Helicobacter pylori stool Antigen ELISA KIT
using a special formulated buffer for extraction
Helicobacter pylori
Stool Antigen ELISA
Kit
Enzyme Immunoassay for Detection of
H. pylori antigen in Human stool
(For in Vitro Diagnostic Use Only)
Catalogue No.PT-H.pylori.S.Ag
PISHTAZTEB DIAGNOSTIC
Introduction
Helicobacter pylori (H. pylori) is a gram-negative
spiral bacterium found in the gastric mucosa layer.
Many studies have shown the relationship between
presence of Helicobacter pylori and various
gastrointestinal diseases including chronic gastritis,
peptic ulcer, duodenal ulcer and gastric
adenocarcinoma. H. pylori is present in 95-98% of
patients with duodenal ulcer and 60-90% of gastric
ulcer patients. The prevalence of the infection
identified by the bacteriologic, histologic and
serologic methods in individuals with clinical
symptoms is about 90%. However, many patients
(more than 50% over the age of 50) are only
colonized by the bacteria and do not show clinical
symptoms for the rest of their lives. It should be
noted that evidences such as the diagnosis of
specific antibodies against H. pylori antigens,
positive urea test, and positive culture or biopsy
without clinical symptoms can only indicate the
bacterial colonization and the infection is only
confirmed when clinical sign and symptoms are also
present. There are invasive and non-invasive
techniques for the diagnosis of Helicobacter pylori.
The invasive procedure is performed using
endoscopy and biopsy, histopathology and rapid
urease testing which are not only expensive, but
also unpleasant for the patients. Non-invasive
techniques Includes urea breath test (UBT),
serologic tests, and detection of Helicobacter pylori
antigen in the stool specimen. Presence of H.pylori
antigen in the stool is a definite indicator of bacterial
colonization in the gastrointestinal tract, and ELISA
is the technique of choice for this antigen.
Test Principle
To perform this test, the amount of H. pylori
antigen present in the stool sample is measured
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process in specific ELISA method. This H. pylori
ELISA Test Kit is based on the principle of
sandwich enzyme-linked immune sorbent assay.
In this method, the extracted samples and controls
are allowed to react with the solid phase
monoclonal antibodies against anti-H.pylori stool
antigen coated on the microtiter wells.
Simultaneously, second anti-H.pylori conjugated
with HRP will be added, resulting in the sandwich
form Immune complex which proportional to the
stool antigen concentration of Helicobacter pylori
in each well. After incubation and following wash
step, chromogen solution containing Hydrogen
peroxide (H2O2) and chromogenic substrate is
added and incubated for 15 minutes, resulting in
the development of a blue color. The color
development is stopped with addition of stop
solution, and the color is changed to yellow and
measured by ELISA Reader at 450 nm.
Materials provided with the kit
1. Antibody coated wells: microtiter wells
coated with monoclonal anti-H. Pylori
antigen.(Anti-H. pylori Antigen Coated
Plate)
2. Extraction Buffer, ready to use.
3. Enzyme conjugate: Anti-H.pylori Antigen
labeled with HRP in buffer, ready to use.
4. Positive control solution: Contains certain
amount of H.pylori whole cell lysate diluted
in buffer containing 0.05% Kathon as
preservative, ready to use.
5. Negative control solution: diluted buffer
without H.pylori whole cell lysate and 0.05%
Kathon as preservative, ready to use.
6. Chromogen substrate reagent contains tetra
methyl benzidine (TMB) and hydrogen
peroxide, ready to use solution.
7. Wash solution (concentrated 20x): contains
phosphate buffer solution with 0.05%
Tween 20. Note: for preparing of the wash
solution dilute the concentrated wash
solution 1:20 with distilled water.
8. Stop solution: contains hydrochloric acid
(1M) Ready to use
9. Cardboard sealer.
MA_H. pylori.S.Ag
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 Helicobacter pylori stool Antigen ELISA KIT
Materials required but not
Provided with the kit
 Precise micro pipettes:25,100 and 1000 µl.
 Microtiter plate ELISA reader with 450 nm
filter and 630 nm filter as a reference.
 Distilled water
 Timer
 37°C incubator
 Digital scale
 Centrifuge (minimum RCF=3000xg)
 Fecal sample extraction tube
 Disposable applicator stick
Important points for Users
1. Do not mix kit reagents from different
batch/lot numbers.
2. All kit components must be used only in
their original kit.
3. Patient stool samples, controls and wells
used for test should be regarded as
infectious waste. All reagents and
solutions should be disposed in
accordance with national regulations on
disposal of infectious waste.
Storage Conditions
o
1. Kit should be stored at 2-8 C upon receipt
and when it is not in use.
2. Keep Un-used wells in their sealed bag
with desiccants.
3. Do not use expired date reagents.
4. Once the wash solution 20X is diluted with
o
distilled water, it is stable 1 week at 2-8 C.
Specimen Collection and Preparation
1. The kit is just for use with human stool
specimen. After collection, the stool
specimen should be kept in the refrigerator
and in the first possible time, the extraction
process should be performed. (Maximum 2
days)
2. Avoid keeping the specimens exposed to
temperatures above 30°C.
3. If extraction is not possible for less than 2
days, it is necessary that the specimen is
kept at -20°C freezer and again reached to
the room temperature prior to extraction.
4. The extracted sample can be stored for
maximum up to 2 days in the refrigerator.
2
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If extra time is required, it can be stored in
