U.S. patent application number 09/400564 was filed with the patent office on 2002-02-28 for fluorescence polarization- based diagnostic assay for equine infectious anemia virus.
Invention is credited to JOLLEY, MICHAEL E., MONTELARO, RONALD C., NASIR, MOHAMMAD S., TENCZA, SARA B..
Application Number | 20020025512 09/400564 |
Document ID | / |
Family ID | 26798382 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025512 |
Kind Code |
A1 |
MONTELARO, RONALD C. ; et
al. |
February 28, 2002 |
FLUORESCENCE POLARIZATION- BASED DIAGNOSTIC ASSAY FOR EQUINE
INFECTIOUS ANEMIA VIRUS
Abstract
A fluorescence polarization assay for Equine Infectious Anemia
Virus utilizes a short peptide reagent probe derived from a
conserved immunodominant region of gp45. The probe is N-terminally
labeled, preferably with 6-carboxyfluorescein, and purified by
HPLC, which reacts in a homogenous assay with anti-EIAV antibodies
contained in the serum of field infected horses and ponies. The
assay has a sensitivity of about 90 percent with a specificity
approaching 100 percent.
Inventors: |
MONTELARO, RONALD C.;
(WEXFORD, PA) ; TENCZA, SARA B.; (PITTSBURGH,
PA) ; JOLLEY, MICHAEL E.; (ROUND LAKE, IL) ;
NASIR, MOHAMMAD S.; (GRAYSLAKE, IL) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
26798382 |
Appl. No.: |
09/400564 |
Filed: |
September 21, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60101553 |
Sep 23, 1998 |
|
|
|
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
Y10S 435/968 20130101;
G01N 33/533 20130101; G01N 2333/155 20130101; G01N 33/56983
20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70; C12P
013/14 |
Claims
What is claimed is:
1. A synthetic fluorescent antigen probe comprising: a peptide
comprising a sequence of amino acids selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
and SEQ ID NO:5; and a fluorophore conjugated to said peptide,
wherein said synthetic fluorescent antigen probe binds to serum
antibodies to equine infectious anemia virus to produce a
detectable change in fluorescence polarization.
2. The synthetic fluorescent antigen probe of claim 1, wherein said
peptide is 9 to 50 amino acids in length.
3. The synthetic fluorescent antigen probe of claim 2, wherein said
fluorophore is selected from the group consisting of
5-carboxyfluorescein and 6-carboxyfluorescein.
4. The synthetic fluorescent antigen probe of claim 3, wherein said
fluorophore is conjugated to the N-terminal amino acid of said
peptide.
5. The synthetic fluorescent antigen probe of claim 4, wherein said
fluorophore is 6-carboxyfluorescein.
6. The synthetic fluorescent antigen probe of claim 5, wherein said
peptide consists of the amino acid sequence of SEQ ID NO:1.
7. An assay for serum antibodies reactive with an antigen common to
a number of field strains of equine infectious anemia virus
comprising the steps of: diluting a serum specimen suspected of
containing antibodies reactive with an antigen of equine infectious
anemia virus with a buffer solution, to provide a buffered
specimen; adding to said buffered specimen a synthetic fluorescent
antigen probe comprising a fluorophore conjugated to a peptide
comprising a sequence of amino acids selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4
and SEQ. ID NO:5; incubating for a time sufficient to permit
binding in solution of said antibodies to said antigen probe to
provide a reaction product; and comparing the fluorescence
polarization of said reaction product to a blank control.
8. The assay of claim 7, wherein said peptide is 9 to 50 amino
acids in length.
9. The assay of claim 8, wherein said fluorophore is selected from
the group consisting of 5-carboxyfluorescein and
6-carboxyfluorescein.
10. The assay of claim 9, wherein said fluorophore is conjugated to
the N-terminal amino acid of said peptide.
11. The assay of claim 10, wherein said fluorophore is
6-carboxyfluorescein.
12. The assay of claim 11, wherein said peptide consists of the
amino acid sequence of SEQ ID NO:1.
13. The assay of claim 10, wherein said buffer solution is
substantially free of sodium chloride.
14. The assay of claim 13, wherein said buffer solution has a pH in
the range of 6.8 to 7.0.
15. The assay of claim 14, wherein said buffer solution contains
sodium phosphate in a concentration in the range of about 20
millimolar to about 50 millimolar.
16. A diagnostic assay kit for detecting serum antibodies to a
number of field strains of equine infectious anemia virus
comprising a synthetic fluorescent antigen probe in an amount
suitable for at least one assay and suitable packaging, said
synthetic fluorescent antigen probe comprising a fluorophore
conjugated to a peptide comprising a sequence of amino acids
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
17. The kit of claim 16, wherein said peptide is 9 to 50 amino
acids in length.
18. The kit of claim 17, wherein said fluorophore is selected from
the group consisting of 5-carboxyfluorescein and
6-carboxyfluorescein.
