U.S. patent application number 14/451847 was filed with the patent office on 2015-02-12 for outer membrane protein 18 as a diagnostic marker for campylobacter.
The applicant listed for this patent is TECHLAB, INC.. Invention is credited to JANICE E. BUSS, DAVID M. LYERLY, TRACY D. WILKINS.
Application Number | 20150044698 14/451847 |
Document ID | / |
Family ID | 52448968 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150044698 |
Kind Code |
A1 |
BUSS; JANICE E. ; et
al. |
February 12, 2015 |
OUTER MEMBRANE PROTEIN 18 AS A DIAGNOSTIC MARKER FOR
CAMPYLOBACTER
Abstract
Accurate and fast detection of the presence of Campylobacter
disease is important for the proper treatment of patients with
Campylobacter infection. Present tests depend upon culture of
viable bacteria and identification by microscopy, which requires
care, skill, and two or more days for conclusive results. The
current invention improves the ease of use and overcomes the
limitations of loss of viability and delay inherent in
Campylobacter bacterial culture and provides a more rapid
alternative for the identification and diagnosis of Campylobacter
and campylobacteriosis. The invention provides a new method of
detecting Campylobacter by utilizing an outer membrane protein (OMP
18) to develop antibodies for use in immunoassays of bacterial
cultures or human fecal samples.
Inventors: |
BUSS; JANICE E.; (DUBLIN,
VA) ; LYERLY; DAVID M.; (RADFORD, VA) ;
WILKINS; TRACY D.; (RINER, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TECHLAB, INC. |
Blacksburg |
VA |
US |
|
|
Family ID: |
52448968 |
Appl. No.: |
14/451847 |
Filed: |
August 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61862787 |
Aug 6, 2013 |
|
|
|
Current U.S.
Class: |
435/7.32 |
Current CPC
Class: |
G01N 33/56922
20130101 |
Class at
Publication: |
435/7.32 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A method for detecting Campylobacter in a fecal sample from a
person, the method comprising: obtaining the fecal sample from the
person; determining the presence of Outer Membrane Protein 18
(OMP18) in the fecal sample by contacting said fecal sample with a
purified antibody that reacts specifically with Omp 18 or reactive
fragment thereof under suitable conditions to form a complex, of
said Omp 18 or reactive fragment thereof and the purified antibody,
wherein the purified antibody specifically reacts with Omp 18 amino
acid sequence of SEQ. ID NO: 1 or any amino acid sequence having at
least 70% identity with SEQ. ID NO:1; detecting the presence of
said complex, wherein the presence of said complex is diagnosing
the person with Campylobacter infection.
2. The method of claim 1, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against a full length
sequence or a portion of a native OMP18 having at least 70%
identity with SEQ. ID NO:1 purified from various Campylobacter
species.
3. The method of claim 1, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against recombinant
OMP18 having at least 70% identity with SEQ. ID NO:1 expressed in
bacteria, yeast, or mammalian cells.
4. The method of claim 1, wherein one or more antibodies are
polyclonal or monoclonal antibodies, or fragments of monoclonal
antibodies that specifically recognize OMP18 amino acid sequence of
SEQ. ID NO: 1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO: 1.
5. The method of claim 1, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against one or more
variants of OMP18 from one or more Campylobacter species.
6. The method of claim 1, wherein the presence of OMP18 is
identified using lateral flow techniques and immunochemical
reactions on a membrane.
7. The method of claim 1, wherein the presence of OMP18 is
identified using an enzyme-linked immunoassay.
8. The method of claim 1, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against a full length
sequence or a portion of a native OMP18 purified from various
Campylobacter species or recombinant OMP18 expressed in bacterial,
yeast, or mammalian cells.
9. The method of claim 1, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against one or more
variants of OMP18 from one or more Campylobacter species.
10. The method of claim 1, wherein one or more antibodies are
polyclonal or monoclonal antibodies, or fragments of monoclonal
antibodies that specifically recognize OMP18 amino acid sequence of
SEQ. ID NO: 1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO: 1.