a freezer at -20°C and again reached to
the room temperature prior to use.
5. Before extraction, it is necessary to
homogenize the specimen using an
applicator stick.
Sample extraction process by weighing
method
1. Reset the digital scale to zero by placing
an empty screw cap tube together with a
disposable fecal sampling applicator stick
inside it on the weighing platform.(Tare
weight)
2. Take between 50 and 100 mg of
homogenized stool samples using the
sampling applicator stick and place it in the
provided tube. Avoid picking hard particles
and undigested food fragments.
3. Place the used applicator stick into the
tube and record the sample weight.
4. Break the unwanted top part of the
applicator stick to close tube cap.
5. Add the extraction buffer to a stool sample
at the ratio of approximately 1 to 10. For
example, if the stool sample weight in the
tube is 100 mg, it is necessary to add 10
times (1000 = 10 x 100)1000µl extraction
buffer to the tube.
6. Close the tube cap and vortex the contents
strongly for 1 to 2 minutes until the
specimen is completely dissolved in the
extraction buffer.
7. Centrifuge the tubes for 3 minutes at
3000x g.
8. Transfer the supernatant to a new tube
and discard the sediment. The extracted
sample can be stored for a maximum of 2
days at 2 to 8ºC and maintained at -20ºC
or below for a longer time.
Sample extraction process by use of
fecal sample extraction tubes
1. When the samples and the extraction
buffer in the kit reach at room temperature
add 1 ml of extraction buffer into each
tube.
2. Use the grooved part of the sampling stick
attached to the screw cap to dip into stool
at five different spots(up to a depth of 5
mm) and collect about 100 mg of the
homogenous stool approximately the size
of a large lentil seed (Fig. 1).Insert the
sampling stick back into the tube and close
the screw cap tightly. Shake or vortex the
MA_H. pylori.S.Ag
Edition-No. 1
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 Helicobacter pylori stool Antigen ELISA KIT
tube to suspend the specimen in the buffer
and get completely dissolved.
Fig. 1: sample collection by collecting tube
Note: Avoid picking up hard particles and
undigested food fragments
3. The approximate ratio of sample weight to
extraction buffer is about 1 to 10. For
example, 100 mg of stool should be mixed
with about 1000 µl (1 ml) of extraction
buffer. If the fecal sample is liquid, use an
adjustable sampler at about 100 µl volume
to pick up the sample and mix it with 10
times of extraction buffer.
4. Break the top part of the tube cap (A
breakable small part at upper end of the
cap) in order to use the tube as a dropper;
and then add 3 drops of the contents into
each sample well.
General information
1. All reagents should be allowed to reach
o
room temperature (22-28 C) before use.
2. All test steps should be done sequentially
without delay.
3. Use disposable pipette tips.
4. After adding of stop solution, read
absorbance at 450 nm within 30 minutes.
5. In order to obtain best result, washing
step should be done completely and tap
all residual droplets onto absorbent
paper.
6. One of the major factors in obtaining best
result is incubation time. It is
recommended to prepare all reagents
before starting the test to reduce delay
time and achieve accurate results.
7. The run time of adding samples, and
controls into the wells should not exceed
more than 5 minutes.
Assay Procedure
1. Secure the desired number of coated wells
in the holder and keep the remaining with
desiccants in tightly closed special bag.
2. Dispense 100 µl of controls and extracted
sample or (3 droplets of extracted
samples) in appropriate wells (duplicate
recommended, run each sample in two
wells and use the mean optical
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absorbance of both wells for final result).
3. Add 25 µl of Anti-H.pylori antigen-
conjugated with enzyme (Conjugate
solution) into the wells and Mix the
contents by gently shaking the wells for
30-60 seconds.
4. Cover the plate with cardboard sealer
firmly and incubate the wells for 60
minutes at 37ºC temperature.
5. Discard the microplate well contents into a
waste container. Rinse and dispense the
wash solution in the microtiter wells 5
times (each time with 300 µl of working
wash solution). Tap the wells sharply onto
absorbent paper or paper towels to remove
all residual water droplets.
6. Dispense 100 µl of chromogen-substrate
solution into the microplate wells.
7. Incubate the microplate wells at room
temperature in darkness for 15 minutes, to
develop blue color.
8. Stop the reaction by adding 100 µl of stop
solution to the microplate wells.
9. Measure absorbance at 450 nm by ELISA
reader. Use 630 nm filter as a reference
filter if it’s available.
Validity of the Assay
The assay is to be considered valid if:
1. The OD value for the negative control is
lower than 0.1. Higher values indicate an
incorrect washing procedure. In such a
case, check the efficiency of the washing
device.
2. The mean OD value of positive control is
higher than the 1.0. Lower values indicate
kit reagents decay. In such a case, check
expiry date of the kit before repeating the
assay.
Calculation of results
Use any ELISA readers with 450 nm
reading OD capability
.
1. Read absorbance value of controls and
samples at 450 nm (Use 630 nm filter as
reference filter if it’s available).
2. Calculate Cut off value using following
formula:
Cut off value =Mean OD of negative
control solution + 0.25
3. To determine positive and negative results
calculate cut off index by dividing sample
OD to cut off value:
MA_H. pylori.S.Ag
Edition-No. 1
160220
 Helicobacter pylori stool Antigen ELISA KIT