19. The kit of claim 18, wherein said fluorophore is conjugated to
the N-terminal amino acid of said peptide.
20. The kit of claim 19, wherein said fluorophore is
6-carboxyfluorescein.
21. The kit of claim 20, wherein said peptide consists of the amino
acid sequence of SEQ ID NO:1.
22. The kit of claim 19, further comprising a buffer solution.
23. The kit of claim 22, wherein said buffer solution is
substantially free of sodium chloride.
24. The kit of claim 23, wherein said buffer solution has a pH in
the range of 6.8 to 7.0.
25. The kit of claim 24, wherein said buffer solution contains
sodium phosphate in a concentration in the range of about 20
millimolar to about 50 millimolar.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/101,553, filed on Sep. 23, 1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is related to the field of veterinary
diagnostics and, more particularly, to a homogeneous fluorescence
polarization-based assay to detect specific antibodies contained in
the blood of horses and ponies infected with the lentivirus,
aetiologic for Equine Infectious Anemia (EIA).
[0004] 2. Description of Related Art
[0005] Equine Infectious Anemia Virus (EIAV) is a lentivirus
genetically related to human immunodeficiency virus type 1 (HIV-1)
that infects horses, ponies, and other equids (for a recent review
see Montelaro, et al., "Equine Retroviruses, in J. A. Levy, Ed.,
The Retroviridae, Vol. 2, p. 257 (Plenum Press: 1993 N.Y.). It
causes a chronic disease characterized by a period of cyclic fevers
and viremia, followed by clinical quiescence. The animals generally
survive this disease but remain infected, becoming lifelong
inapparent carriers; they appear to be healthy but in fact still
may have virus in their blood. There are thousands of EIAV-positive
horses in the US; most of them reside in the "hot zone", a group of
18 states along the Gulf coast and Mississippi valley (see Cordes,
"Equine Infectious Anemia", USDA 91-55-032 (1996)). The disease is
most prevalent there due to the humid environment that favors
growth of horse flies, the major vector of transmission of EIAV. In
an attempt to control the spread of this virus, horses are tested
before showing, breeding, or crossing state lines. If a horse is
found to be seropositive, its movement is severely restricted; the
horse must be euthanized or quarantined with a 200-yard barrier for
the rest of its life. However, because testing is not yet mandatory
for all horses, it is estimated that over 80% have never been
tested; this pool of horses may be a major reservoir for the virus.
Efforts are underway to encourage, and in some states mandate,
testing of all equids to better control this disease and reduce the
rate of infection.
[0006] EIAV-infected animals mount a vigorous immune response to
the viral infection. This results in reduction of viremia during
clinical quiescence to very low, often undetectable, levels. This
immune response is characterized by high-titer antibodies directed
to three major viral antigens: the envelope glycoproteins, gp90 and
gp45, and the capsid protein or core antigen, p26. Due to the
presence of high levels of antibody and low levels of virus during
most of the disease course, diagnostic assays have focused on
detection of viral antibodies.
[0007] One way to improve testing compliance is to develop better,
faster assays. Current official diagnostic assays for EIAV include
agar gel immunodiffusion (AGID) as reported in Coggins, et al.,
Cornell Vet USA LX: 330 (1970), competitive ELISA (C-ELISA), and
synthetic antigen ELISA (SA-ELISA). The first two assays detect
antibodies to the major core protein p26, which has a well
conserved structure but is a relatively poor immunogen compared to
the envelope proteins, gp90 and gp45. SA-ELISA detects antibodies
to gp45 and is approved for use, but can have a lower sensitivity.
The major drawbacks of the AGID test are the length of time it
takes to test the samples and the technical difficulty in
interpreting the results. ELISA-based tests can be completed in
several hours, but in a recent study the C-ELISA had a 2% false
positive rate, as reported in Issel, EIA-Hotzone Project, U of
Kentucky.
[0008] Fluorescence polarization (FP) has been used as a tool to
monitor protein-protein, protein-peptide, and other intermolecular
interactions, as described in Jolley, J. Biomol, Screen 1: 33
(1996). First described by Perrin (1926), it is the property of
many fluorophores that they emit light in the same direction in
which it is absorbed. When a fluorophore is freely rotating in
solution, the light is emitted in all directions by virtue of the
molecule's rotation during the lifetime of the fluorescence
emission; it is non-polarized. If, however, the fluorophore is part
of a slowly rotating molecule (one that is large or in a viscous
environment), the molecule does not rotate quickly with respect to
the lifetime of the fluorescence, and the emission will occur in
roughly the same direction as the absorption; it is polarized. This
property of fluorescence can therefore be used to distinguish small
molecules (e.g. fluorescent-labeled peptides) from large ones (e.g.
peptide bound to antibody). Relatively recent advances in
instrumentation have allowed the use of this phenomenon to develop
rapid immunoassays; for a large number of analytes including
therapeutic drugs and metabolites as well as antibodies to
infectious agents as, for example, Nielsen, et al., J. Immunol.
