Immunoassay for H. pylori in fecal specimens

A process for the determination of H. pylori in a fecal specimen comprising (a) dispersing a fecal specimen suspected of carrying H. pylori in a sample diluent; (b) contacting the fecal specimen in the diluent with a first polyclonal antibody for H. pylori antigen to form a complex of the antibody and the antigen; (c) separating said specimen and said complex; (d) exposing the complex to a second polyclonal antibody for said antigen and a portion of the antibody reacting with said complex, one of said first and second antibody being bound to a solid carrier and the other being labeled with a detection agent; and (e) determining the amount of the labeled antibody and in turn determining the presence of H. pylori antigen in said fecal specimen.

This invention relates to a method for detecting Helicobacter pylori in 
fecal specimens. 
H. pylori is a bacterium that is found in the upper gastrointestinal tract 
of humans which has been implicated in gastroduodenal diseases such as 
peptic ulcers, gastritis and other maladies. The bacterium was originally 
classified as a Campylobacter and then reclassified as a Heliobacter based 
on more detailed information regarding its ultrastructure and fatty acid 
composition. 
A number of different techniques, both invasive and noninvasive, have been 
used to detect H. pylori. The invasive techniques involve gastric biopsies 
and cultures. The noninvasive techniques include a urea breath test, in 
which the patient is given C-13 or C-14 labelled urea with a beverage, and 
the detection of H. pylori antibody in sera using antigens in 
enzyme-linked immunosorbent assays (ELISA). Examples of the latter 
techniques are found in U.S. Pat. No. 5,262,156 to Aleonohammad and 
European Patent Application 0 329 570 to Blaser. 
Several major antigens have been identified and used in immunoassays in the 
detection of H. pylori antibodies. However, these assays have not 
exhibited the specificity and sensitivity that are desired in 
serodiagnosis. Newell, D. G., et al. Serodian. Immunother. Infec. Dis.,, 
3:1-6 (1989). One problem with of these immunoassays is cross-reactivity. 
Studies of the dominant antigens in H. pylori, in particular, the putative 
flagellar protein, which has a molecular weight of 60 Da, have shown that 
some of these antigen are not specific to H. pylori and also found in 
other bacteria such as C. jeuni and C. coli. A second problem that has 
been encountered in designing immunoassays for H. pylori is strain 
variation. Substantial differences in the antigens has been observed in 
different strains of H. pylori. These problems preclude designing an assay 
around the use of a single antigen. They also rule out the use of 
monoclonal antibodies. One approach that has been taken to improving the 
specificity and selectivity of antibody immunoassays for H. pylori has 
been to use a mixture of antigens from different H. pylori strains which 
mixture is enriched with certain antigen fragments. One ELISA which 
detects H. pylori antibodies in a blood sera is commercially available 
from Meridian Diagnostics. This assay uses a bacterial whole cell lysate 
as the antigen. 
There are certain disadvantages to using an ELISA which employs antigens to 
detect the presence of H. pylori antibodies. In particular, the antibody 
titer in human sera remains high for a prolonged time (in some cases as 
much as six months) after the infection has been treated. Consequently, a 
positive test using this ELISA does not necessarily mean that the patient 
is currently infected and requires treatment for H. pylori infection. When 
confronted with a positive ELISA, treating physicians often order a 
gastric biopsy to confirm the presence of the bacteria before initiating 
antibiotic therapy. Therefor, the antigen-based ELISA does not eliminate 
the need for the invasive procedure. By contrast, if an immunoassay could 
be designed for detecting H. pylori antigen instead of the antibody, the 
need to obtain gastric biopsies to confirm infection could be reduced 
significantly because the antigen generally can not be detected in a 
patient within days of its treatment. Thus, there is a need for an ELISA 
which detects H.pylori antigen and, more particularly, there is a need for 
an ELISA for detecting H. pylori directly from fecal specimens. 
While ELISA's for detecting microorganisms such as C. difficile and 
adenovirus in fecal specimens are known, in studies of patients with 
gastric biopsies which are positive for H. pylori, the bacteria ordinarily 
can not be cultured and isolated from the fecal specimens. This and the 
problems of cross reactivity and strain variation raised serious doubts 
that an ELISA could be designed that would be specific for H. pylori and 
sensitive enough to reliably detect H. pylori antigen directly from a 
fecal specimen. 