11. A method for detecting Campylobacter in a bacterial culture
that is derived from a fecal sample from a person, the method
comprising: obtaining the fecal sample from the person; preparing
and growing Campylobacter bacteria from the fecal sample by
standard methods; determining the presence of Outer Membrane
Protein 18 (OMP18) in the bacterial culture by contacting said
bacterial culture with a purified antibody that reacts specifically
with Omp 18 or reactive fragment thereof under suitable conditions
to form a complex, of said Omp 18 or reactive fragment thereof and
the purified antibody, wherein the purified antibody specifically
reacts with Omp 18 amino acid sequence of SEQ. ID NO:1 or any amino
acid sequence having at least 70% identity with SEQ. ID NO:1;
detecting the presence of said complex, wherein the presence of
said complex indicates a diagnosis of the person with Campylobacter
infection.
12. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against a full length
sequence or a portion of a native OMP18 amino acid sequence of SEQ.
ID NO: 1 or any amino acid sequence purified from various
Campylobacter species or any amino acid sequence having at least
70% identity with SEQ. ID NO:1.
13. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against recombinant
OMP18 expressed in bacterial, yeast, or mammalian cells.
14. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against one or more
variants of OMP18 from one or more Campylobacter species.
15. The method of claim 11, wherein one or more antibodies are
polyclonal or monoclonal antibodies, or fragments of monoclonal
antibodies that specifically recognize OMP18 amino acid sequence of
SEQ. ID NO: 1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO: 1.
16. The method of claim 11, wherein the presence of OMP18 is
identified using lateral flow techniques and immunochemical
reactions on a membrane or an enzyme-linked immunoassay.
17. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against a full length
sequence or a portion of a native OMP18 amino acid sequence of SEQ.
ID NO: 1 or any amino acid sequence purified from various
Campylobacter species or any amino acid sequence having at least
70% identity with SEQ. ID NO:1.
18. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against recombinant
OMP18 expressed in bacterial, yeast, or mammalian cells.
19. The method of claim 11, wherein one or more antibodies against
OMP18 or reactive fragment thereof are raised against one or more
variants of OMP18 from one or more Campylobacter species.
20. The method of claim 11, wherein one or more antibodies are
polyclonal or monoclonal antibodies, or fragments of monoclonal
antibodies that specifically recognize OMP18 amino acid sequence of
SEQ. ID NO: 1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO: 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application No. 61/862,787, filed Aug. 6, 2013, entitled "Outer
Membrane Protein 18 (CJ0113) as a Diagnostic Marker for
Campylobacter," the entire contents of which are herein
incorporated by reference.
BRIEF SUMMARY
[0002] The present invention provides a method to develop
immunoassays for the detection of Campylobacter. The invention
includes a method for use of a recombinant form of a Campylobacter
outer membrane protein (OMP18) as an antigen and use of antibodies
produced to the antigen in antibody-based diagnostic assays for
Campylobacter. The object of the present invention is to overcome
the difficulty of the prior art, which is identification or
diagnosis of Campylobacter based on the culture of the
bacteria.
SEQUENCE LISTING
[0003] A text file in compliance with ASCII and having a ".txt"
extension has been electronically submitted via EFS-Web. The text
file named "Sequence Listing" was created on Aug. 1, 2014, and is
1.78 KB. The text file is expressly incorporated by reference
herein in its entirety.
BACKGROUND
[0004] The Center for Disease Control estimates that, as of 2011,
almost 48 million people in the USA experience some form of
food-borne illness every year. Of these, 128,000 are hospitalized
and 3000 die. Worldwide, Campylobacter species are the most common
cause of bacterial gastroenteritis, with 400-500 million cases of
diarrhea each year. Infants in developing countries are at even
greater risk, as are travelers to those countries.
Campylobacter-associated gastroenteritis is estimated to affect
nearly 1 million people a year in the USA. In approximately 1 in
1000 cases, Campylobacter jejuni is closely linked to the
subsequent development of Guillian-Barre Syndrome, an acute,
auto-immune paralysis. C. jejuni infection has also been associated
with reactive arthritis in both children and adults.
[0005] In addition to Campylobacter spp., other common causes of
bacterial food-borne gastroenteritis are enteropathogenic
Escherichia coli, and Salmonella spp.; norovirus is a significant
viral cause of food-borne disease. Although many cases of "food
poisoning" self-resolve without the need for medical treatment,
infants and the elderly are at higher risk for dehydration and more
serious complications.