Methods 195: 161 (1996). These assays can be performed in a matter
of minutes (vs. hours or days for the other tests) and usually do
not require extensive sample preparation. In addition, the
materials required for the assay are relatively simple and highly
stable, making this technique attractive for field use.
[0009] In light of the need for a more rapid assay that can be used
in the field to detect EIAV-infected horses, we pursued FP as a
medium on which to develop a new diagnostic for anti-EIAV
antibodies. We selected, labeled, and evaluated several candidate
peptides for their ability to detect the presence of antibodies to
three EIAV proteins. This investigation has led to the development
of an FP-based assay which uses a well-conserved, immunodominant
region of gp45 transmembrane protein. The test is rapid and
possesses both high sensitivity and very high specificity. It
reacts with antibodies in serum or plasma from both experimentally-
and field-infected animals from various geographic areas.
SUMMARY OF THE INVENTION
[0010] In a first principal aspect, the present invention provides
a synthetic antigen probe comprising a fluorophore conjugated to a
peptide comprising a sequence of amino acids selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, and SEQ ID NO:5, wherein the synthetic fluorescent antigen
probe binds to serum antibodies to equine infectious anemia virus
to produce a detectable change in fluorescence polarization.
[0011] In a second principal aspect, the present invention provides
an assay for serum antibodies reactive to an antigen common to a
number of field strains of equine infectious anemia virus that
comprises the following steps. First, a serum specimen suspected of
containing antibodies reactive with an antigen of equine infectious
anemia virus is diluted with a buffer solution to provide a
buffered specimen. Next, a synthetic fluorescent antigen probe is
added to the buffered specimen. The synthetic fluorescent antigen
probe comprises a fluorophore conjugated to a peptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. The
buffered specimen with added antigen probe is incubated for a time
sufficient to permit binding in solution of the EIAV antibodies
with the antigen probe to provide a reaction product. The
fluorescence polarization of the reaction product is then compared
to a blank control.
[0012] In a third principal aspect, the present invention provides
a diagnostic assay kit for detecting serum antibodies reactive to a
number of field strains of equine infectious anemia virus. The kit
is comprised of a synthetic fluorescent antigen probe in amount
suitable for at least one assay and suitable packaging. The
synthetic fluorescent antigen probe comprises a fluorophore
conjugated to a peptide comprising a sequence of amino acid
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
[0013] In accordance with preferred embodiments of the present
invention, the fluorescence polarization-based diagnostic assay,
utilizing a synthetic fluorescent antigen probe, is rapid, easy to
use, and has a high sensitivity to and specificity for a number of
field strains of equine infectious anemia virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the reactivity of the original panel of
peptides with anti-EIAV IgG.
[0015] FIG. 2 shows the influence of peptide length and fluorescein
linkage on FP reactivity of peptide R51.
[0016] FIG. 3 shows the influence of the peptide length on FP
reactivity of peptide R32.
[0017] FIG. 4 shows the reactivity of R51-6CF with field-infected
and uninfected sera.
[0018] FIG. 5 shows relative peptide reactivity measured by
antifluorescein-capture ELISA.
[0019] FIG. 6 shows early three-week detection of newly
seroconverted animals.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0020] The initial panel comprised seven peptides, each derived
from one of the three major proteins of EIAV: peptide 1 and peptide
12 from gp90 (surface unit), R51, R32 and R51/32 from gp45
(transmembrane), and Sam50 and Sam51 from p26 (capsid). Candidate
peptides were chosen based on previous work showing regions of
broadly reactive antigenicity in certain proteins of EIAV, namely,
the p26 capsid, as described in Chong et al., J. Virology, 65: 1007
(1991), the gp45 transmembrane, as described in Chong, et al., J.
Virology, 65: 1013 (1991), and the gp90 surface unit, as described
in Ball, et al., J. Virology, 66: 732 (1992).
[0021] Table 1 lists these peptides and cross-references the
peptide name with the SEQ. ID. NO., the amino acid sequence, and
the source protein. These sequences were based on the Prototype
(cell-adapted Wyoming) strain of EIAV, described in Rushlow, et
al., Virology, 155: 309 (1986), and correspond to conserved regions
of the envelope proteins, as shown in Payne, et al., Virology, 172:
606 (1989).
