Title:  Method for diagnosis of Epstein-Barr virus associated disease

United States Patent:  6,506,553

Issued:  January 14, 2003

Inventors:  Smith; Richard S. (Del Mar, CA); Parks; D. Elliot (Del Mar, CA)

Assignee:  Ortho Diagnostics Systems, Inc. (Raritan, NJ)

Appl. No.:  413233

Filed:  March 30, 1995

A novel assay utilizing Epstein-Barr virus (EBV) specific peptides is disclosed. The assay is particularly useful for detecting early antigen antibodies in a blood sample from an individual having an EBV-associated disease, the disease preferably being infectious mononucleosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an assay for the presence and quantification of anti-EA antibodies associated with an EBV-associated disease in a specimen from an individual. Preferably, the method of the invention is used for diagnosis of infectious mononucleosis (IM) and the preferred individual is a human.

In a preferred embodiment, the present invention provides a method for detecting antibodies which bind to a peptide having the amino acid sequence [XKQKHPKKVKQAFNPLY]_n or [XPARPETPSPAIPS]_n, wherein X and Y are independently from about 0 to 5 naturally occurring amino acids, wherein n is about 1 to 1000, and wherein the peptide is capable of binding antibody in a specimen from an individual with Epstein-Barr virus (EBV) associated disease. The method comprises contacting a specimen from the individual with the peptides, incubating the specimen and the peptides for a period of time and under conditions sufficient for the antibodies to bind to the peptides, and detecting the presence of the antibodies to the peptides.

The method of the invention comprises the epitopic polypeptides KQKHPKKVKQAFNPL (SEQ ID NO:1) and PARPETPSPAIPS (SEQ ID NO:2), and conservative variations and mixtures of these peptides. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Thus, by using a routine screening method, such as by testing a conservative variant polypeptide with sera from a patient with EBV-associated disease, one of skill in the art can readily determine if the variant polypeptide has the requisite biological activity of the polypeptide of the invention without resort to undue experimentation.

The method of the invention further includes the addition of the polypeptide ETFTETWNRFITHTEY (SEQ ID NO:3) to polypeptides KQKHPKKVKQAFNPL (SEQ ID NO:1) and PARPETPSPAIPS (SEQ ID NO:2), for diagnosis of between 95% and 100% of IM patients. SEQ ID NO:1 is the 50.10 peptide of EA-D, SEQ ID NO:2 is the K7B peptide of EA-D (U.S. Pat. No. 4,879,213) and SEQ ID NO:3 is the 17.1 peptide of EA-R. Reactivity of a sample with EA-D, 50.10 peptides or anti-EA-D peptide antibodies is preferably associated with infectious mononucleosis and nasopharyngeal carcinoma. Likewise, reactivity with EA-R, 17.1 peptides or anti-EA-R peptide antibodies is preferably associated with lymphonias. These specific peptides and their corresponding monoclonal antibodies are also useful for detecting the EA-D and EA-R transitions associated with a particular disease state.

The epitopic polypeptides of the invention may contain additional amino acids at the amino and carboxy termini in order to increase their serological reactivity. Preferably, the additional amino acids are the naturally occurring amino acids of the protein, or conservative variations of these amino acids, and range in number from about 0 to about 5 independently. For example, a variation of SEQ ID NO:1 comprises the epitopic polypeptide, ARQKQKHPKKVKQAFNPLI, wherein the underlined amino acids represent extensions of the original polypeptides. The polypeptides of the invention can also be utilized as repeating units ranging from 1 to about 1000 units in length. These units can be homogeneous, for example, where all of the units are repeats of the same polypeptide or can be mixtures of the polypeptides of the invention.

The peptides used in the method of the invention can be used singularly, in mixtures, or as multimers such as aggregates, polymers, and the like, in various combinations. Thus, the invention embraces polypeptides which comprise one or more of the same, or different, polypeptides of the invention to produce a homogeneous or heterogeneous polymer with respect to the particular polypeptides of the invention which are contained therein. Appropriate techniques for producing various mixtures, aggregates, multimers and the like will be known to those of skill in the art. For example, the invention includes a polypeptide comprising SEQ ID NO:1 and SEQ ID NO:2, and optionally SEQ ID NO:3, or any combination of these, wherein the sequences may be linked either directly or indirectly, for example, by using a spacer or linker moiety.

Peptides of the invention can be synthesized by such well known solid phase peptide synthesis methods described by Merrifield, J. Am. Chem. Soc. 85:2149, 1962, and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969, pp. 27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0^oC. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on SEPHADEX G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid-phase Edman degradation.

During or after the synthesis, reactive amino acids may be protected by various blocking groups, for example, cysteines may be blocked by 3,4-dimethylbenzyl (DMB) groups, arginines and histidines by tosyl (TOS) groups, aspartic acid and glutamic acids by benzyl (Bzl) groups, and lysines the 2-chloro-benzyloxycarboxyl (2-CBZ) groups. Other protective blocking groups are well-known, and can be used in the present invention. Those of ordinary skill in the art will know of other techniques for peptide synthesis, or can readily ascertain such techniques, without resorting to undue experimentation.