[0006] When individuals with severe symptoms of gastroenteritis
seek medical help, the clinician is faced with multiple possible
causes that can present with similar clinical features (e.g.,
diarrhea, nausea, vomiting, fever, abdominal pain) but that require
very different treatment. For viruses, use of antibiotics is
ineffective and can contribute to development of antibiotic
resistance in bacteria. Ineffective empirical treatment can release
selective pressure on resident resistant bacteria and convert
non-symptomatic infection into illness. Treatment of
enteropathogenic E. coli with antibiotics is contraindicated, as
this may actually increase the chances of the patient experiencing
hemolytic uremic syndrome (HUS). Antibiotic treatment of
Salmonella-caused gastroenteritis may prolong the carrier state and
is associated with relapse. Conversely, erythromycin treatment of
Campylobacter infection may shorten illness and the time during
which bacteria are shed, but only if the antibiotic is begun within
4 days of the onset of symptoms. Post-infectious irritable bowel
syndrome has been associated with a longer duration of untreated
infection. Identifying, without delay, which organism may be
causing the gastroenteritis is important for diagnosis and
selection of the appropriate treatment.
[0007] For Campylobacter, the current standard for identification
is bacterial culture followed by microscopic examination of the
organisms. Although this traditional method is straightforward, it
has three major limitations. First, pathogenic species of
Campylobacter are microaerophilic or strictly anaerobic, so that
exposure of culture or stool to environmental oxygen conditions
leads to death or inactivation of the bacteria. Thus, during
transport or storage of specimens the number of viable organisms
can decrease, leading to potentially inaccurate culture results.
Second, while the standard culture medium and microaerophilic
incubation conditions have been improved for two common species,
Campylobacter jejuni (Cj) and Campylobacter coli (Cc), other
Campylobacter species such as C. lari, C. upsaliensis, C.
ureolyticus and C. hyointestinalis have also been linked to human
disease, but are more difficult to grow and require different
culture media and anaerobic incubation that are not routinely
available in clinical laboratories. As a result, illnesses caused
by these more fastidious, but never-the-less pathogenic organisms,
are likely to go unrecognized and unreported. Third, even with
appropriate culture conditions, each of these species is
slow-growing, requiring from 48-72 hours (e.g., Cj or Cc) up to 7
days before reaching a point where the culture can safely be
reported out as negative. Prolonged culture lengths are
particularly true in the food safety industry, in which rinses of
poultry carcasses or samples of raw milk (the most commonly
identified sources of Campylobacter illnesses) are sampled for
surveillance of sanitation measures or during outbreaks. Such
delays can leave the clinician in a quandary and the patient with
non-specific, ineffective, or even inappropriate treatment.
[0008] To provide a more rapid alternative to growth of cultures
and microscopy for the identification and diagnosis of
Campylobacter and campylobacteriosis, the present invention
utilizes an outer membrane protein (OMP 18) as a biomarker for use
in immunoassays of bacterial cultures or human fecal samples. The
fecal samples used in the present invention may be unpreserved or
preserved.
[0009] OMP 18 is an 18 kilodalton protein found in the outer
membrane of many Campylobacter species. The amino acid sequence of
OMP 18 is partially homologous to a similar protein, PAL in E.
coli. The E. coli PAL protein is a peptidoglycan-associated
lipoprotein, and the Campylobacter OMP18 is predicted to share
these properties.
[0010] Several properties of OMP18 make it a good candidate for a
diagnostic marker. Outer membrane proteins are typically subject to
significant variation even among strains of the same species. The
flagellin protein (FlaA), major outer membrane protein (MOMP/PorA)
and fibronectin-binding protein (CadF) show large variability even
between Campylobacter jejuni strains. However, from the genomic
sequences of various strains of Cj that are currently available,
the amino acids of OMP 18 (C jejuni subsp jejuni, ATTC 33560;
GenBank: EIB41509.1) (FIG. 1) are completely identical among 51
strains of C. jejuni and 93% identical to the OMP18 protein of C.
coli. OMP 18 thus provides a potential immunogen that is
well-conserved among many strains of Cj and Cc. Conversely, OMP 18
bears little similarity to outer membrane proteins of Campylobacter
species that are non-pathogenic or that are not clearly associated
with human illness. Additionally, as a proposed antigen, OMP 18 has
been shown to be highly immunogenic, a requirement for robust
immunoassays. Serum from individuals that have had confirmed
campylobacteriosis contains antibodies that recognize OMP 18 in
immunoblots of bacterial cultures.