Cut off index (COI) = OD of sample/Cut-

off value.
3. Precision: To assess test precision, both intra-
Test Interpretation assay (within the same working run) and inter-
assay (between several working runs) were
Based on above formula, those results tested by two positive, one negative and two
greater than 1.1 are considered as weakly positive samples.
positive and those lower than 0.9 are
considered as negative. Test Results are shown in table 1 and 2:
Samples with COI between 0.9-1.1 are
considered as suspicious and should be Table no. 1 (Intra-assay)
repeated with fresh stool sample after a
while. In order to report the results
quantitatively, refer to the conversion table Sample No. of tests Mean CV
provided with the brochure inside the kit. SD
No. performed OD (%)
According to this table, the results of the
Positive
samples in Sample/Cut-off can be 20 1.35 0.064 4.7
sample 1
converted to ng/ml unit.
- A single negative result for untreated Negative
20 0.032 0.0022 6.87
patient does not rule out H. pylori infection sample
and may be due to undetectable Weakly
Helicobacter pylori antigen. Positive 20 0.297 0.025 8.42
- Positive results should be repeated again sample 1
to be confirmed. Weakly
- Positive samples that become negative in Positive 20 0.372 0.028 7.52
second repeated test must be reported as sample2
negative result. False positive results in Positive
20 2.43 0.16 6.58
first run of the test can be due to a random sample 2
error during wash step or sampling.
- As with all diagnostic tests, a definitive Table no. 2 (Inter-assay)
clinical diagnosis should not be based on
the results of a single test, but should only
be made by the physician after all clinical Sample No. of tests Mean CV
and laboratory findings have been SD
No. performed OD (%)
evaluated.
Positive
- Taking some drugs such as antibiotics and 10 1.39 0.073 5.25
sample 1
proton pump inhibitors (antacids) can
cause false-negative results, so it is Negative
10 0.038 0.003 7.89
recommended that at least one to four sample
weeks before testing, depending on the Weakly
drug type, avoid taking these drugs (four Positive 10 0.311 0.027 8.68
weeks for antibiotics and one week for sample 1
antacids) Weakly
Positive 10 0.398 0.031 7.79
Performance Characteristics sample2
Positive
10 2.48 0.17 6.85
1. Sensitivity:105 positive samples which were sample 2
*
confirmed by reference methods were tested Each test has been run in duplicate
by our developed ELISA kit and 104 samples
were detected as positive. Therefore, based on 4. Cross reactivity
obtained results, our test sensitivity was
99.04% which is comparable to other available To investigate the cross-reactivity in negative stool
8
commercial kits. samples, we added 10 CFU of the different
bacteria to the extraction buffer solution and
2. Specificity: 285 negative samples were tested calculated the S/C for each test, all of which were
simultaneously by our ELISA kit and reference observed negative and showed no cross reactivity
methods. Results showed that our developed
(table No. 3).
ELISA kit can correctly detect 283 of negative
sample. These findings represent 99.29%
specificity.
4
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 Helicobacter pylori stool Antigen ELISA KIT

Table no. 3 (Cross reactivity) References:

