in length, about 60 to 1000 amino acids in length5, about 70 to 1000 amino acids in length. PDZ domain peptides usually have no more than about 200 amino acids (e.g. 50 to 200 amino acids, 60 to 180 amino acids, 80 to 120 amino acids or 90 to 110 amino acids) and encode a PDZ domain.

Suitable antibodies for detecting HPV E6 protein are described in 20050142541 (published June 30, 2005), for example. Detailed methods for identifying HPV cepasoncogenic E6 protein are found in published U.S. patent application US20040018487, which methods are incorporated herein in their entirety. These published methods are readily adapted for application to the present methods.

In certain embodiments, an anti-E6 antibody may be attached to a solid support and a protein extract produced by the subject methods is contacted with the antibody attached to the solid support. Binding of oncogenic E6 protein in protein extract can be detected using a protein containing the PDZ domain. In other embodiments, a PDZ domain containing protein may be bound to a solid support and a protein extract produced by the object methods is contacted with aprotein containing the solid support bound PDZ domain. Oncogenic E6 protein binding in protein extract can be detected using an anti-E6 antibody. In alternative methods, binding between the PDZ domain containing protein deprotein may occur in solution (i.e., in the absence of binding of the PDZ domain containing antibody or protein to a solid support), and, after binding, the domain containing antibody or protein PDZ may be attached to a solid support (e.g. pearls or the like). In these embodiments, the PDZ domain-containing protein may be a fusion protein having an affinity domain that binds to the solid support. The presence of the E6 protein can be detected using a second capture agent that recognizes the E6 protein.

The results obtained from the assay methods described above may be compared to the results obtained from appropriate controls, for example, a positive control (in which a protein extract known to contain the protein to which the capture agent binds may be used) or a negative control ( for example, where a protein extraction reagent was not contacted with a cell sample may be used).

Results obtained from the assay methods described above may indicate the presence, absence or, in certain embodiments, the amount of a protein in a protein extract. In certain embodiments, the results obtained from the assay methods described above may be reported from returns to a remote location, for example by telephone, fax, email, mail or any other means. Results can be communicated to the patient or a patient's physician, for example.

The above protein detection methods may be performed in combination with a different test, such as a technology test, for example, a Pap test to identify cervicaiscancerous or precancerous cells or other molecular tests. In these realization forms, the cell sample can be divided into parts prior to use. The first part may be used in cytological assays and the second part may be used in the methods described above.

According to the above, certain embodiments of the invention also provide a system for producing a protein extract.

The system generally contains: a) a cell sample containing fixed cells; b) an extraction reagent having a pH of at least about pH 10.0; and c) a neutralization reagent, where fixed cells, extraction reagent and neutralization agent may be used in the above methods to produce a protein extract suitable for use in a deletion assay. The extraction reagent and / or neutralization reagent contains a nonionic detergent.

KITS

In another aspect, the present invention provides kits for practicing the object methods, for example, for producing a deprotein extract from fixed cells, in certain embodiments, for testing for the presence of a protein in the protein extract. Object kits include at least one extraction reagent that has a pH of at least about pH 10.0 and a neutralizing reagent. The extraction reagent and / or neutralization reagent contains a nonionic detergent. In addition, kits may include a capture agent for detecting a protein, and, in certain embodiments, reagents (e.g., buffers and detection reagents) for detecting this protein using the decapture agent. The above components may be present in separate containers or one or more components may be combined into a single container, for example a glass or plastic bottle.

In addition to the above components, object kits may also include instructions for practicing object methods. These instructions may be present in object kits in a variety of ways, one or more of which may be present in the kit. One way in which these instructions may be present is as information printed on a suitable medium or substrate, for example, a piece or pieces of paper on which the information is printed, on the kit packaging, on a packaging insert, etc. Another medium would be a computer readable medium, for example, floppy disk, CD, etc., on which the information was recorded. Another means that may be present is a website address that can be used by the Internet to access information at a remote site.

Any convenient means may be present in the kits.

UTILITY

The method and system described above are readily employed in a variety of research and diagnostic methods, including methods of diagnosing a particular disease or condition or infection with an infectious agent, such as a virus or bacteria. In a derealization form, the method is used as part of a diagnosis for the detection of cells infected with HPV. Since the presence of HPV strains is associated with cancerous and precancerous cells, the present methods can be used to detect cervicaiscancerous or precancerous cells.