[0011] The present invention is directed towards improving ease of
use and overcoming the limitations of delay inherent in
Campylobacter bacterial culture. As previously stated, the current
standard for identification of Campylobacter is through culturing
and microscopy, which has several limitations, such as decreased
viability with exposure to aerobic conditions. In contrast, in an
embodiment of the present invention, a new method of detecting
Campylobacter in stool samples using antibodies to OMP18 in
immunoassays is provided. Antibody-based tests, such as
Enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests,
are rapid and cost-effective tests for detection of
pathogen-specific antigens. Furthermore, this new method allows for
the identification of Campylobacter in aerobic conditions, without
culturing. Thus, the identification of Campylobacter can take place
even when the sample is not alive. This is beneficial, for example,
if a sample is brought into the lab under prolonged aerobic
conditions. We propose to use OMP18 as an antigen, and antibodies
developed using OMP18, for the detection of Campylobacter in
stool.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Illustrative embodiments of the present invention are
described in detail below with reference to the attached drawing
figures, wherein:
[0013] FIG. 1 depicts the amino acid sequence of OMP18 from
Campylobacter jejuni subspecies jejuni strain ATTC 33560; GenBank:
EIB41509.1.
[0014] FIG. 2 depicts a Coomassie Blue-stained SDS-polyacrylamide
gel of recombinant OMP18, which was used as antigen.
[0015] FIG. 3 depicts an immunoblot in which proteins from C.
jejuni bacteria were separated by SDS-polyacrylamide gel
electrophoresis, transferred to a membrane, and incubated with
HRP-conjugated, affinity-purified anti-OMP18.
[0016] FIG. 4 depicts a graphical representation of a response
curve of recombinant OMP18 on an anti-OMP18 ELISA.
[0017] FIG. 5 depicts a graphical representation of a response
curve of several strains of Campylobacter jejuni and Campylobacter
coli on an anti-OMP18 ELISA.
[0018] FIG. 6 shows a graphical representation of the response
curve of the anti-OMP18 ELISA to bacterial cultures of four species
of Campylobacter.
DETAILED DESCRIPTION
[0019] Embodiments of the present invention may be embodied as,
among other things, a method, a device, and the like for testing
fecal samples and identifying the presence of the Outer Membrane
Protein 18 (OMP18).
[0020] Accordingly, in one aspect, the present invention is
directed to a method for testing a fecal sample from a person. The
method includes obtaining a fecal sample from the person,
determining whether Outer Membrane Protein 18 (OMP18) is present in
the fecal sample, and upon determining OMP18 is present in the
fecal sample, diagnosing the person with Campylobacter
infection.
[0021] In another aspect, the present invention is directed to a
method for testing a bacterial culture that is derived from a fecal
sample from a person. The method includes obtaining a fecal sample
from the person, preparing and growing Campylobacter bacteria from
the sample by standard methods, determining whether OMP18 is
present in the bacterial culture sample, and upon determining OMP18
is present in the bacterial culture sample, diagnosing the person
with Campylobacter infection.
[0022] In yet another aspect, the present invention is directed to
a device to test a fecal sample for the presence of OMP18 to be
utilized to diagnose a person with Campylobacter infection. The
device comprises a receiving portion to receive a fecal sample from
a person, and a testing portion to detect OMP 18 in the fecal
sample to be utilized to diagnose a person with Campylobacter
disease.
[0023] In yet another embodiment, the present invention is directed
to a method for detecting Campylobacter in a fecal sample from a
person. The method includes obtaining the fecal sample from the
person, determining the presence of OMP18 in the fecal sample by
contacting said fecal sample with a purified antibody that reacts
specifically with Omp 18 or reactive fragment thereof under
suitable conditions to form a complex of said Omp 18 or reactive
fragment thereof and the purified antibody, wherein the purified
antibody specifically reacts with Omp 18 amino acid sequence of
SEQ. ID NO: 1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO:1, and detecting the presence of said
complex, wherein the presence of said complex results in diagnosing
the person with Campylobacter infection.