SUMMARY OF THE INVENTION 
The present invention provides a method for detecting H. pylori in fecal 
specimens which comprises: 
(a) dispersing a fecal specimen suspected of carrying H. pylori in a sample 
diluent; 
(b) contacting the fecal specimen in the diluent with a first polyclonal 
antibody for H. pylori antigen to form a complex of the antibody and the 
antigen; 
(c) separating said specimen from said complex; 
(d) exposing the complex to a second polyclonal antibody for said antigen 
and a portion of the antibody reacting with said complex, one of said 
first and second antibody being bound to a solid carrier and the other 
being labelled with a detection agent; and 
(e) determining the amount of the labelled antibody and in turn determining 
the presence of H. pylori antigen in said fecal specimen. 
In the preferred embodiment of the invention, the first antibody is bound 
to a carrier and the second is labelled with an enzyme. Triple sandwich 
assays are also provided. 
The immunoassay will be supplied in the form of a kit including a plate of 
antibody-coated wells, sample diluent, the labeled antibody, e.g., an 
enzyme-antibody conjugate, wash buffer and, in the case of an ELISA, a 
substrate solution. 
DETAILED DESCRIPTION 
The immunoassay of the present invention employs polyclonal antibodies for 
H. pylori. These antibodies can be obtained from the sera of a sensitized 
animal. Sensitization can be accomplished by injecting the antigen into an 
antibody producing species, typically a mammal and preferably a rabbit, 
goat or cow. Usually an initial injection is given followed by subsequent 
booster injections to maximize the response. Optimally, the injection 
regime is in multiple doses given to White New Zealand rabbits. The amount 
of antigen injected must be adequate to elicit a sufficient amount of 
antibody to be detectable. Antibody production is verified using a trial 
bleed and Indirect Fluorescent Assay. 
H. pylori cells from ATCC strain 43504 have been found to be particularly 
useful in producing the polyclonal antibody. As previously mentioned, 
substantial strain variation has been observed in H. pylori. Differences 
in the organism have been observed in different geographic regions as well 
as dietary groups. However, antibodies obtained through sensitization 
using cells from strain 43504 have been found to be useful in detecting 
the organism across geographic regions and dietary groups. If necessary, 
for example, if it is found that the ELISA is not effective in detecting 
the organism in certain populations, cells from more than one strain of H. 
pylori could be used to produce the antibody. 
The same labels used in known immunometric assays can be used to label the 
polyclonal antibody used in the present invention. Among these may be 
mentioned fluorogenic labels for detection by fluorimetry as described in 
U.S. Pat. No. 3,940,475, enzymatic markers as described in U.S. Pat. No. 
3,654,090, and radioisotopes such as Iodine-125. One of the most common 
enzymatic markers is horseradish peroxidase (HRP) and alkaline phosphatase 
enzyme. Example 3 below illustrates labeling polyclonal antibodies with 
HRP. 
The unlabeled polyclonal antibody used in the process of the present 
invention to extract the antigenic substance from the fecal specimen being 
tested can be immobilized on any of the supports commonly used in 
immunometric assays. Among those that may be used are filter paper, 
plastic beads, polyethylene, polystyrene, polypropylene or other suitable 
test tube. The techniques for bonding antibodies to such materials are 
well known to those skilled in the art. 
To prepare the fecal specimen for use in the assay, the specimen is 
dispersed in a protein-based sample diluent. The diluent be formulated and 
buffered to minimize cross-reactivity. As examples of sample diluents, 
mention can be made of fetal bovine serum, normal goat serum, guinea pig 
serum, horse serum, casein, albumin, gelatin, and bovine serum albumin 
(BSA). A dilution of one part fecal specimen and four parts diluent has 
been found to be useful. In addition to using the protein based additives, 
cross-reactivity can be reduced by the addition of detergents and 
increasing or decreasing pH or ionic strength of the diluent buffer. For 
example, many sample diluents contain Triton X-100 and/or Tween 20 at 
concentrations ranging between 0.05% and 2%. NaCl can be added in the 
ranges between 0-2.9% to alter the ionic strength of the buffer system. 
These changes lead to greater specificity by reducing the likelihood of 
weak or non-specific interactions from forming. 