Name Strain S/C Result 1. Andy Darma, BagusSamsu, Tri Nugroho,

VinnyYoanna, Indah Sulistyani, Alpha
ATCC FardahAthiyyah, Reza Gunadi, RanuhSubijanto,
Escherichia coli 0.68 Negative
25922 MartoSudarmo.Comparison of Helicobacter pylori
Staphylococcus ATCC stool antigen, salivary IgG, serum IgG, and serum
0.79 Negative IgM as diagnostic markers of H. pylori infectionin
aureus 25923
childrenDOI:https://doi.org/10.18502/ijm.v11i3.1316
Pseudomonas ATCC 2019-08-06
0.84 Negative
aeruginosa 27853 2. Mei‐Jyh, Chen Yu‐Jen, Fang Ming‐Shiang, Wu
Enterococcus ATCC Chieh‐Chang, Chen Yen‐Nien, Chen Chien‐Chun,
0.81 Negative
faecalis 29212 Yu Chia‐Chi, Kuo Min‐Chin, Chiu Wen‐Hao, Hu
ATCC Min‐Horn, Tsai Cheng‐Lin, Hsieh Hsin‐Hung, Chen
Proteus vulgaris 0.76 Negative Ming‐Jong, Bair Jyh‐Ming Liou. Application of
6380
Helicobacter pylori stool antigen test to survey the
updated prevalence of Helicobacter pylori infection
5. Interference in Taiwan for the Taiwan Gastrointestinal Disease
and Helicobacter Consortium First published: 13
To evaluate the effect of interfering substances on August 2019 https://doi.org/10.1111/jgh.14828
sample results, potential interfering agents 3. Moon HW, Lee SY, Hur M, Yun YM.
Characteristics of Helicobacter pylori-seropositive
(hemoglobin, Human Albumin and hCG) were
subjects according to the stool antigen test findings: a
added to the sample matrix at following prospective study. Korean J Intern Med.
concentration. The H.pylori Stool Antigen S/C was 2018;33(5):893–901. doi:10.3904/kjim.2016.353
compared in both forms before and after the adding 4. Kakiuchi T, Okuda M, Hashiguchi K, Imamura I,
interfering substances (table No. 4). Nakayama A, Matsuo M. Evaluation of a Novel
Stool Antigen Rapid Test Kit for Detection of
.
Helicobacter pylori Infection. J ClinMicrobiol. 2019
Table no. 4 (Interference) Mar;57(3) . doi:10.1128/JCM.01825-18. PMID:
30567746; PMCID: PMC6425190.
Result difference
After adding (S/C)
Concentration

Before adding
substances
Interfering

(S/C)

(%)

1.5 1.47 -2.0

Hemoglobin 1 mg/ml 0.6 0.62 3.34
8.2 8.3 1.21
1.5 1.54 2.67
Human
1 mg/ml 0.6 0.58 -3.34
Albumin
8.2 8.0 -2.44
1.5 1.55 3.34
hCG 1000 IU/ml 0.6 0.62 3.34
8.2 7.9 -3.66

6. Hook Effect

No hook effect was observed with this kit at

Helicobacter pylori Antigen concentrations up to
1000 µg/ml.

5
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JTC Diagnosemittel UG Schulweg 8 Edition-No. 1
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 Helicobacter pylori stool Antigen ELISA KIT

Helicobacter pylori stool Antigen

Kit Contents table
TEST PROCEDURE
Kit Content 48 Test Format 96 Test Format
Step 1 Plate 1 × 48 Wells 1 × 96 Wells

Extracted Extraction Buffer 1× 50 ml 2×50 ml

Control
Sample
Conjugate 1×2 ml 1×3.5 ml
Reagents
Negative and Positive
2×1.0 ml 2×2.0 ml
Control Solutions
Stop Solution 1×6 ml 1×12 ml
Control 100 µl None
Extracted Wash Solution 1× 25 ml 1× 50 ml
None 100 µl
Sample
Anti H.pylori Chromogen - Substrate 1×6 ml 1×12 ml
Antigen
25 µl 25 µl Cardboard Sealer 1 1
enzyme
conjugate

Mix by gently tapping for 30 seconds and cover the

microplate wells with cardboard sealer. Incubate
them for 60 minutes at 37ºC temperature.

Step 2

Remove contents of the wells. Rinse and flick the

microtiter wells 5 times with working wash solution.

Step 3

Chromogen 100 µl 100 µl

-substrate
solution

Incubate the micro plate wells for 15 minutes at room

temperature and dark.

Step 4

100 µl 100 µl
Stop Solution

Read the absorbance of the wells at 450 nm

(Use a 630 nm filter as reference filter if it’s available).

6
PishtazTeb Diagnostics European authorized representative MA_H. pylori.S.Ag
JTC Diagnosemittel UG Schulweg 8 Edition-No. 1
D-34516 Voehl/GERMANY 160220