Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-06-03
2002-10-01
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S007900, C435S007910, C435S007920, C435S007930, C435S007940, C435S007950, C435S975000, C436S518000, C436S531000, C530S326000, C530S327000, C530S328000, C530S826000
Reexamination Certificate
active
06458528
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the general field of diagnosing feline immunodeficiency virus (“FIV”) infection.
BACKGROUND OF THE INVENTION
Infection with a variety of lentiviruses is associated with immunodeficiency disease. Cats infected with FIV (Pedersen et al., Science 235:790-793, 1987; U.S. Pat. No. 5,037,753), for example, show a number of pathogenic symptoms reminiscent of acquired immunodeficiency disease (“AIDS”) (Yamamoto et al., Am. J. Vet. Res. 49:1246-1258, 1988; Ackley et al., J. Virol. 64:5652-5655, 1990; Siebelink et al., AIDS Res. Hum. Retroviruses 6:1373-1378, 1990). FIV-associated feline AIDS is an important feline disease, with incidences as high as 15% in populations of sick animals (O'Connor et al., J. Clin. Microbiol. 27:474-479, 1989). The transient, low level viremia often seen in connection with the persistence of intracellular proviruses makes detection of the antibody response to infection a reliable assay for infection by FIV.
Enzyme-linked immunosorbent assays (“ELISA”) for detecting FIV antibodies use purified, inactivated FIV virions and/or antigens as solid-phase reagents to bind FIV antibodies in samples. Antibodies recognizing epitopes on the gag-encoded p24 capsid (“p24”) and p15 nucleocapsid proteins and on the env-encoded gp40 transmembrane (“gp40”) and gp100 surface proteins have been detected by radioimmunoprecipitation analysis (“RIPA”) or immunoblot (Hosie et al., AIDS 4:215-220, 1990; Steinman et al., J. Gen. Virol. 71:701-706, 1990; Andersen et al., U.S. Pat. No. 5,656,732; Kemp et al., U.S. Pat. No. 5,591,572; Mermer et al., Similarities between the Transmembrane Proteins of FIV and HIV, Cold Spring Harbor Symposium, RNA Tumor Viruses, 1991; Tilton et al., J. Clin. Microbiol. 28:898-904, 1990). Various anti-FIV antibodies have also been generated (O'Connor et al., U.S. Pat. Nos. 5,219,725 and 5,177,014).
IDEXX Laboratories, Inc. markets a FIV diagnostic device under the trademark SNAP® COMBO, which detects FIV antibodies in feline samples. The device includes recombinant p24 as a solid-phase capture reagent. FIV antibody captured by the solid-phase reagent is detected with disrupted FIV conjugated to horseradish peroxidase. See U.S. Pat. Nos. 5,726,010 and 5,726,013.
SUMMARY OF THE INVENTION
Applicants have discovered that detection of FIV antibodies indicative of FIV infection is improved by using a polypeptide marker composition that is enhanced for the presence of both FIV env polypeptides and FIV gag polypeptides. By “enhanced” is meant that the FIV env and gag polypeptides are present in the marker composition at higher weight percentage levels than in a simple mixture of FIV proteins obtained from disrupted virus. Enhancement can be achieved, e.g., by spiking the viral mixture with a purified or partially purified preparation of the env and gag polypeptides, or by using such a preparation as a marker composition without inclusion of the viral mixture.
Accordingly, the invention features a diagnostic method for determining FIV infection by contacting a feline sample (e.g., a serum or blood sample) with an antibody-binding capture composition that includes both enhanced FIV env and gag polypeptides. An enhanced (e.g., purified) polypeptide can be a recombinant or synthetic polypeptide, or a polypeptide isolated from FIV virions. The reaction of antibodies in the sample with the capture composition indicates that the donor of the sample is infected with FIV.
An immunogenic fragment of a polypeptide is a polypeptide fragment that can bind to one or more antibodies that are specific to the polypeptide in its native conformation. Immunogenic fragments of a FIV gag precursor p55 include, but are not limited to, p55 itself, p55 cleavage products such as p24, p15, and p10, and any p55 fragments recognized by monoclonal antibody (“mAb”) 2D4 (American Type Culture Collection (“ATCC”) HB9890), 3H8 (ATCC HB12531), 4F2 (ATCC HB9888), 2H4 (ATCC HB12530), or 6E6 (ATCC HB9899). Immunogenic fragments of a FIV env precursor gp130 include, but are not limited to, gp130 itself, gp130 cleavage products such as gp40 and gp110; they also include any gp130 fragments containing a cysteine loop of gp40 and any gp130 fragments that bind to mAb 2F11 (ATCC HB10295), 1C9 (ATCC HB12529), or 3H9 (ATCC HB12528). An exemplary immunogenic fragment of gp130 is ELGCNQNQFFCK (SEQ ID NO:1). An exemplary second capture polypeptide is CELGCNQNQFFCK (SEQ ID NO:2).
In one embodiment of the above-described method, the binding composition is attached to a phase (e.g., a solid phase) immiscible with the sample. An antibody-binding detection composition can be applied to detect reaction of antibodies in the feline sample with the capture composition. This detection composition may include two detection polypeptides that respectively contain immunogenic fragments of p55 and gp130. For instance, the detection composition can contain disrupted FIV (e.g., a mixture of viral proteins obtained by disrupting native FIV virions with a detergent) spiked with a peptide having the sequence of SEQ ID NO:1or 2. The polypeptides in the detection composition are preferably labeled with a detectable moiety, such as an enzyme that catalyzes a detectable reaction, colloidal gold, a radionuclide, and a fluorophore.
Also embraced by the invention is a device for performing an assay that determines whether a feline is infected with FIV. This device contains the above-described antibody-binding capture and detection compositions. In one embodiment, the detection composition is held in a container.
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. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
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Groat Randall G.
Mermer Brion
O'Connor Thomas P.
Fish & Richardson P.C.
Idexx Laboratories Inc.
Stucker Jeffrey
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