|
H. Pylori
Test
A Rapid Visual Test for the qualitative detection
of IgG antibodies specific to Helicobacter pylori CLIA COMPLEXITY:
Moderately Complex
This test is NOT AVAILABLE FOR RETAIL SALE.
FOR HEALTHCARE PROFESSIONAL PURCHASES ONLY.
INTENDED USE
The H. pylori test is a rapid test for the qualitative detection
of IgG antibodies specific to Helicobacter pylori in human serum,
plasma and whole blood specimens. This test kit is intended as an
aid in the diagnosis of H. pylori infection in patients with gastrointestinal
symptoms.
<< back to top
SUMMARY
Gastritis and peptic ulcers are two of the most common human diseases.
Since the discovery of H. pylori (Warren & Marshall, 1983),
many reports have suggested that this organism is one of the major
causes of ulcer diseases (Anderson & Nielsen, 1993; Hunt &
Mohammed, 1995; Lambertet al. 1995) and stomach cancer. Although
the exact role of H. pylori is not fully understood, the eradication
of H. pylori has been associated with elimination of ulcer diseases
(Hunt & Mohammed, 1995).
The human serological responses to infection with H. pylori have
been demonstrated (Varia & Holton, 1989; Evans, et al., 1989).
The detection of the specific IgG antibodies to H. pylori has been
used as an aid in the diagnosis of symptomatic patients. H. pylori
may also colonize in some asymptomatic persons. A serological test
may be used either as an adjunct to endoscopy or as an alternative
measure in symptomatic patients (Peura, 1997; Dubois, 1995).
The H. pylori test is intended for use in the detection of the
IgG antibodies specific to H. pylori in serum, plasma or whole blood
samples. This information can be used by the physician and the patient
for ulcer disease management.
<< back to top
TEST PRINCIPLE
The H. pylori test is a chromatographic immunoassay for the qualitative
determination of anti-H. pylori. IgG antibodies in human serum,
plasma and whole blood. The test device contains a membrane strip
which is pre-coated with H. pylori antigens on the test band region
and H. pylori specific monoclonal antibody on the control band region.
The H. pylori antigens-colloid gold conjugate pad is placed at the
end of the membrane. When the H. pylori specific IgG antibodies
are present in patient samples, the mixture of colloid gold conjugate,
patient sample and developer buffer moves along the membrane chromatographically
to the test region (T) and forms a visible line as the antigen-antibody-antigen
gold particle complex forms. Therefore, the formation of a visible
line in the test region (T) indicates a positive result for the
detection of H. pylori specific IgG antibodies. When the H. pylori
specific IgG antibodies are absent in the sample, no visible color
band will form in the test region (T). Therefore, the absence of
a color band in the test region (T) indicates a negative result
for the detection of H. pylori specific IgG antibodies. A color
band will always appear in the control region (C). This control
band serves as a procedural indicator that 1. verification that
sufficient volume has been added, 2. verification that proper flow
is obtained, and 3. reagent control.
<< back to top
STORAGE AND STABILITY
The test kit (including the positive and negative controls) is
to be stored refrigerated (2-8°C) or at room temperature (up
to 30°C). The test device should remain in the sealed pouch
for the duration of the shelf-life.
<< back to top
PRECAUTIONS
- FOR IN-VITRO DIAGNOSTIC USE ONLY.
- For Professional and Laboratory use only.
- Do not use test kit beyond expiration date.
- Do not mix reagents from different lots.
- Do not open the foil pouch until ready to perform the test.
- Do not use whole blood specimens which have been stored for
more than three days.
- Heat treated and/or contaminated sera may cause erroneous results.
- Developer buffer, positive and negative controls contain sodium
azide which may react with lead or copper plumbing to form potentially
explosive metal azides. When disposing of these solutions, always
flush with copious amounts of water to prevent azide buildup.
- Standard guidelines for handling infectious agents and chemical
reagents should be observed throughout all procedures. All contaminated
waste such as patient samples and used devices should be properly
disposed of.
- Warning: Potential Biohazardous Material
Each donor unit of human plasma or serum used in the preparation
of the Positive and Negative Controls was tested by FDA-approved
methods for the presence of anti-HIV-1/HIV-2, HBsAg and anti-HCV,
and found to be negative. However, caution should be used when
handling and disposing of these items at biosafety level 2, as
recommended in the U.S. Department of Health and Human Services
Public Health Service, Center for Disease Control/National Institutes
of Health Manual, Biosafety in Micobiological and Biomedical Laboratories,
1999.