1TABLE 1 Summary of EIAV Diagnostic peptides Peptide Name SEQ ID NO
Sequence Source R51 1 IGCIERTHVFCHTG gp45 (env 534-547) R51G 2
GCIERTHVFCHTG gp45 (env 535-547) R51C 3 CIERTHVFCHTG gp45 (env
536-547) R51L 4 LIGCIERTHVFCHTG gp45 (env 533-547) R51CysCys 5
CIERTHVFC gp45 (env 536-544) R32 6 KERQQVEETFNLI gp45 (env 522-534)
R32ER 7 ERQQVEETFNLI gp45 (env 523-534) R32R 8 RQQVEETFNLI gp45
(env 524-534) R32QQ 9 QQVEETFNLI gp45 (env 525-534) R32Q 10
QVEETFNLI gp45 (env 526-534) R32V 11 VEETFNLI gp45 (env 527-534)
R32E 12 EETFNLI gp45 (env 528-534) R32/51 13
KERQQVEETFNLIIGCIERTHVFCHTG gp45 (env 522-547) Sam50 14
ADDWDNRHPLPNAPLVAPPQGPIPMT p26 (170-201) Sam50H 15
HPLPNAPLVAPPQGPIPMT p26 (177-201) Sam50A 16 APLVAPPQGPIPMT p26
(182-201) Sam51 17 VDCTSEEMNAFLDVVPGQAGQKQILLDA- IDKI p26 (202-227)
Peptide 12 18 LETWKLVKTSGVTPLPISSEANTGL gp90 (env 408-434) Pep12S
19 SGVTPLPISSEANTGL gp90 (env 419-434) Pep12 20 PISSEANTGL gp90
(env 425-434) Peptide1 21 YGGIPGGISTPITQQSEKSK gp90 (env 1-20)
[0022] Probes based on all three proteins were explored because
whereas p26 is more conserved among EIAV strains, the level of
antibody induced is 10-to-100-fold lower to this protein compared
to the envelope proteins, gp90 and gp45. The peptides correspond to
conserved regions of the proteins that have been shown to react
broadly with equine sera in an ELISA-based format.
[0023] Due to problems encountered with testing horse serum, the
initial evaluation made use of purified IgG from a reference
long-term, field-infected horse (Lady). Purified IgG from field
infected horse serum (100 .mu.g/ml) was incubated with the
candidate probe peptides (2 nM) in PBS for 20 min. The results are
shown in FIG. 1, wherein black bars indicate probe added to IgG and
gray bars indicate probe in buffer alone. All peptides were the
5-carboxyfluorescein derivatives. Most of the peptides were found
to be insensitive to the presence of 60-100 .mu.g/ml Lady IgG in
PBS; however K51-5CF, derived from gp45, did undergo an increase in
FP in the presence of Lady IgG (see FIG. 1) from a free-peptide
polarization level of about 60 mP to around 140 MP. The other
peptides in the panel had only slight changes in polarization in
the presence of Lady IgG. Based on these results, we used R51-5CF
to explore the proper buffer conditions for interaction with
antibodies in whole serum.
[0024] It was observed that phosphate-buffered saline (10 mM Na, K
phosphate, 150 mM NaCl, pH 7.4) with Tween 20, Triton X-100, or
lithium dodecyl sulfate often caused precipitation of serum
proteins and resulted in low, and occasionally even negative,
polarization values due to severe background intensities and low
lamp feedback. Several different buffer compositions and detergents
were therefore tested for compatibility with horse serum. When
horse serum was diluted 1:50 or 1:100 into 20-50 mM sodium
phosphate without NaCl, this problem was virtually eliminated. Low
salt conditions also obviated the need for a detergent in the
buffer, although signal-to-noise ratios were slightly improved when
0.05% Tween-20 was added to the buffer. Under the low-salt
conditions, the polarization of peptide R51-5CF increased from 50
mP to over 200 mP with a 1:100 dilution of a strong positive EIAV
antiserum from an experimental infection (Pony 95). Thus it was
determined that the optimal buffer composition for the FP assay was
50 mM sodium phosphate, pH 6.8-7.0.
EXAMPLE 1
General Methods
[0025] Horse Sera. Serum from EIAV field-infected and uninfected
horses were generous gifts from the Texas Animal Health Commission,
Missouri Department of Agriculture, and University of Kentucky
(Utah, Florida,and Oklahoma field-infected sera). Prior to use and
after a freeze-thaw cycle, the sera were centrifuged at
12000.times.g for 2 minutes to pellet any precipitated protein.
[0026] Peptide Synthesis and Labeling. Peptides were produced on a
0.2-mmol scale using a Millipore Automated Peptide Synthesizer and
standard Fmoc chemistry, as described previously in Fontenot, et
al., Peptide Res., 4: 19 (1991). Peptides were labeled with 5- or
6-carboxyfluorescein (Molecular Probes, Eugene, Oreg.) while still
on the resin, thus placing the fluorophore on the N-terminus of the
peptide. The Fmoc protecting group was removed from the N-terminus
of the peptide-resin by 25% piperidine in dimethylformamide (DMF)
followed by four washes with DMF. The fluorescent probe was
dissolved in DMF to a concentration of 0.3 M and this solution was
mixed with 0.9 M DIPEA and 0.6 M HOBT/TBTU in a 5:4:2 ratio. The
dye mixture was added to the resin and incubated overnight with
shaking. Following four washes, each with DMF and dichloromethane,
the resin was dried under vacuum. The dye-conjugated peptides were
cleaved from the resin using standard TFA cleavage procedures
followed by multiple ether extractions. Peptides were purified by
reverse-phase HPLC and analyzed by mass spectrometry to confirm
that the desired product was obtained.
