WO1993001304A1 - Synthetic peptides related to fiv-env proteins - Google Patents

Abstract

Peptides are disclosed which are novel and useful for diagnostic tests for feline immunodeficiency virus (FIV).

Description

SYNTHETIC PEPTIDES

RELATED TO FIV-ENV PROTEINS

This application is a continuation of United States application Serial No. 07/728,134 filed 10 July 1991 which is still pending.

FIELD OF THE INVENTION

This invention relates to non-naturally occurring or synthetic peptides which mimic an antigenic site on the surface of feline immunodeficiency virus (FIV) envelope protein.

BACKGROUND OF THE INVENTION

A retrovirus termed feline immunodeficiency virus (FIV) is now known to be the etiological agent in feline acquired immunodeficiency syndrome (FAIDS). Testing for FIV is important in diagnosing exposure to the virus in order to to limit the spread of infection.

Detection of exposure to FIV is accomplished by measuring levels of FIV antibodies in serum, plasma, whole blood or saliva. In the first generation of tests for FIV antibodies, inactivated crude or purified viral protein from lysates of FIV-infected cells are used to coat a solid phase. Biological samples suspected of containing FIV antibodies are incubated with the solid phase and after an

appropriate period washed away. Then anti-feline antibody tagged with a detectable label is added to the solid phase. The label, which may be an enzyme, radioisotope or fluorescent molecule is measured to determine the presence of FIV antibodies in the sample. The difficulty with using viral lysates as a source of FIV antigens is the high level of

impurities which interfere with testing for FIV antibodies. In particular, serum proteins derived from cell culture media are difficult to remove.

Many animals have antibodies to these contaminants which are also contained in most vaccines. When samples from such animals are tested, the can give rise to false positive test results. See, e.g., Hosie, et al. AIDS 4:215-300 (1990). There is a need for FIV antigens which are well defined and do not cross react with other non-FIV antibodies and other interfering materials contained in the sample to be tested.

SUMMARY OF THE INVENTION

This invention provides a highly sensitive and accurate detection or diagnostic procedure that does not require the use of virus or lysates thereof as a test reagent. In the procedure of the invention a polypeptide test reagent prepared by chemical means, is used to detect the presence of antibodies to FIV in body fluids.

BRIEF DESCRIPTION OF THE INVENTION

A test reagent comprising a peptide having about twenty seven amino acids arranged in a specific sequence, preferably prepared by solid phase

synthesis is provided. The 27mer peptide is useful in a highly sensitive and accurate method for the detection of antibodies to FIV in serum. A shorter 13mer peptide contained within the 27mer is similarly useful. Peptides having specific immunoreactivity to FIV antibodies are selected from the group consisting of peptides containing from twelve to twenty seven amino acids, the sequence of which corresponds to a part or the entirety of the following sequence

(27mer): Lys-Val-Glu-Ala-Met-Glu-Lys-Phe-Leu-Tyr-Thr-Ala-Phe- Ala-Met-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe- Phe-Cys; or the sequence (13mer):

Lys-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe-Phe-Cys and analogues thereof wherein the amino acids in the sequence are substituted, deleted or added which retain the immunoreactivity to FIV antibodies is preserved.

The highly sensitive and accurate method for the detection of antibodies to FIV in serum comprises the following steps:

(a) Preparing a peptide selected from the group consisting of peptides containing from twelve to twenty seven amino acids, the sequence of which corresponds to a part or the entirety of the

following sequence:

Lys-Val-Glu-Ala-Met-Glu-Lys-Phe-Leu-Tyr-Thr-Ala-Phe- Ala-Met-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe- Phe-Cys; or the sequence:

Lys-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe-Phe-Cys and analogues of such sequences wherein the amino acids in the sequence are substituted, or deleted, but which retain immunoreactivity to FIV antibodies.

(b) Coating a solid phase, such as polystyrene with a buffered solution containing from about 0.5 ug/ml to about 20 ug/ml of peptide, in an appropriate solvent such as phosphate buffered or borate buffered saline. (c) Attaching to the peptide a detectable label which may be an enzyme, radioisotope or fluorescent molecule.

(d) Combining a portion of the sample fluid with the peptide-coated solid phase and the conjugated peptide for sufficient time, e.g., 5 minutes, to permit FIV antibody linkage to the solid phase and labelled peptide.

(e) Determining the extent of bound labelled peptide which will form in direct proportion to the concentration of FIV antibody in the sample fluid.

EXAMPLE I

Detection of Antibodies to FIV by an

Enzyme-Linked Immunosorbent Assay Using 27mer Peptide

Wells of 96-well plates were coated at 22-24°C overnight with the 27 mer peptide, prepared as

described, at 0.5 ug per well in 100 ul 10 mM borate buffer, pH 9.6. To the peptide-coated wells was added 200 ul of a solution containing 1% (w/v) bovine serum albumin, 10% (w/v) sucrose in 10 mM phosphate pH 7.4 to block non-specific protein binding sites. Following an overnight incubation at 2-8 °C the wells were emptied and dried under laminar flow at 22-24 °C for 16-24 hours.

