Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-05-06
2002-05-07
Budens, Robert D. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S007940, C435S007950, C530S395000
Reexamination Certificate
active
06383765
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to purification of polypeptides from feline T-cell lymphotropic lentivirus (FIV).
FIV is a retrovirus originally isolated from cats which exhibit an AIDS-like syndrome. Pederson et al., 235 Science 790, 1987. The virus belongs to the same group as the human immunodeficiency virus (HIV), the causative agent of human AIDS. Pederson et al., describe detection of antibody to FIV by an immunofluorescent assay, and by Western blots, using virus purified by centrifugation on sucrose gradients in Tris base, pH 7.4, containing 0.1M NaCl and 1 mM EDTA. A few protein bands were detected and, although antigenic comparison was not made, the positions of these bands were tentatively said to correspond to the major core protein, p24 the gag precursor protein, p55, and the endonuclease protein, p32, of HIV.
Pederson et al., U.S. Patent Application Filed Aug. 26, 1987 entitled “Feline T-Lymphotropic Lentivirus” (which is not admitted to be prior art to the present application) describes the results presented by Pederson et al., supra, and states that Western blotting of FIV infected cell lysates yielded major protein bands at approximately 22-26 kD, usually about 24 kD; 50-60 kD, usually about 55 kD; and 28-36 kD, usually about 32 kD.
SUMMARY OF THE INVENTION
In a first aspect, the invention features a purified polypeptide having an epitope of an antigenic polypeptide of FIV. The polypeptide may be glycosylated or unglycosylated. By antigenic polypeptide is meant a polypeptide which is able to raise (with the aid of an adjuvant if necessary) an antibody response in cats. The polypeptide may be a polypeptide fragment of at least 5 amino acids, or a polypeptide naturally occurring in FIV particles. The fragment may be obtained from a naturally-occurring polypeptide, for example, by enzymatic digestion of a naturally occurring polypeptide, or may be produced by isolation or synthesis of a gene encoding a desired polypeptide and expression of that polypeptide within a desired expression system, for example, a bacterial, yeast, or mammalian expression system.
By epitope is meant a single antigenic site of an antigenic polypeptide. Such an epitope is recognized specifically by a monoclonal antibody to an antigenic polypeptide of FIV.
By purified is meant that the polypeptide is separated from other cell components with which it naturally occurs, for example, FIV polypeptides. Preferably, the polypeptide is sufficiently pure to permit its use to prepare a monoclonal antibody to the polypeptide, and even more preferably, pure enough to allow the amino acid sequence of the polypeptide to be determined by standard procedure. Generally, the purified polypeptide is biologically active in that it is suitable for preparation of a monoclonal antibody, or is suitable for detection of naturally-occurring antibodies within the serum of a cat.
In preferred embodiments, the purified polypeptide has at least 75% amino acid homology to a polypeptide fragment of at least 20 amino acids obtained from an FIV gag or env polypeptide, most preferably the purified polypeptide includes an amino acid sequence having at least 75% homology to a whole of a gag or env polypeptide, even more preferably, the purified polypeptide is an entire gag or env amino acid sequence. Examples of gag and env polypeptides include p10, p15, p26, gp40, gp100, and gp130.
In a second aspect, the invention features a method for detecting an antibody to FIV within a sample, including the steps of providing a purified polypeptide as described above, and contacting that polypeptide under conditions suitable to allow an antibody/polypeptide complex to form between antibodies within the sample and the purified polypeptide, and detecting the formation of such complexes. The presence of antibody/polypeptide complexes is indicative of antibody to FIV present within the sample.
In a third aspect, the invention features a purified nucleic acid including a 50 nucleotide sequence having at least 90% homology with a 50 nucleotide sequence naturally occurring in an FIV particle. By purified is meant that the nucleic acid is substantially separated away from all of the components with which it naturally occurs, e.g., polypeptides and other nucleic acids. Preferably, the nucleic acid is completely separated from such components, and is a pure solution of nucleic acid, or is held within a cell in which it does not naturally occur, e.g, a bacterial cell, another viral particle or a non-feline eucaryotic cell. By 90% homology is meant that the nucleotide sequence is identical at at least 45 of the 50 nucleotides.
In preferred embodiments, the nucleic acid encodes a polypeptide including an epitope of an antigenic polypeptide of FIV, e.g., an epitope of a gag or env polypeptide of FIV. Most preferably the nucleic acid is carried in an expression vector and can be expressed in a bacterial, fungal or other eucaryotic cell, e.g., a mammalian cell.
In a related aspect the invention features purified polypeptide including ten or more contiguous amino acids taken from the sequence (using standard letters to represent amino acids) V-Q-S-R-G-S-G-P-V-C-F-N-C-K-K-P-G-H-L-A-R-Q-S-H or P-I-Q-T-V-N-G-V-P-Q-Y-V-A-L-D-P-K-M-V-S or S-V-Q-S-R-G-Q-G-P-V-A-F-N.
Applicants have provided polypeptides suitable for specific detection of FIV antibodies and thus have allowed accurate detection of infection with FIV. Applicants have also provided polypeptides useful for production of vaccines to prevent disease caused by FIV in cats.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
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Andersen Philip R.
O'Connor Thomas P.
Tonelli Quentin J.
Budens Robert D.
Idexx Corporation
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