[0027] Anti-Fluorescein Capture ELISA. In order to measure antibody
binding to test peptides without regard to their suitability for
FP, an anti-fluorescein capture ELISA was used. To each well of an
Immulon 2 HB 96-well plate (Dynex, Chantilly, Va.) was added 50
.mu.L rabbit anti-fluorescein antibody (Molecular Probes), 3.5
.mu.g/ml in 50 mM sodium bicarbonate, pH 9.6; the plates were
sealed and incubated overnight. The wells were blocked with Blotto
(5% nonfat dry milk, 5% normal bovine serum, 0.025% Tween 20 in PBS
(PBST). The plates were then incubated with test horse sera,
diluted 1:100 in Blotto, for 1 h at RT, washed as above, then
incubated with anti-horse IgG(Fc)-HRP (United States Biochemical),
diluted 1:10.sup.5 in Blotto, for 1 h at RT and washed. The
substrate, TM Blue Soluble reagent (200 .mu.L/well; Intergen,
Milford, Mass.) was added and incubated for 20 minutes with
shaking, and the reaction stopped with the addition of 50
.mu.L/well 1.0 N H.sub.2SO.sub.4 for 5 minutes with shaking.
Absorbance at 450 nm was measured on a Dynex MR5000 microplate
reader. Because each peptide caused a slightly different background
absorbance, control wells containing no horse serum were included
for each peptide tested.
[0028] Fluorescence Polarization (FP) Measurements. The
fluorescein-labeled peptides were evaluated for their suitability
as probes for FP using an FPM-1 Fluorescence Polarization Analyzer
(Jolley Consulting and Research, Grayslake, Ill.) batch mode with
the following settings: PMT gain 80, heater off, single read. Serum
was diluted 1:100 or 1:50 into 2 mL of buffer in 12.times.75 mm
borosilicate glass tubes (VWR). After reading the blank,
fluorescently labeled peptide was added to a final concentration of
1-2 nM (100K-200K total intensity) and incubated for at least 15
minutes. The FP of the sample was measured and expressed as
millipolarization units (mP). Some of the sera were very dark,
presumably due to hemolysis. If such a serum sample had low lamp
feedback (<0.63), a two-fold further dilution was tested.
Polarization data was output to a computer running the FPM-1 data
collection software, then converted to an ASCII text file and
imported into the Quattro Pro spreadsheet program (Corel, Ottawa,
Ontario) for data analysis and graphing.
EXAMPLE 2
[0029] Once serum testing was enabled, we tested the panel of
peptides with sera from both experimentally and field-infected
horses. Although some reactivity was observed with peptides R32 and
peptide 12 against Pony 95, R51-5CF again was the only peptide from
the original panel that was sensitive to serum from field infected
horses. This result was in contrast to our ELISA results, in which
these two peptides reacted very strongly with both Pony 95 and Lady
sera. Thus, ELISA reactivity was not a good predictor of FP
reactivity. None of the peptides reacted with EIAV-negative horse
serum in either the FP or ELISA assays.
[0030] Based on these data the R51 peptide was optimized for
maximum FP signal by exploring the effects of alterations in
peptide length and fluorescein linkage. Because different
fluorescein linkages can result in differences in sensitivity in
the FP assay, R51 peptide was labeled with 6-carboxyfluorescein so
the difference between the two labels could be ascertained. Analogs
of R51 were also synthesized possessing 0-3 amino acid residues
between the N-terminal cysteine and the fluorescein probe. Peptides
(approx. 2 nM) were incubated with a 1:100 dilution of serum in 50
mM sodium phosphate, pH 6.8, for 20 minutes. The results are shown
in FIG. 2, in order of decreasing peptide length. In FIG. 2, black
bars show the results for experimentally-infected (pony 95),
hatched bars for field infected (Lady), gray bars for uninfected
(Petite), and white bars for no serum added. It was found that
neither reducing nor increasing peptide length improved signal but
changing from a 5- to 6-carboxyfluorescein label did significantly
improve the signal of R51 with positive sera (220 mP for 5CF vs.
>300 for 6CF) without increasing background as shown in FIG. 2.
As the R51-6CF probe was the most sensitive to the positive sera
tested, 6-carboxyfluorescein is the preferred fluorophore. However,
other fluorophores, such as rhodamine, BODIPY.TM. M, Texas Red.TM.
and Lucifer yellow, could also be used. For a detailed listing of a
variety of commercially available fluorophores, see Handbook of
Fluorescent Probes and Research Chemicals, ed. Karen Larison, by
Richard P. Haughland, Ph.D., 5th ed., 1992, published by Molecular
Probes, Inc.