HPV is known to be a causative agent in the following diseases: epidermodysplasia verruciformis (EV), a vidalonga skin disorder that results in a high risk for skin cancer (eg, squamous cell); cervical neoplasms such as intraepithelial-cervical neoplasia (CIN) and invasive cervical carcinoma (CHF); vaginal neoplasms such as vaginal intraepithelial neoplasia (VAIN) and vaginal carcinoma (VC); vulvar neoplasms such as vulvar intraepithelial neoplasia (VIN) and vulvar ecarcinoma; penile carcinoma (including Bowenoid papulosis); anal (AC) and perianal (PC) carcinomas; oropharyngeal carcinomas (OS), esophageal carcinomas (CE); non-melanoma skin cancers (e.g., BCC basal cell carcinoma and SCC squamous cell carcinoma); Emelanoma. As such, in one embodiment, the present methods may be used as a diagnosis for any of these diseases.

In one embodiment, the cells are obtained (e.g., exfoliated or dissected) from a patient and deposited in a fixative-containing meioliquid which, in certain embodiments, may be a transport medium for cytological testing. Cells are usually obtained from the physician or clinic, the cell sample is sent and received by a test facility in which the above protein detection methods and, optionally, cytology assays are performed. The results of this test are communicated to the patient in some embodiments by the physician and an associate thereof.

The patient from which the cells are used may be a mammal, for example, a dog or cat, a rodent (e.g., mouse, guinea pig or rat) or primate (e.g., a human, chimpanzee or monkey). In many embodiments, the patient will be a human, particularly a male or female. In certain embodiments, the patient may have symptoms of HPV infection (for example, may have warts on one or more parts of the body), may be suspected of being infected with HPV (for example, may contain cells that are cytologically compatible with such an infection). ) or may have already been tested positive for HPV. In certain embodiments, the patient may have no indication of HPV infection and the above methods may be used as part of a routine screening.

In one embodiment, the present methods may be used to detect any oncogenic HPV strain, for example HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68, or HPV 69, (particularly any of the most prevalent HPV strains, for example, HPV 16, HPV 18, HPV 31, HPV 33 and HPV 45) by detecting the E6 protein destacepa. In one embodiment, at the point of initiation of the present methods, it is not known whether the fixed cells contain the oncogenic E6 protein or of which strain an oncogenic E6 protein is. If a detection assay indicates the presence of an oncogenic E6 protein in fixed cells, then the identity of the HPV strain that has infected these cells can be determined by other molecular assays, for example those using antibodies specific to a particular E6 protein or other protein encoded by virus or viral DNA sequencing.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Extraction of blocked clinical specimens

Cells transfected with the HPV16 E6 gene (C33A +) were fixed with THINPREP® medium and added (i.e., "blocked") portions of the THINPREP® fixed clinical samples as listed below.

Cells were blocked in half of each of the five clinical samples (each half of the clinical sample having 20 million C33 A + cells).

C33A (+) ThinPrep cells / 20M cells per ml

1 to 20 M C33A (+) ThinPrep cells in 1/2 # 229 negative clinical (1.0 ml extraction)

2 to 20 M C33A (+) ThinPrep cells in 1/2 of # 230negative negative (1.0 ml extraction)

3 to 20 M C33A (+) ThinPrep cells in 1/2 # 231 negative clinical (1.0 ml extraction)

4 to 20M C33A (+) ThinPrep cells in 1/2 # 232 negative clinical (1.0 ml extraction)

5 to 20 M C33A (+) ThinPrep cells in 1/2 # 233 negative clinical (1.0 ml extraction)

6 to 20 M C33A (+) ThinPrep cells (1.0 ml extraction)

Extraction Reagent:

Triton X-100 / Lot 092K0171 - (1% = 250 μΐ) 5M NaCl / Lot 5701-53 - (0.15M = 750 μΐ) 0.5 M Tris Base / Lot 5708-20 - (0.1 M = 5 ml)

0.5 M Glycine / Lot 5708-9 - (0.1 M = 5 ml) 10% SDS / Lot 5708-8 - (0.05% = 125 μΐ) 8 M Urea / Lot 5678-83 - (0 , 25 M = 781 μΐ) Add RO / Dl to 20 ml - (8.1 ml)

5 N NaOH / Lot A09522 - (525 μΐ) Add 25 ml RO / Dl - (4.475 ml) Final pH - 11.48

Final formulation: 0.1 M Tris / 0.1 M Glycine / 0.15 M NaCl / 1% Triton X-100 / 0.05% SDS / 0.25 M urea pH 11.48 protein:

1. Add cell suspension to 50 ml2 centrifuge tube. Spin at 3000 rpm for 10 to 15 minutes

3. Carefully remove supernatant

4. Transfer contents to a 1.5 ml nunc tube.

5. Spin at 3000 rpm for 10 to 15 minutes

6. Carefully remove supernatant

7. Add the required amount of extraction reagent to the pellet.

8. Resuspend to break cell pellet. Additives (DTT @ 1: 100)

9. Check pH, adjust to 11.5

10. Mix at room temperature (or appropriate temperature for extraction) for 30 minutes.

11. Spin at 14,000 rpm for 10 to 15 minutes

12. Remove clarified supernatant

13. Add DTT @ 1: 100

14. Neutralize to pH 8.0 with 5 N HCl and ELISA test (Neutralize to pH 8.0 with 31.0 μΐ 5 N HCl / 1 ml) 100 mM DTT / NR 5701-90 / DOM 2/7 / 05