[0024] In an additional embodiment, the present invention is
directed to a method for detecting Campylobacter in a bacterial
culture that is derived from a fecal sample from a person. The
method includes obtaining the fecal sample from the person,
preparing and growing Campylobacter bacteria from the fecal sample,
determining the presence of OMP 18 in the bacterial culture by
contacting said bacterial culture with a purified antibody that
reacts specifically with Omp 18 or reactive fragment thereof under
suitable conditions to form a complex, of said Omp 18 or reactive
fragment thereof and the purified antibody, wherein the purified
antibody specifically reacts with Omp 18 amino acid sequence of
SEQ. ID NO:1 or any amino acid sequence having at least 70%
identity with SEQ. ID NO:1, and detecting the presence of said
complex, wherein the presence of said complex indicates a diagnosis
of the person with Campylobacter infection.
[0025] The following are examples of procedures which have been
utilized to establish the preferred assays according to the present
invention. The following examples are merely exemplary and not
presented by way of limitation.
EXAMPLE 1
[0026] DNA encoding amino acids 29-165 of OMP 18 (C. jejuni
subspecies jejuni, strain ATTC 33560; GenBank: EIB41509.1) was
synthesized commercially and expressed in E. coli. Recombinant Omp
18 (either untagged or synthesized so that it contained an affinity
tag) was purified from the E. coli lysates by chromatography (as
illustrated in FIG. 2) and used as the antigen to make polyclonal
antibodies in goats. The OMP18 protein was also coupled to
NHS-Sepharose for use in affinity-based purifications of the
resulting antibodies. The ability of the purified antibodies to
bind specifically to the native form of the OMP18 protein when the
OMP18 is present in a lysate of Campylobacter jejuni bacteria was
tested and confirmed by immunoblotting (as illustrated in FIG.
3).
[0027] Specifically, FIG. 2 illustrates a preparation of
recombinant OMP18 that had been purified by chromatography, was
separated by SDS-polyacrylamide gel electrophoresis and stained
with Coomassie Blue. The right hand lane displays molecular weight
markers. The arrow points to OMP18 at the appropriate 18 kilodalton
size. The lack of other significant protein bands indicates that
the preparation is highly purified.
[0028] FIG. 3 illustrates a sample of Campylobacter jejuni bacteria
that was separated by SDS-polyacrylamide gel electrophoresis,
transferred to a membrane, and incubated with HRP-conjugated
anti-OMP18. The right hand lane displays molecular weight markers.
The arrow points to OMP18. Among the thousands of proteins present
on the immunoblots from the bacterial sample, only the native 18
kilodalton OMP18 protein was recognized by the purified anti-OMP18
antibodies.
[0029] An ELISA (enzyme-linked immunosorbent assay) was developed
using polyclonal anti-OMP18 antibodies as capturing antibodies and
horseradish peroxidase (HRP)-conjugated polyclonal anti-OMP18
antibodies as detection antibodies. The recombinant OMP18 antigen
was tested on the ELISA and produced a response with an excellent
fit (R.sup.2=0.99) to a linear trend line as illustrated in FIG. 4.
The ability of the assay to detect sub-nanogram amounts of OMP18
(<0.04 ng recombinant OMP18) confirms that the antibodies
produced to the OMP18 antigen are highly sensitive.
EXAMPLE 2
[0030] Twenty-eight human fecal samples that were positive and 198
that were negative by standard laboratory culture for Campylobacter
jejuni and Campylobacter coli were tested using this anti-OMP18
ELISA. Samples were diluted five-fold, added to the ELISA wells,
HRP-conjugated polyclonal Anti-OMP18 antibody added, and the plate
incubated for 20 minutes at 37 degrees C. Wells were washed,
standard HRP substrate added for 10 minutes, followed by a standard
solution to stop the color development reaction. The cutoff of this
ELISA was set as an absorbance of 0.080 by dual wavelength
(OD450-620 nm) spectrometry. Twenty-six of the 28 culture-positive
samples were positive on the anti-OMP ELISA, for a sensitivity of
93%. One hundred eighty-eight of the 198 culture-negative samples
were negative on the anti-OMP ELISA, for a specificity of 95%.
Table 1 below illustrates this result.
TABLE-US-00001 TABLE 1 Culture Positive Culture Negative Total = 28
Total = 198 OMP18 Total = 36 26 10 ELISA Positive OMP18 Total = 190
2 188 ELISA Negative Sensitivity = 93% Specificity = 95%
EXAMPLE 3
[0031] The OMP18 ELISA can also be used to detect Campylobacter
jejuni and Campylobacter coli from bacterial cultures. Using the
ELISA assay described in Example 2, individual cultures of 2
strains of Campylobacter jejuni, and 2 strains of Campylobacter
coli were tested. Bacterial cultures were scraped from agar growth
plates (CAMPY CVA plates, Hardy Diagnostics) into
phosphate-buffered saline (PBS). Protein concentrations of
appropriate dilutions of the bacterial suspensions were determined
using a commercial Coomassie Blue-based (Bradford) assay.