[0031] Because R51 contains two Cys residues that may form a loop
in the native protein, the differences in reactivity were assessed
between linear or cyclized peptide (cyclic by virtue of an
intramolecular disulfide bond). In particular, the cyclized peptide
was more sensitive to field isolates than the linear form of the
probe. However, the probe was prone to precipitation under
conditions that allow cyclization, which caused an increase in the
polarization of the free probe and reduction of sensitivity;
therefore, the peptide stock solutions contained dithiothreitol
(DTT) to prevent aggregation. The peptide was found to be stable
upon dilution, and probably spontaneously cyclizes under those
conditions.
[0032] Because of the loop formed by the two Cys residues in R51,
it is believed that the sequence of amino acids between and
including the two Cys residues, i.e., the R51CysCys peptide, SEQ.
ID. NO:5 (see Table 1), constitutes the minimum peptide length
useful for detecting serum antibodies in field-infected equines.
The maximum useable peptide length is not known. However, other
experimental work has shown that peptides as large as 50 amino
acids in length, that include the R51 peptide, have been found to
react to such serum antibodies.
EXAMPLE 3
[0033] In addition to R51, peptides R32 and pepl2 were engineered
in an effort to improve their sensitivity in FP. These peptides
showed strong and broad reactivity in the anti-fluorescein ELISA,
but did not exhibit an increase in FP upon mixing with purified
antibodies from a field-infected animal. A series of peptides of
different lengths was synthesized and labeled at their N-terminal
by fluorescein-6-isothiocyana- te. The complete R32 series was
tested for reactivity to positive and negative sera as set forth in
FIG. 3. We observed a bell-shaped curve, with a maximum FP of
>200 mP with a 1:100 dilution of pony 95. The most sensitive
peptide was R32QQ, a 10-amino acid peptide. The R32 peptides all
showed good reactivity with strongly positive experimentally
infected animals (pony 95, for example) but little reactivity with
serum from the field-infected horse (Lady). Likewise, neither of
the pepl2 analogs displayed a large change in FP in the presence of
Lady serum (data not shown). Therefore, it was concluded that under
the conditions of the assay, these peptides are sensitive only to
experimentally infected horse sera and are not appropriate for a
diagnostic assay for field infected equids.
EXAMPLE 4
[0034] Focusing on our highly sensitive probe, R51-6CF, 258 sera
from both uninfected and field infected horses from Texas,
Missouri, Utah, and Florida were tested. The specificity of the
probe was examined by testing serum samples that were negative by
AGID (FIG. 4, open circles). Testing at a 1:100 dilution, the 110
negative serum samples had very low and consistent polarization
values (73.6.+-.3.0 mP), indicating that specificity was very high
for R51-6CF. Out of the 110 negative samples tested, only two
initially reacted in the assay, and both of these had signs of
bacterial contamination. Upon sterile filtration and re-testing,
these two samples gave consistently negative readings. Thus
provided that the samples were kept in good condition our assay had
a specificity of 100%. This represents a practically perfect
correlation with a negative AGID result and is an improvement in
specificity over the C-ELISA. In addition to the high specificity,
the polarization values were so consistent that one could
distinguish a positive from a negative sample by as few as 5 mP
units.
[0035] In order to determine the sensitivity of this assay, 153
sera from field-infected animals were tested at a 1:100 dilution.
These sera were obtained from geographically distinct regions
throughout the United States: Texas, Utah, Missouri, and Florida.
The probe reacted well with most of the sera: the distribution of
values is represented in FIG. 4, showing the results for peptide
(.about.2 nM) incubated with a 1:100 dilution of sera from
field-infected horses. Sera are grouped by geographic region. The
measurable sera caused the polarization of R51-6CF to increase to
an average mP value of 150.+-.55, a clear and significant
difference from the average of the negative sera. The probe reacts
well with antibodies from diverse geographic regions, indicating
that the epitope is well conserved and is thus suitable for a
diagnostic antigen. The overall percent reactivity of this serum
panel in the FP assay was found to be 93%. This represents the
correlation between reactivity in the two assay formats; actual
percent sensitivity to true positives may need to be determined by
Western blotting of the discrepant samples. In two other studies,
the average sensitivity of the FP assay was 95% and the specificity
was 100%.
EXAMPLE 5
[0036] Some of the sera from Missouri (4/10) could not be tested
due to interference from a high level of hemolysis, resulting in
low lamp feedback values. However, we found 14 samples out of 123
positive Texas sera that did not react with this probe in the FP
assay (FIG. 4), even at a 1:50 dilution. In order to confirm the
serological status of FP-unreactive sera, they were tested in a
western blot (data not shown) as well as in the
antifluorescein-capture ELISA using the seven original peptides
derived from the three major antigens mentioned above (FIG. 5).
Sera (1:50 dilution) were tested for reactivity to four
EIAV-derived, fluorescein-labeled peptides in an ELISA format as
described in the methods. NHS, normal (uninfected) horse serum;
Tx43 through Txll7, FP-nonreactive, Tx47 through pony 95,
FP-reactive sera. Black bars, peptide R51F; hatched bars, R32; gray
bars, pepl2; white bars, Sam50. These data indicated that several
of the FP-nonreactors have no measurable antibody to either R51 or
R32 in the ELISA format, and bind only weakly to the other peptides
(pepl2 from gp90 and Sam50 from p26). Thus these sera do not appear
to have antibody to the gp45 antigen.