The term "EBV-associated disease" means any disease caused, directly or indirectly, by EBV as well as diseases which predispose a patient to infection by EBV. Examples of diseases falling into the former category include infectious mononucleosis, nasopharyngeal carcinoma, and Burkitt's lymphoma. Diseases in the latter category (i.e., those which place the patient at risk of EBV infection) include Sjorgren's syndrome and, generally, any condition that causes a state of immunosuppression or decreased function of the immune system such as patients who receive organ transplants and certain cancer therapies.

The peptides described herein are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the peptides in these immunoassays can be detectably labeled in various ways. Alternatively, a detectably labeled protein which binds to antibody can be utilized to detect binding of the peptide and EA-D or EA-R antibody. For example, a preferred detectably labeled protein is a second antibody which specifically binds to IgM, IgG, or IgA antibody.

Examples of types of immunoassays which can be utilized in the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antibodies using the peptides of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention: include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the peptides or second antibody for example, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the peptides of the invention or a second antibody can be done using standard techniques common to those of ordinary skill in the art.

"ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify. the amount of an antigen present in a sample. (A description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P. Sites, et al., published by Lange Medical Publications of Los Altos, Calif. in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043, which are all incorporated herein by reference.)

For an ELISA, typically used enzymes linked to a polypeptide as a label include horseradish peroxidase, alkaline phosphatase and the like. Each of those enzymes is used with a color-forming reagent or reagents (substrate) such as hydrogen peroxide and o-phenylenediamine; and p-nitrophenyl phosphate, respectively. Alternatively, biotin linked to a polypeptide can be utilized as a label to signal the presence of the immunoreactant in conjunction with avidin that is itself linked to a signalling means such as horseradish peroxidase.

For purposes of the invention, an anti-EA-D or anti-EA-R antibody specific for a peptide of the invention may be detected by the method of the invention when present in biologicai fluids and tissues. Any specimen containing a detectable amount of such antigen can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis. An especially preferred sample is blood, and most preferably serum. Preferably, the specimen is a human blood or serum sample.

The specific concentrations of the antibody and antigen, the temperature and time of incubation, as well as other assay conditions, can be varied, depending on such factors as the concentration of the antibody in the sample, the nature of the sample and the like. Those of skill in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Typically, the time period is predetermined for a given set of reaction conditions by well known methods prior to performing the assay.

For example, the immunoassay of the invention may be run at 4^o-45^oC. Under biological assay conditions, the maintenance time period is usually from minutes to hours, such as 30 minutes to 2 hours to overnight, however, these time periods will vary. Other steps such as washing, stirring, shaking, filtering, or pre-assay extraction of antigen, and the like, may, of course be added to the assay, as may be desired or necessary for a particular situation. The immunocomplex formed can then be detected by means described herein.

The method of the invention is well suited for preparation of a diagnostic kit for detecting anti-EA antibodies in a specimen. Therefore, in another embodiment, the invention provides a diagnostic kit for detecting the presence of antibodies which bind to a peptide having the amino acid sequence [XKQKHPKKVKQAFNPLY]_n or [XPARPETPSPAIPSY]_n, wherein X and Y are independently from about 0 to 5 naturally occurring amino acids, wherein n is about 1 to 1000, and wherein the peptide is capable of binding antibody in a specimen from an individual with Epstein-Barr virus (EBV) associated disease. The kit comprises a first container containing the peptide(s) and a second container containing a detectable label for detecting binding of the peptides and the antibodies. The kit may further include a container comprising a peptide having the amino acid sequence [XETFTETWNRFITHTEYY]_n.

A kit of the invention comprises a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise the peptides of the invention which are or can be, detectably labelled. The kit may also have containers containing any of the other above-recited immunochemical reagents used to practice the diagnostic methods.

The diagnostic system or kit described herein can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably, the specific binding agent binds the reagent species when that species is present as part of a complex and most preferably, the binding agent is an IgG or IgM antibody or antibody binding fragment. In preferred embodiments, the specific binding agent is labeled.

The diagnostic kit of the present invention can be used in an "ELISA" format to detect the quantity of an EA antibody in a vascular fluid sample, such as blood, serum, or plasma. Thus, a K7B and 50.10 peptide of the present invention, alone or in combination with a 17.1 peptide, can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems. A reagent is typically affixed to a solid matrix by absorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used.

The peptides described for use in the method of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising a polypeptide of the invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding peptides, or will be able to ascertain such, using routine experimentation.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene, cross-linked polyacrylanide, nitrocellulose- or nylon-based webs, such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

The reagent species or labeled specific binding agent of a diagnostic kit described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support, such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. The term "package" refers to a solid matrix or material, such as glass, plastic (e.g., polyethylene, polypropylene and polycarbonate), paper, foil and the like capable of holding within fixed limited a diagnostic reagent such as a peptide or second antibody of the present invention. Thus, for example, a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody or polypeptide to be detected.

Claim 1 of 19 Claims

What is claimed is:

1. A method for detecting antibodies which bind to a first peptide consisting essentially of the amino acid sequence KQKHPKKVKQAFNPL, or a second peptide consisting essentially of the amino acid sequence PARPETPSPAIPS, wherein the first and the second peptides bind to an antibody in a body specimen from an individual with an infectious mononucleosis (IM) disease, the method comprising the steps of:

(a) providing the first and second peptides;

(b) contacting a specimen from the individual with the peptides;

(c) incubating the specimen and the peptides for a period of time and under conditions sufficient for the antibodies to bind to the peptides; and

(d) detecting the presence of the antibodies to the peptides.
 

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