Suspensions were adjusted to 3 mg protein/ml and, from a starting
concentration of 300 micrograms protein per milliliter, one hundred
microliters of serial two-fold dilutions of each bacterial
suspension was added to wells of the ELISA plate. Incubation,
development and reading of the plate were performed as in Example
2. FIG. 5 illustrates the results of titration of these
Campylobacter cultures. Each of the four Campylobacter strains from
bacterial culture was detected strongly by the OMP18 ELISA assay.
Thus OMP18 can be used as a marker to detect multiple strains of
Campylobacter jejuni or Campylobacter coli.
EXAMPLE 4
[0032] Positive ELISA test results for OMP18 were also compared to
known PCR markers that are specific to Campylobacter. The 28 fecal
samples that were culture positive were further tested using
in-house PCR assays that detect 16S ribosomal RNA of C. jejuni and
C. coli, the hipO gene of C. jejuni and the CadF gene of C. coli.
Of these 28 culture-positive samples, 26 were also PCR-positive,
while 2 samples could not be confirmed as positive by PCR analysis
as illustrated below in Table 2. These 26 samples were also
positive for Campylobacter by the OMP18-based ELISA assay. The
results confirm that the anti-OMP ELISA detects true positives
accurately.
[0033] A subset of 107 of the 198 culture-negative samples were
also tested using the PCR assay as illustrated in Table 2. All 107
of the negative fecal samples were also found to be negative by
PCR. Among these 107 negative samples, 106 were indicated as
negative and one was positive by the anti-OMP ELISA assay. This
indicates that the anti-OMP ELISA can also distinguish negative
samples with high accuracy.
[0034] The high degree of correlation between positive PCR results
and positive ELISA results verified the validity of the ELISA assay
for the presence of OMP18.
TABLE-US-00002 TABLE 2 PCR Positive PCR Negative Total = 26 Total =
107 OMP18 Total = 24 23 1 ELISA Positive OMP18 Total = 109 3 106
ELISA Negative Sensitivity = 88% Specificity = 99%
EXAMPLE 5
[0035] In addition to the OMP18 ELISA, a rapid enzyme-linked
immunoassay (rapid EIA) using anti-OMP18 antibodies striped onto
membranes and horseradish peroxidase (HRP)-conjugated anti-OMP18
antibodies as detection antibodies was developed. The recombinant
OMP18 antigen was tested on the membrane-based rapid EIA and,
similar to the OMP18 ELISA, could detect sub-nanogram amounts of
OMP18 (<0.15 ng recombinant OMP18). The OMP18 EIA was used to
test 37 human fecal samples that were Campylobacter positive by
standard culture techniques, and 643 samples that were
culture-negative. Samples were diluted five-fold, mixed with a drop
of HRP-conjugated anti-OMP18 antibody, and added to the EIA
membrane device. Devices were incubated for 15 minutes at room
temperature, washed with 300 .mu.L of buffer, one drop of the
HRP-conjugated anti-OMP18 added, and incubation continued another
10 minutes. Visible detection of a blue-colored test line in the
reaction window indicated a positive reaction for the presence of
Campylobacter OMP18 in the sample. Of the 37 culture-positive
specimens, the OMP18 rapid EIA detected 36, for a sensitivity of
97%. Among the 643 culture-negative specimens, the OMP18 rapid EIA
detected 8 positives, and 635 negatives, for a specificity of 99%.
Thus, anti-OMP18 can be used accurately in multiple detection
formats.