<< back to top
REAGENTS AND MATERIALS SUPPLIED
- 25 individually wrapped test devices with disposable transfer
pipet. Each test cassette contains one test strip with H. pylori
antigen coated membrane, colored H. pylori antigen pad.
- Developer Buffer (8 ml): 50mM Tris-HCl buffer with 0.02% sodium
azide.
- H. pylori Negative Control (0.4 ml): Normal human serum or plasma
diluted in saline solution with 0.1% sodium azide.
- H. pylori Positive Control (0.4 ml): H. pylori antibody positive
human serum or plasma diluted in saline solution with 0.1% sodium
azide.
- One Instruction Sheet.
MATERIALS REQUIRED BUT NOT PROVIDED
1. Vacutainer tubes: Plain for serum procedure. EDTA, heparin or
citrate for plasma or whole blood procedure.
2. Finger lancet for fingerstick blood procedure.
<< back to top
SPECIMEN COLLECTION AND HANDLING
Fingerstick
1. Clean the area to be lanced with an alcohol swab.
2. Squeeze the end of the fingertip and pierce with a sterile lancet.
3. Wipe away the first drop of blood with sterile gauze or cotton.
4. Allow the second drop of blood to flow directly into the sample
well. Alternatively, use the disposable transfer pipet provided
to obtain about 40 µl (two full drops) of fresh blood. Hold
the pipet in a vertical position and add two full drops of blood
into the sample well.
Whole Blood
1. Collect whole blood into a purple, blue or green top collection
tube (containing EDTA, citrate or heparin, respectively) according
to standard laboratory procedure.
2. The whole blood may be used for testing immediately or stored
at 2-8°C up to three days.
Plasma
1. Collect whole blood into a purple, blue or green top collection
tube (containing EDTA, citrate or heparin, respectively) by venipuncture.
2. Separate the plasma by centrifugation.
3. Carefully withdraw the plasma for testing, or label and store
at 2-8°C for up to two weeks. Plasma may also be frozen at -20°C
for up to one year.
Serum
1. Collect whole blood into a red top collection tube (containing
no anticoagulants) by venipuncture.
2. Allow the blood to clot at room temperature about 30 minutes
and separate the serum by centrifugation.
3. Carefully withdraw the serum for testing, or label and store
at 2-8°C for up to two weeks. Serum may also be frozen at -20°C
for up to one year.
<< back to top
TEST PROCEDURE
Assay Procedure
1. Test device, developer buffer, patient samples, and controls
should be brought to room temperature (20°C to 30°C) prior
to testing.
2. Bring the device to room temperature before opening the pouch
to avoid condensation of moisture on the membrane. Remove the test
device from its foil pouch when ready to perform the test. Label
the device with patient or control identification.
3. Add the specimen to the sample well.
Serum or plasma sample in collection tube: Hold the provided transfer
pipet in vertical position and add 1 drop (about 20 µl ) into
the sample well.
Whole blood sample in collection tube: Hold the provided transfer
pipet in vertical position and add two drops (about 40 µl)
of whole blood into the sample well. Mix well before using.
Fingerstick blood: Use the provided transfer pipet to collect enough
sample (more than 40 µl) of blood. Hold transfer pipette in
a vertical position and add two full drops (about 40 µl) of
fresh blood into the sample well.
4. Immediately add 3 or 4 drops of developer buffer into the sample
well.
5. After the addition of the developer buffer, wait for the pink-red
colored bands to appear. Depending on the concentration of IgG antibodies
present, positive result may appear as soon as 1 minute. However,
to confirm a negative result, the complete reaction time of 5 minutes
is required. Do not interpret results after 8 minutes.
<< back to top
INTERPRETATION OF RESULTS
1. NEGATIVE: Only one pink-red colored band appears in the control
region (C). No apparent faint pink to red colored band in the test
region (T). A negative result indicates that there is no anti-H.
pylori IgG in the patient sample or that IgG concentration is below
the detection level.