[0037] Of the samples that were non-reactive in FP but confirmed to
be positive, several exhibited ELISA reactivity to the p26-derived
Sam50 peptide that was higher than some of the positive controls
(FIG. 5). These data suggested that although the original Sam50
peptide was insensitive to EIAV-positive sera, a shorter form of
the Sam50 peptide might be more sensitive in the FP assay for these
serum samples. Two shortened analogs of Sam50; Sam50A, a 14-AA
peptide, and Sam50H, a 19-AA peptide, were synthesized. However
when tested in the FP assay, none of these analogs displayed a
measurable interaction with the EIAV-positive sera. This lack of
reactivity may be due to the low levels of antibodies to this
epitope and/or that the peptide is still too long for the
fluorophore to undergo a change in polarization upon antibody
binding. Further testing will be needed to determine whether a
Sam50-based peptide will be able to detect antibodies to EIAV when
the R51-6CF peptide does not react.
EXAMPLE 6
[0038] In addition to testing sera from various geographic areas,
the ability of R51-6CF to detect antibodies early in infection was
examined. Serum samples acquired weekly during an experimental
infection of four ponies were tested for the presence of anti-EIAV
antibodies by FP.
[0039] This assay detected antibody in both 1:100 and 1:50
dilutions of serum at 3 weeks post infection (FIG. 6), which is the
same time at which antibody was first detected by Con A capture
ELISA (Hammond et al., J. Virology 71: 3840 (1997)). These data
indicate that the FP assay is at least as sensitive as an ELISA is
in detecting early antibody responses to EIAV infection. In
addition, the test was as or more sensitive than AGID in detecting
early antibody responses; ponies 561, 562, and 564 were AGID
positive on day 21, and pony 567 was not positive until day 23.
Thus the FP technique may have an advantage over AGID in the
detection of early immune responses; this may be due to the fact
that the immune response to envelope tends to arise earlier and to
higher levels than do the antibodies to p26. In summary, peptides
derived from all three of these proteins were evaluated and found
that R51, the peptide derived from gp45, had the best combination
of high reactivity and broad specificity, as it was able to detect
antibodies from horses infected with many field strains. The R51
peptide is based on a region that is immunodominant in
lentiviruses, yet is well conserved. Although the amino acid
sequences of envelope proteins of lentiviruses generally vary more
than the capsid and other core proteins, it was found that
antigenic variation was not a large problem in this case, since we
have achieved approximately 90% sensitivity with a single
envelope-based peptide antigen. The few samples that did not bind
to this probe may be from animals infected with an unusual strain
of EIAV that bears sequence variation in this region of the
protein. For these few sera, a peptide based on p26 or gp90 may
need to be developed. The R51 non-reactor ponies did show some
reactivity to Sam50 in the peptide ELISA. The R51 nonreactive
horses do show antibody reactivity to all three major proteins in a
Western blot, so efforts are underway to find a peptide epitope
that will react with these field infected sera.
Assay Kit
[0040] The synthetic fluorescent antigen probe of the present
invention is preferably made available in kit form. The kit
includes a quantity of buffer solution for diluting serum specimens
suspected of containing antibodies to EIAV, the synthetic
fluorescent antigen probe in amount suitable for at least one assay
(i.e., about 100 nanograms), along with suitable packaging and
instructions for use. The synthetic fluorescent antigen probe may
be provided in solution, as a liquid dispersion, or as a
substantially dry powder (e.g., in lyophilized form).
[0041] The suitable packaging can be any solid matrix or material,
such as glass, plastic, paper, foil, and the like, capable of
separately holding within fixed limits the buffer and the synthetic
fluorescent antigen probe. For example, the buffer solution and the
synthetic fluorescent antigen probe may be provided in separate
labeled bottles or vials made of glass or plastic.
[0042] The synthetic fluorescent antigen probe comprises a peptide
comprising a sequence of amino acids selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
and SEQ ID NO:5, with a fluorophore conjugated to the peptide.
Preferably, the peptide is no more than 50 amino acids in length.
The fluorophore is preferably selected from the group consisting of
5-carboxyfluorescein and 6-carboxyfluorescein and is preferably
conjugated, i.e., covalently bonded, to the N-terminal amino acid
of the peptide, though other fluorophores and other binding sites
could be used. The most preferred fluorophore is
6-carboxyfluorescein, and the most preferred peptide consists of
the amino acid sequence of SEQ ID NO:1. Thus, the synthetic
fluorescent antigen probe ideally comprises the R51-6CF probe
described herein.