ELISA Method

1 - Coat the plate (Nunc 439454 Maxisorp F96 / lot542043) with 5 μ ^ ηιΐ of GST-Magi-PDZ (batch 88.18 / 0.65 μ§ / μ \) in PBS (batch 021405) - 100 μΐ per well

11 ml χ 5 μ ^ πύ = 55 μg χ 1 μ1 / 0.65 μg = 84.6 μΐ GST-Magi-PDZ

2 - Incubate overnight at 4 ° C

3 - Wash 3x (TBS-Tween) with plate washer

4 - Block the plate with 250 μΐ blocking buffer (lot033005)

5 - Incubate for 2 hours at 25 ° C - Wash 3x (TBS-Tween) with plate washer

7 - Add 100 μΐ MBP-E6 / lysate sample to appropriate reservoirs

8 - Incubate for 1 hour at 25 ° C

9 - Wash 3x (TBS-Tween) with plate washer

10 - Add 100 μΐ anti-E6 antibody (4C6 - 2.85 mg / ml-lot 02) about 5 μ§ / ιη1 to the appropriate wells in 2% HBSN bufferBSA (lot 031805B). N-terminal peptide (HPVI6E6 lot # PN3952-2) is added to appropriate samples at 10 μ ^ ιηΐ to verify signal specificity (peptide is preincubated with anti-E6 antibody for 45 minutes prior to addition).

11 - Incubate for 2 hours at 25 ° C

12 - Wash 3x (TBS-Tween) with plate washer

13 - Prepare a 1: 5000 dilution of goat anti-mouse IgG-HRP (Jackson GxM IgG-HRP / Catalog # 115-035062 / lot60988) in 2% BSA / 0.05% Tween 20 Buffer (Lot 040505) .

10.0 ml χ 1/5000 = 0.002 ml χ 1000 μΐ / ml = 2.0 μΐ goat anti-mouse IgG-HRP

14 - Add 100 μΐ of goat anti-mouse IgG-HRP 1: 5000 dilution to appropriate reservoirs.

(Remove TMB Substrate and place at room temperature)

15 - Incubate for 1 hour at 25 ° C

16 - Wash 5x (TBS-Tween) with plate washer

17 - Add 100 μΐ of Neogen K-Blue TMB Substrate (lot041018)

18 - Incubate for 30 minutes at 25 ° C

19 - Add 100 μΐ Stop Solution (lot 030705) and Read at A450Formulation:

2% BSA / 0.05% Tween Buffer - (lot 040505) 2% BSA Blocker lot 033005 (49.975 ml) Tween 20 lot AO16759301 (0.025 ml)

Results

<table> table see original document page 39 </column> </row> <table>

* Extraction Volume - 1 ml

As can be seen from the results shown in the table above, E6 binding was detected for all blocked clinical specimens.

It is evident from the above results and debates that object methods provide several distinct advantages for fixed cell molecular analysis. In particular, the methods provide a routine method for producing a protein extract from fixed cells where proteins in the protein extract are detectable in deletion assays. Since it is generally difficult to detect certain proteins in fixed cells, the subject invention makes a significant contribution to the art.

All publications and patent applications cited in the descriptive report are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for this disclosure prior to the filing date and should not be construed as an admission that the present invention is not designed to precede such publication by virtue of the prior invention.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary technical skill in view of the disclosures of this invention that certain changes in modifications may be made to it without departing from the spirit or scope of the attached claims.

Claims (44)