2. POSITIVE: Two pink-red colored bands appear. One in the control
region (C) and one in the test region (T). When testing with strong
positive samples, the intensity of the control band may be lighter
than expected. A positive result indicates that there is anti-H.
pylori IgG in the patient sample. Comparison of the line intensities
is not recommended.
3. INVALID: A total absence of pink colored bands in both regions
is an indication of procedural error or that test reagents may have
deteriorated. Repeat the test with a new test device. If the problem
persists, call Applied Biotech at (858) 587-6771 for Technical Assistance.
<< back to top
QUALITY CONTROL
Internal Procedural Control
1. A procedural control is included in the test. A colored band
appearing on the control region (C) of the membrane indicates proper
performance of the test and the device.
2. A clear background in the result window is considered an internal
negative control. However, when whole blood samples are tested,
the background may appear slightly reddish due to the low level
hemolysis of some red blood cells. This is acceptable as long as
it does not interfere with the interpretation of the test result.
The test is invalid if the background fails to clear and obscures
the reading of the result.
External Quality Control
1. Positive Control: Add one drop (about 20 µl) of Positive
Control in the sample well using the provided transfer pipet by
holding it in a vertical position. Immediately add 3 or 4 drops
of developer buffer. A positive signal is indicated by two pink
to red lines, one in the test region (T) and one in the control
region (C). Read results at 5 minutes.
2. Negative Control: Add one drop (about 20 µl) of Negative
Control in the sample well using the provided transfer pipet by
holding it in a vertical position. Immediately add 3 or 4 drops
of developer buffer. A negative signal is indicated by only one
pink to red colored line in the control region (C). Read results
at 5 minutes.
3. It is recommended that both a Positive and a Negative control
be tested with every new shipment or new kit lot number, and as
otherwise required by your state and local regulations.
<< back to top
LIMITATIONS
1. This test kit is to be used for the qualitative detection of
IgG antibodies to H. pylori.
2. This test kit should be used for symptomatic individuals suspected
of having gastrointestinal disorders.It is not intended for use
with asymptomatic patients and is not intended for use with pediatric
patients.Diagnosis of gastritis and/or peptic ulcer should be made
by confirmation with other clinical findings.
3. A positive result suggests the presence of IgG antibodies to
H. pylori. It does not distinguish between active infection and
past exposure to H. pylori and does not necessarily indicate the
presence of gastrointestinal disease.
4. A negative result does not rule out H. pylori infection because
the IgG antibodies to H. pylori may be absent or may not be present
in sufficient quantity to be detected.
<< back to top
EXPECTED RESULTS
The majority of individuals exposed to H. pylori developed IgG
antibodies against the organism. It is reported that H. pylori is
universally distributed and an estimated 50% of the world's population
is colonized by H. pylori (Lambert et. al.,1995). The presence of
H. pylori IgG antibodies is a function of age, race, geography and
clinical conditions. A relatively large proportion of patients who
have positive levels of IgG antibodies are asymptomatic even though
they are colonized with the H. pylori. Therefore, IgG antibodies
levels do not necessarily correlate with the severity of clinical
symptoms (Tytgat & Rauws, 1989).
<< back to top
PERFORMANCE CHARACTERISTICS
A. Accuracy
a. Comparison with biopsy results
The accuracy of the H. pylori Test was evaluated in comparison to
biopsy results of human specimens. Out of the three hundred and
seventeen (317) samples, two hundreds and eighty-three (283) test
results agreed with the biopsy results. Thirty-four (34) samples
gave different results. Out of the thirty four (34) different test
results, eighteen (18) samples obtained positive results with H.
pylori Test and negative biopsy results. Sixteen (16) samples obtained
negative results with H. pylori Test and positive biopsy results.
A commercial EIA kit was used to reanalyze the discrepant samples.
Out of the eighteen (18) positive H. pylori Test results, fifteen
(15) samples were positive, and three (3) were negative. Out of
the sixteen (16) H. pylori negative test results, one (1) was negative,
four (4) were indeterminate and eleven (11) were positive when tested
in comparison with an EIA kit. The biopsy sample comparison results
are summarized in Table 1a:
This comparison study results gave a sensitivity of 92.6% (199/215),
a specificity of 82.4% (84/102), and a total agreement of 89.3%
(283/317). The relatively low specificity of the serological test
results in comparison to the biopsy results may be partially attributed
to a sampling error of the biopsy test.
b. Comparison Study with a Commercially Available Rapid H. pylori
Test
The accuracy of FirstStep H. pylori Test was also evaluated against
a commercially available rapid H. pylori Test using serum/plasma
specimens. Specimens were tested at Applied Biotech, Inc. in a side
by side comparison using the FirstStep H. pylori Test and a commercial
rapid H. pylori test. The discrepant specimens were tested with
a commercial EIA kit.