Cross-reactivity can also be addressed in the formulation of the antibody 
solutions and the washes that are used in the assay. The antibody can be 
provided in a buffered solution in conjunction with one of the protein 
sera mentioned previously. The washes used in the assay can be formulated 
and buffered by the addition of salts and surfactants to control 
cross-reactivity. A preferred wash for reducing cross-reactivity is a 
phosphate buffered saline solution. 
The preparation of the antigen, production of the polyclonal antibodies and 
an ELISA are illustrated in more detail by the following non-limiting 
examples.

EXAMPLE 1 
Preparation of the Helicobacter pylori (H. pylori) antigen 
H. pylori (ATCC strain 43504) was streaked for isolation on Tryptic Soy 
Agar (TSA) supplemented with 5% defibrinated sheep blood. The plate was 
incubated at 37.degree. C. in a microaerophilic environment for 6-7 days. 
The resultant bacterial growth was evaluated by use of colony morphology, 
urease, catalase and oxidase reactions, and gram stain. Acceptable growth 
was subcultured to four TSA with sheep blood agar plates and grown at 
37.degree. C. in a microaerophilic environment for 3-4 days. 
Each plate was flooded with 5 ml of 0.85% NaCl and the bacterial growth was 
harvested by a plate spreader. The bacteria were centrifuged at 10,000 xg 
for 15 minutes at 2.degree.-8.degree. C. Each pellet was resuspended in 3 
ml of 0.85% NaCl and combined to one centrifuge container. The bacterial 
suspension was centrifuged at 10,000 xg for 15 minutes at 
2.degree.-8.degree. C. The pellet was resuspended and centrifuged as 
before. The final pellet was resuspended to 3% of the total original 
volume in 20 mM phosphate buffer. The bacterial cells were transferred to 
an iced container and sonicated 5 times for 3 minutes at the maximum 
setting that does not cause foaming, resting for 30 seconds in between 
cycles. The sonicated bacterial cells were centrifuged at 57,000 xg for 15 
minutes at 2.degree.-8.degree. C. The bacterial supernatant was collected 
and the pellet discarded. 
EXAMPLE 2 
Production of Rabbit Polyclonal 
The bacterial supernatant obtained in Example 1 was diluted in equal parts 
with Freunds complete adjuvant (total immunogen is 1.0 ml) to provide 
1.times.10.sup.8 cells per ml. This solution was mixed thoroughly and 
0.2-0.5 ml of the solution was injected intramuscularly into the right 
hind leg and 0.1-0.25 ml of the solution was injected subcutaneously into 
each of eight to ten sites on the back. Subsequent injections were one 
month apart using Freunds incomplete adjuvant and the injection sites were 
limited to subcutaneous back. 
A trial bleed was taken after three months. The bleed was taken from the 
central ear vein one week after the third injection. This bleed was 
incubated overnight at 2.degree.-8.degree. C. The next day the blood was 
centrifuged at 5,000 xg for 15 minutes at room temperature. The 
supernatant was collected and the pellet discarded. The supernatant was 
tested by an Indirect Fluorescent Assay (IFA). The IFA was performed by 
placing 10 ul of H. pylori suspension on glass slides and heat fixed. The 
slides were blocked with 3% bovine serum albumin (BSA) for 5 minutes then 
washed with a wash of phosphate buffered saline (PBS) and 0.5% Tween 20 
(PBS/Tween wash). 50 ul of the trial bleeds and normal rabbit serum, as a 
control, diluted 1:10 in phosphate buffered saline with sodium azide 
(PBSA) were added and incubated in a humid environment for 30 minutes. 
After washing with PBS/Tween wash, Goat anti-Rabbit conjugated to FITC 
(fluoroescein isothiocyanate) was diluted 1:10 in PBSA and 50 ul was added 
to each well. The slides were incubated for 30 minutes in a dark humid 
environment. The slides were washed again. Fluorescence assay mounting 
media and a cover slip were added and viewed with a fluorescence 
microscope. Rabbits whose sera demonstrated a 4+ fluorescence intensity 
reading were then volume bled. 
The volume bleed was obtained similarly to the trial bleed except 50 ml was 
removed from each rabbit. The blood was incubated and centrifuged as the 
trial bleed was. 