[0043] The buffer solution provided in the kit is preferably
substantially free of sodium chloride because, as described herein,
this has been found to produce the best results. Preferably, the
buffer solution is a sodium phosphate solution with a concentration
in the range of about 20 millimolar to about 50 millimolar, to
provide a pH in the range of 6.8 to 7.0.
[0044] The diagnostic assay kit is intended to be used in the
following way, as should be described in the instructions for use.
A serum specimen suspected of containing antibodies to EIAV is
diluted with a quantity of the buffer solution provided in the kit
to provide a buffered specimen. A dilution of about 1:100 is
preferred. Next, enough of the synthetic antigen probe is added to
the buffered specimen to yield a probe concentration of about 2 nM.
The buffered specimen with added probe is then incubated for a time
sufficient to permit binding in solution of EIAV antibodies with
the antigen probe to provide a reaction product. An incubation time
of about 20 minutes is typically sufficient. The fluorescence
polarization of the reaction product is then compared to a blank
control, i.e., compared to a buffered solution of the synthetic
antigen probe at about the same concentration without added serum.
Sequence CWU 1
1
21 1 14 PRT Equine infectious anemia virus 1 Ile Gly Cys Ile Glu
Arg Thr His Val Phe Cys His Thr Gly 1 5 10 2 13 PRT Equine
infectious anemia virus 2 Gly Cys Ile Glu Arg Thr His Val Phe Cys
His Thr Gly 1 5 10 3 12 PRT Equine infectious anemia virus 3 Cys
Ile Glu Arg Thr His Val Phe Cys His Thr Gly 1 5 10 4 15 PRT Equine
infectious anemia virus 4 Leu Ile Gly Cys Ile Glu Arg Thr His Val
Phe Cys His Thr Gly 1 5 10 15 5 9 PRT Equine infectious anemia
virus 5 Cys Ile Glu Arg Thr His Val Phe Cys 1 5 6 13 PRT Equine
infectious anemia virus 6 Lys Glu Arg Gln Gln Val Glu Glu Thr Phe
Asn Leu Ile 1 5 10 7 12 PRT Equine infectious anemia virus 7 Glu
Arg Gln Gln Val Glu Glu Thr Phe Asn Leu Ile 1 5 10 8 11 PRT Equine
infectious anemia virus 8 Arg Gln Gln Val Glu Glu Thr Phe Asn Leu
Ile 1 5 10 9 10 PRT Equine infectious anemia virus 9 Gln Gln Val
Glu Glu Thr Phe Asn Leu Ile 1 5 10 10 9 PRT Equine infectious
anemia virus 10 Gln Val Glu Glu Thr Phe Asn Leu Ile 1 5 11 8 PRT
Equine infectious anemia virus 11 Val Glu Glu Thr Phe Asn Leu Ile 1
5 12 7 PRT Equine infectious anemia virus 12 Glu Glu Thr Phe Asn
Leu Ile 1 5 13 27 PRT Equine infectious anemia virus 13 Lys Glu Arg
Gln Gln Val Glu Glu Thr Phe Asn Leu Ile Ile Gly Cys 1 5 10 15 Ile
Glu Arg Thr His Val Phe Cys His Thr Gly 20 25 14 26 PRT Equine
infectious anemia virus 14 Ala Asp Asp Trp Asp Asn Arg His Pro Leu
Pro Asn Ala Pro Leu Val 1 5 10 15 Ala Pro Pro Gln Gly Pro Ile Pro
Met Thr 20 25 15 19 PRT Equine infectious anemia virus 15 His Pro
Leu Pro Asn Ala Pro Leu Val Ala Pro Pro Gln Gly Pro Ile 1 5 10 15
Pro Met Thr 16 14 PRT Equine infectious anemia virus 16 Ala Pro Leu
Val Ala Pro Pro Gln Gly Pro Ile Pro Met Thr 1 5 10 17 32 PRT Equine
infectious anemia virus 17 Val Asp Cys Thr Ser Glu Glu Met Asn Ala
Phe Leu Asp Val Val Pro 1 5 10 15 Gly Gln Ala Gly Gln Lys Gln Ile
Leu Leu Asp Ala Ile Asp Lys Ile 20 25 30 18 25 PRT Equine
infectious anemia virus 18 Leu Glu Thr Trp Lys Leu Val Lys Thr Ser
Gly Val Thr Pro Leu Pro 1 5 10 15 Ile Ser Ser Glu Ala Asn Thr Gly
Leu 20 25 19 16 PRT Equine infectious anemia virus 19 Ser Gly Val
Thr Pro Leu Pro Ile Ser Ser Glu Ala Asn Thr Gly Leu 1 5 10 15 20 10
PRT Equine infectious anemia virus 20 Pro Ile Ser Ser Glu Ala Asn
Thr Gly Leu 1 5 10 21 20 PRT Equine infectious anemia virus 21 Tyr
Gly Gly Ile Pro Gly Gly Ile Ser Thr Pro Ile Thr Gln Gln Ser 1 5 10
15 Glu Lys Ser Lys 20
* * * * *