Out of the one hundred and seventy (170) samples, one hundred and
fifty eight (158) gave the same results. Twelve (12) samples gave
negative results with FirstStep H. pylori Test and positive results
with the commercial rapid H. pylori test. When tested with an EIA
kit, out of these twelve (12) samples, three (3) were positive,
seven (7) were negative, and two (2) were indeterminate. The comparison
results are summarized in Table 1b:
These results gave a relative sensitivity of 84.0 % (63/75), a
relative specificity of 100% (95/95), and a total agreement of 92.9
% (158/170).
B. Test Sensitivity:
Since there is no sensitivity standard established for H. pylori
IgG antibodies, the following dilution (test sensitivity) studies
were performed for comparison purposes. Six (6) H. pylori positive
human specimens (serum/plasma) purchased from suppliers were diluted
with a H. pylori negative human serum. The diluted samples were
tested with FirstStep H. pylori Test and a commercially available
rapid H. pylori Test. The results of the test sensitivity study
are summarized in Table 2.
GMSI: H. pylori test.
C: commercially available rapid H. pylori test.
These results indicated that the sensitivity of ABI H. pylori Test
was determined to be comparable to the commercial rapid H. pylori
Test.
C. Specificity:
Cross Reactivity
D. Specimen Matrix Study
The H. pylori Test can be used with serum/plasma and whole blood
specimens. A comparison study was conducted to verify the performance
of H. pylori Test in the three types of specimens. One hundred and
twelve (112) matched sets of serum/plasma and venous whole blood
specimens were collected and evaluated with the H. pylori Test.
Three different anticoagulants were used for whole blood samples:
EDTA (50 samples, at the concentration of 1-2 mg/ml), heparin (22
samples, at the concentration of 20-30 unit/ml) and citrate (40
samples, at a concentration of 10-15 mM). A total of thirty-six
(36) samples tested positive for H. pylori antibodies in serum/plasma
specimens and thirty-five (35) tested positive for H. pylori antibodies
in whole blood specimens. Only one set of samples tested differently,
positive in serum/plasma specimen and negative in whole blood specimen.
A comparison study was also performed using twenty-two (22) matched
venous whole blood and capillary blood samples. Of the twenty-two
(22) samples tested, one (1) gave discordant result (positive with
venous whole blood and negative with capillary whole blood). The
results of the specimen matrix study illustrates that an excellent
agreement exists between serum/plasma and venous whole blood specimens,
and between venous whole blood and capillary whole blood. No significant
difference in performance was observed. E. Reproducibility / Site
Study
The precision of the H. pylori Test has been evaluated at Applied
Biotech, Inc. and three independent clinical sites. Three human
whole blood specimens with different level of H. pylori antibodies
were diluted and blind labeled into twenty (20) vials each and used
for the study. Of the forty (40) positive samples with two levels
of H. pylori antibodies, the results were all positive. Test results
of twenty (20) negative samples rendered 100% agreement with expected
results. Fifteen (15) volunteers were also sent to three sites to
test for anti-H.pylori IgG with fingerstick blood. Seven (7) people
tested positive and eight (8) people tested negative by all three
sites. The simplicity of the H. pylori WB Test has been evaluated
at the three different sites. Twenty people at each site, most of
whom were non- professionals with the equivalent of a high school
education, were asked to read the instructions and perform three
tests using blind labeled samples. The results from these studies
are summarized in Table 3. For the one hundred twenty (120) samples
with different levels of H. pylori antibodies, results were all
positive. For the sixty (60) negative samples, the results were
all negative. The results obtained from all sites demonstrate 100
% ageement. The field results demonstrate that the ABI H. pylori
WB test is easy to perform and can be used to detect a visual qualitative
result of IgG antibodies specific to H. pylori. The results are
summarized in Table 3.
<< back to top
|