TABLE-US-00003 TABLE 3 Culture Positive Culture Negative Total = 37
Total = 643 OMP18 Total = 44 36 8 rapid EIA Positive OMP18 Total =
636 1 635 rapid EIA Negative Sensitivity = 97% Specificity =
99%
EXAMPLE 6
[0036] The OMP18 ELISA can also be used to detect OMP18 in
Campylobacter species which are closely related to C. jejuni and in
which the amino acid sequence of the species' OMP18 is homologous
as illustrated in Table 4. Using the ELISA assay described in
Example 2, individual cultures of Campylobacter lari and
Campylobacter upsaliensis were tested. Bacterial cultures were
grown on AnaeroGro Campy Selective Agar plates for 48-72 hours at
37 degrees C. in a microaerophilic atmosphere generated by CampyGen
Sachets. Bacterial cultures were scraped from agar growth plates
into phosphate-buffered saline (PBS). Protein concentrations of
appropriate dilutions of the bacterial suspensions were determined
using a commercial Coomassie Blue-based (Bradford) assay.
Suspensions were adjusted to 3 mg protein/ml and, from a starting
concentration of 300 micrograms protein per milliliter, one hundred
microliters of serial two-fold dilutions of each bacterial
suspension was added to wells of the ELISA plate. Incubation,
development and reading of the plate were performed as in Example
2. FIG. 6 illustrates the results of titration of these
Campylobacter cultures along with titrations of cultures of C.
jejuni and C. coli. The two additional Campylobacter species from
bacterial culture were detected strongly by the OMP18 ELISA assay.
Thus OMP18 and antibodies to the protein can be used to detect
multiple species of Campylobacter.
TABLE-US-00004 TABLE 4 Accession No. Amino Acid Species (strain)
for OMP 18 Identity to Cj Campylobacter jejuni (81116) YP_1781132.1
Campylobacter coli (RM2228) ZP_00367850.1 94% Campylobacter
upsaliensis (RM3195) ZP_00371247.1 89% Campylobacter lari (RM2100)
YP_002574815.1 71%
[0037] Table 4 illustrates the results of alignment of the amino
acid sequences of the OMP18 proteins of the four indicated
Campylobacter species using the NCBI BLAST protein algorithm
2.2.29. The percent identity of the amino acids calculated by the
algorithm are given in the table above.
[0038] In summary, the present invention provides OMP18 as a
diagnostic marker for detecting Campylobacter in stool samples and
bacterial cultures. The examples and embodiments of the present
invention have been described in relation to particular embodiments
which are intended in all respects to be illustrative rather than
restrictive. Alternative embodiments will become apparent to those
skilled in the art to which the present invention pertains without
departing from its scope.
[0039] In its most basic form, the device of the invention
comprises (1) a receiving portion to receive a fecal sample from a
person and (2) a testing portion to detect the presence of OMP18 in
the fecal sample, where the testing portion comprises a specific
binding pair member that is specific for OMP18. Any additional
components that permit the practice of the method of the invention
may also be included in the device.
[0040] From the foregoing, it will be seen that this invention is
one well adapted to attain all the ends and objects herein above
set forth together with other advantages which are obvious and
which are inherent to the method. It will be understood that
certain features and sub-combinations are of utility and may be
employed without reference to other features and sub-combinations.
This is contemplated by and is within the scope of the claims.
Sequence CWU 1
1
11165PRTCampylobacter jejuni ATCC 33560 1Met Lys Lys Ile Leu Phe
Thr Ser Ile Ala Ala Leu Ala Val Val Ile 1 5 10 15 Ser Gly Cys Ser
Thr Lys Ser Thr Ser Val Ser Gly Asp Ser Ser Val 20 25 30 Asp Ser
Asn Arg Gly Ser Gly Gly Ser Asp Gly Trp Asp Ile Asp Ser 35 40 45
Lys Ile Ser Gln Leu Asn Asp Thr Leu Asn Lys Val Tyr Phe Asp Phe 50
55 60 Asp Lys Phe Asn Ile Arg Pro Asp Met Gln Asn Val Val Ser Thr
Asn 65 70 75 80 Ala Asn Ile Phe Asn Thr Glu Val Ser Gly Val Ser Ile
Thr Val Glu 85 90 95 Gly Asn Cys Asp Glu Trp Gly Thr Asp Glu Tyr
Asn Gln Ala Leu Gly 100 105 110 Leu Lys Arg Ala Lys Ala Val Lys Glu
Ala Leu Ile Ala Lys Gly Val 115 120 125 Asn Ala Asp Arg Ile Ala Val
Lys Ser Tyr Gly Glu Thr Asn Pro Val 130 135 140 Cys Thr Glu Lys Thr
Lys Ala Cys Asp Ala Gln Asn Arg Arg Ala Glu 145 150 155 160 Phe Lys
Leu Ser Arg 165
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