The total volume of sera was determined and an equal volume of phosphate 
buffer saline (PBS) was added. A 40% ammonium sulfate precipitation was 
performed to remove unnecessary protein and incubated at 
2.degree.-8.degree. C. for 24 hours. The mixture was transferred to a 
centrifuge tube and centrifuged at 10,000 xg for 30 minutes at room 
temperature. Resuspend the pellet in PBS to approximately one third of the 
original volume. The suspension was dialyzed against 200 times the total 
suspension volume of 0.0175M potassium phosphate, pH 6.5 at 
2.degree.-8.degree. C. After the dialysis, the suspension was centrifuged 
at 10,000 xg for 20 minutes at room temperature. The supernatant was 
collected and the pellet was discarded. 
A DEAE (diethylaminoethyl cellulose) column was equilibrated with 0.0175M 
potassium phosphate, pH 6.5 at room temperature. The supernatant was 
placed over the column and the effluent fractions collected. A protein 
concentration (OD.sub.280) was determined and all fractions greater than 
0.200 were pooled. The pooled antibody was tested in the ELISA. 
EXAMPLE 3 
Horseradish Peroxidase Conjugation 
The conjugation used 10 mg of DEAE purified rabbit anti-H. pylori antibody. 
The antibody was brought to a final volume of 2.5 ml by concentration or 
by the addition of 10 mM sodium bicarbonate pH 9.6. A PD-10 column 
(Pharmacia) was equilibrated with 10 mM sodium bicarbonate pH 9.6. The 
antibody was added to the column and nine fractions of 1.0 ml were taken. 
A protein concentration (OD.sub.280 E.O.=1.4) of each fraction was taken 
and those reading above 0.200 were pooled. 
A separate PD-10 column was equilibrated with 1 mM sodium acetate 
trihydrate pH 4.3. The minimum amount of Horseradish Peroxidase (HRP) used 
was 1.172 mg HRP for each 1 mg of antibody. 11/2 times the calculated 
minimum HRP was weighed out and added to 1.0 ml of deionized water. A 
protein concentration (OD.sub.403 E.O.=2.275) was performed and HRP 
diluted to 10 mg/ml with deionized water. 0.1M sodium m-periodate was 
added at a concentration of 0.2 ml for every 4 mg HRP. This reaction was 
allowed to proceed for 20 minutes at room temperature with gentle rocking. 
The reaction was stopped by the addition of 50 ul of 2M ethylene glycol 
for every ml of HRP at 4 mg. The HRP was eluted through the PD-10 with 1 
mM sodium acetate trihydrate pH 4.3. 
The conjugation ratio was 1 mg antibody to 1.172 mg HRP. The antibody was 
adjusted with 10 mM sodium bicarbonate pH 9.6 and the HRP with 1 mM sodium 
acetate trihydrate pH 4.3. The two were combined in a dedicated flask and 
pH adjusted with 0.2M sodium bicarbonate, pH 9.6 to 9.6. Protected from 
light, the mixture was incubated for two hours on a rotator at 85-95 rpm 
at room temperature. After two hours, 0.1 ml of 4 mg/ml of sodium 
borohydride was added for every 8 mg of antibody. The new mixture was 
incubated at 4.degree. C. for two hours on a rotator. The conjugate was 
passed through a PD-10, equilibrated with PBS, and fractions containing 
the conjugate were collected. The fractions were pooled and concentrated 
to approximately 1.0 ml. The concentrate was placed over a Sephracryl 
S-200 column equilibrated with PBS at a flow rate of 10 ml/hr. 2.0 ml 
fractions were collected and a protein concentration of both the antibody 
and the HRP were performed. The fractions with a simultaneous peak in both 
the OD.sub.280 and OD.sub.403 were pooled and concentrated to 
approximately 1.0 mg/ml. 
Example 4 below illustrates a so called "forward" assay in which the 
antibody bound to the support is first contacted with the specimen being 
tested to extract the antigen from the sample by formation of an 
antibody/antigen complex and contacting the complex with a known quantity 
of labelled antibodies. However, those skilled in the art will appreciate 
that the immunometric assay can also be conducted as a so called 
"simultaneous" or "reverse" assay. A simultaneous assay involves a single 
incubation step as the antibody bound to the solid support and the 
labelled antibody are both added to the sample being tested at the same 
time. After the incubation is complete, the solid support is washed to 
remove the residual sample and uncomplexed labeled antibody. The presence 
of the labeled antibody associated with the solid support is then 
determined. A reverse assay involves the step wise addition first of a 
solution of labeled antibody to the fecal specimen followed by the 
addition of the unlabeled antibody bound to the support. After a second 
incubation, the support is washed in a conventional fashion to free it of 
the residual specimen and the unreacted labelled antibody. 