Method for producing a protein extract from fixed cells, characterized in that it comprises: a) contacting said fixed cells with an extraction reagent to produce an intermediate composition having a pH at least around pH 10.0; and b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and producing said protein extract, wherein one or both of said extraction reagent and neutralizing ditoreagent comprises a nonionic detergent. Method according to claim 1, characterized in that it comprises: a) contacting said fixed cells with an extraction reagent to produce an intermediate composition having a pH at least around pH 10.0; and b) contacting said intermediate composition with a neutralizing reagent comprising a nonionic detergent, to neutralize said pH of said intermediate composition and to produce said protein extract. A method according to claim 1, characterized in that it comprises: a) contacting said fixed cells with an extraction reagent comprising a nonionic detergent to produce an intermediate composition having a pH of at least about pH 10.0; and b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and producing said protein extract. A method according to claim 1, further comprising: receiving a cell sample comprising said fixed cells prior to step a). A method according to claim 1, characterized in that said cells are fixed cervical cells. Method according to claim 1, characterized in that said fixed cells are present in the transport mediumSUREPATH®, CYTOLYT® or PRESERVCYT®. Method according to claim 4, characterized by the fact that said cellular sample is received from a remote location. A method according to claim 1, characterized in that said pH is in the range from pH 11.0 to about pH 13. A method according to claim 1, characterized in that said extraction reagent comprises a denaturant. A method according to claim 9, characterized in that said denaturant is sodium dodecyl sulfate (SDS), urea or sarkosyl. A method according to claim 1, characterized in that said nonionic detergent comprises a TRITON® or TWEEN® detergent. A method for detecting a protein, characterized in that it comprises: a) producing a protein extract from fixed cells according to the method as defined in claim 1; and b) testing for the presence of said protein in said protein extract. A method according to claim 12, characterized in that said test utilizes a capture agent for said protein. A method according to claim 12, characterized in that said test includes an immunoassay. A method according to claim 12, characterized in that said assay is an enzyme-linked immunosorbent assay (ELISA). A method according to claim 12, characterized in that said protein is a human papillomavirus (HPV) protein. A method according to claim 12, characterized in that said protein is an HPV E6 protein. A method according to claim 12, characterized in that said fixed cells are exfoliated cervical cells. A method according to claim 12, characterized in that said fixed cells are received from a remote location prior to said contacting step a). The method of claim 12 further comprising: d) communicating said test results to a remote location. A system for producing a protein extract, comprising: a) a cell sample comprising fixed cells, b) an extraction reagent that has a pH at least around pH 10.0, and c) a reagent wherein one or both of said extraction reagent and neutralization ditoreagent comprises a nonionic detergent and wherein the extraction ditoreagent and neutralizing agent may be used in the method as defined in claim 1 to produce a suitable protein extract for use in a binding assay. A system according to claim 21, further comprising reagents for detecting a protein in the protein extract. 23. Kit for producing a protein extract from fixed cells, characterized in that it comprises: a) an extraction reagent having a pH at least around pH 10.0, b) a neutralizing reagent; and c) instructions for carrying out the method as defined in claim 1 using said extraction reagent and said neutralization reagent, wherein one or both of said extraction reagent and neutralization ditoreagent comprise a nonionic detergent. Kit according to claim 23, characterized in that it further comprises reagents for detecting a protein in said protein extract. A kit according to claim 24, characterized in that said reagents include a capture agent for said ditaprotein. A kit according to claim 24, characterized in that said protein is HPV E6 protein. A method for extracting a target viral protein from a cell sample, comprising: a) contacting a cell sample containing cells in which a target viral protein is present or suspected to be present with an extraction reagent to produce a composition. intermediate having an pH of at least around pH 10.0; and b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and producing an extract of the target viral protein. A method according to claim 27, characterized in that one or both of said extraction reagent and said neutralizing reagent comprises a nonionic detergent. A method according to claim 27, characterized in that the cells in the cell sample are fixed with a chemical fixative. A method according to claim 29, characterized in that the chemical fixative is selected from the group consisting of alcohols, aldehydes, ketones, osmium tethoxide, acetic acid, picric acid, heavy metal ionic salts and propylene glycol. A method according to claim 30, characterized in that the alcohol is methanol or ethanol; aldehyde is glutaraldehyde or formaldehyde; and the ketone is acetone. The method according to claim 1, further comprising: receiving the cell sample comprising fixed cells prior to step a). A method according to claim 27, characterized in that the viral protein is encoded by a pathogenic virus. The method according to claim 33, wherein the pathogenic virus is selected from the group consisting of HIV, Ebola virus, Marburg virus, hepatitis virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papilloma virus (HPV). A method according to claim 27, characterized in that the viral protein is HPV E6 or E7 protein. A method according to claim 35, characterized in that HPV is strain 4, 11, 20, 24, 28, 36, 48, 50, 16, 18, 31, 35, - 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 73 or 82 of the HPV. A method according to claim 35, characterized in that HPV is a strain of oncogenic HPV selected from the group consisting of HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31. HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68, HPV 69, and HPV 82. A method according to claim 27, further comprising: detecting the presence of the target viral protein in the extract. A method according to claim 38, characterized in that the detection utilizes a capture agent for the target viral protein. A method according to claim 39, characterized in that the viral protein is HPV E6 protein; and the decapture agent is an antibody against the E6 protein. A method according to claim 39, characterized in that the viral protein is the HPV E6 protein; and the decapturing agent comprises a polypeptide containing a PDZ domain. Method according to claim 41, characterized in that the PDZ domain is the second domain of MAGI-1, the DLG or TIP1 PDZ domain. A method according to claim 27, characterized in that the cell sample still contains mucus or blood. A method according to claim 27, characterized in that the cell sample contains exfoliated cervical cells.