Horseradish peroxidase was activated by periodate oxidation (20 mM sodium metaperiodate) for two hours at 25°C. Peptide was added to the activate enzyme and allowed to react for two hours at 25 °C and the resulting conjugate was stabilized by sodium

borohydride reduction resulting in a molar

conjugation ratio of peptide to enzyme of 15-35. The conjugated peptide is diluted for use into a solution containing 10 mM phosphate, 6% (w/v) bovine serum albumin and 0.1% (v/v) Tween 20. The working

conjugate concentration is from about 0.5 ug/ml to about 10 ug/ml.

Fifty microliters of sample fluid was added to a peptide-coated well along with 50 ul of diluted conjugate and the mixture was allowed to incubate for 5 minutes. The wells were washed five times with deionized water and 50 ul of 3,3',5,5'-tetramethylbenzidine(0.2 g/l in citrate/acetate buffer pH 5.0) and 50 ul of urea peroxide (0.55 g/1 in citrate buffer pH 5.0) were added to each well and incubated for 5 minutes at 22-24 °C. Color generated by the peroxidase label was measured in an ELISA reader at 630nm. The results are shown in Table 1. The indication of status is based on confirmatory testing by Western blot analysis and/or indirect

immunofluorescence assay (IFA) on FIV infected cells.

The results in Table 1 show that the ELISA test procedure according to the present invention with 98 serum samples is very accurate and highly specific.

EXAMPLE 2

Detection of Antibodies to FIV by an

Enzyme-Linked Immunosorbent Assay Using 13mer Peptide The procedure of Example 1 was repeated using the 13mer peptide

(Lys-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe-Phe-Cys) for both the solid phase coating and enzyme

conjugate. The results are presented in Table 2.

The results in Table 2 show that the ELISA test procedure according to the present invention using the 13mer peptide with 98 serum samples is very accurate and highly specific.

Claims

WE CLAIM:

1. Peptide compositions having specific

immunoreactivity to FIV antibodies selected from the group consisting of peptides containing from twelve to twenty seven amino acids, the sequence of which corresponds to a part or the entirety of the

following sequence:

Lys-Val-Glu-Ala-Met-Glu-Lys-Phe-Leu-Tyr-Thr-Ala- Phe-Ala-Met-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln- Phe-Phe-Cys; analogues thereof wherein the amino acids in the sequence are substituted, deleted or added as long as the immunoreactivity to FIV antibodies derived from the three-dimensional conformation of the sequence is preserved; and conjugates of the peptides or

analogues thereof, wherein:

Ala = alanine

Asn = asparagine

Cys = cysteine

2. A non-naturally occurring peptide composition having specific immunoreactivity to FIV antibodies selected from the group consisting of peptides containing from twelve to twenty seven amino acids, the sequence of which corresponds to a part or the entirety of the following sequence:

Lys-Val-Glu-Ala-Met-Glu-Lys-Phe-Leu-Tyr-Thr-Ala-Phe- Ala-Met-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe- Phe-Cys; analogues thereof wherein the amino acids in the sequence are substituted, deleted or added, but which retain immunoreactivity to FIV antibodies.

3. A peptide composition according to claim 1 wherein the peptide is:

Lys-Val-Glu-Ala-Met-Glu-Lys-Phe-Leu-Tyr-Thr-Ala- Phe-Ala-Met-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln- Phe-Phe-Cys.

4. A peptide composition according to claim 1 wherein the peptide is:

Lys-Gln-Glu-Leu-Gly-Cys-Asn-Gln-Asn-Gln-Phe-Phe-Cys.

5. Labelled peptide compositions according to claim 1 or claim 2 wherein the label is chosen from the group consisting of enzymes, radioisotopes or fluorescent molecules.

6. An immunoassay method for detection of antibodies to FIV comprising:

(a) coating a solid support with an effective amount of the peptide composition according to claim 1;

(b) adding to said coated solid support a test serum and the labelled peptide composition of claim 4 wherein the FIV antibodies in the test serum from a peptide-antibody-labelled peptide complex;

(c) incubating the mixture at room temperature; and

(d) detecting the presence of the

peptide-antibody-labelled peptide complex.

7. A solid support bearing an immunoadsorbent comprising a peptide composition according to claim 1 or claim 2.

8. A test kit comprising:

(a) a solid support as defined by claim 7;

(b) an enzyme labeled peptide composition as

defined by claim 5; and

(c) a substrate that reacts with said enzyme

labeled peptide to form a colored product.