EXAMPLE 4 
ELISA Test 
The antibody was diluted in PBS serially between 20 ug/ml and 2.5 ug/ml. A 
0.100 ml aliquot of each dilution was added to an Immunlon-II strip 
(Dynatech), covered and incubated overnight at room temperature. The plate 
was washed once with PBS/Tween wash. It was blocked with 1%BSA/PBS for 1 
hour at room temperature. Again washed once with PBS/Tween wash. Several 
positive and negative samples were diluted 1:5 in 0.1%BSA/PBS. Each sample 
(0.100 ml) was added to a well of the strips, covered and incubated 1 hour 
at room temperature. The plate was then washed 5 times with Merifluor C/G 
wash. A previously accepted rabbit anti-H. pylori conjugated to 
horseradish peroxidase was diluted to 10 ug/ml and 0.100 ml added to each 
well. The plate was covered and incubated at room temperature for 1 hour. 
Again washed 5 times with PBS/Tween wash then developed for 10 minutes at 
room temperature with 0.100 ml of trimethylbensidine (TMB) solution. 
Stopped with 0.050 ml 2NH.sub.2 SO.sub.4 and read after two minutes. The 
dilution yielding the maximum signal and lowest background was chosen as 
the optimal dilution. 
Quantitative determinations can be made by comparing the measure of labeled 
antibody with that obtained for calibrating samples containing known 
quantities of antigen. Table 1 below shows the optical density obtained 
when four samples, each containing a predetermined number of organisms, is 
run through the assay. 
TABLE 1 
______________________________________ 
No. of organisms 
per ml OD.sub.450/630 
______________________________________ 
3 .times. 10.sup.7 
2.547 
1.9 .times. 10.sup.7 
0.662 
4.6 .times. 10.sup.6 
0.182 
1.1 .times. 10.sup.6 
0.038 
______________________________________ 
The results of running six clinical specimens through the assay are shown 
in Table 2. 
______________________________________ 
Sample OD.sub.450/630 Result 
______________________________________ 
1 0.301 Positive 
2 0.713 Positive 
3 0.284 Positive 
4 0.005 Negative 
5 0.033 Negative 
6 0.008 Negative 
______________________________________ 
So-called triple sandwich assays can also be used for detecting H. pylori 
in fecal specimens in accordance with the invention. Triple assays are 
know in the art and the basic methodology can be applied to the detection 
of H. pylori in fecal specimens. A triple assay is typically conducted by 
dispersing a fecal specimen suspected of carrying H. pylori in a sample 
diluent which minimizes cross-reactivity and adding the diluted sample to 
an immobilized antibody for H. pylori that has been obtained from a first 
species of an antibody producing animal. The sample is incubated to form 
the antibody-antigen complex. After washing excess specimen from the 
immobilized support, an H. pylori antibody known as a primary antibody and 
obtained from a second species of an antibody producing animal is added to 
the antibody-antigen complex and incubated to form an 
antibody-antigen-antibody complex. After forming this complex and removing 
the unreacted antibody, the complex is reacted with an antibody known as a 
secondary antibody which is an antibody to the second antibody producing 
species such as anti-(rabbit, cow or goat) immunoglobulin. The secondary 
antibody is labelled in a conventional manner, typically with an enzyme, 
and incubated with the antibody-antigen-antibody complex to form a triple 
antibody complex or sandwich. After removing the unreacted secondary 
antibody, the antigen is assayed in a conventional manner. In using an 
enzyme label, a substrate is added to the complex of the antigen and the 
three antibodies and the reaction of the substrate with the linked enzyme 
is monitored to determine the amount of the antigen present in the 
specimen. In the triple sandwich assay, as in the basic sandwich assay, 
the washes and the antibody solutions are formulated or buffered to 
control cross-reactivity as needed. 
Having described the invention in detail and by reference to the preferred 
embodiments it will be apparent to those skilled in the art that 
modifications and variations are possible without departing from the scope 
of the invention as defined in the following appended claims.