RE IFU en Helicobacter pylori IgG quantitative

Document Sample
RE IFU en Helicobacter pylori IgG quantitative Powered By Docstoc
					                                                Instructions for Use




    Helicobacter pylori
        IgG ELISA
            Enzyme immunoassays for the quantitative
determination of IgG antibodies against Helicobacter pylori in human
                         serum and plasma.




                                  RE58041
                                  12x8

                                            2-8°C




   I B L       I N T E R N A T I O N A L                             G M B H
   Flughafenstrasse 52a       Phone: +49 (0)40-53 28 91-0   IBL@IBL-International.com
   D-22335 Hamburg, Germany   Fax: +49 (0)40-53 28 91-11    www.IBL-International.com
2. INTENDED USE
The Helicobacter pylori EIA kit is an enzyme immunoassay for the detection of Helicobacter pylori
specific IgG antibodies in human serum and is used as an aid in the diagnosis of Helicobacter pylori
(H.pylori) infections and for the detection of immunity. The assay must be performed strictly in
accordance with the instructions set out in this instructions for use. No responsibility can be held for any
loss or damage (except as required by statute) how so ever caused by or arising out of non-compliance
with the instructions provided.


3. INTRODUCTION
Helicobacter pylori is known to be associated with chronic antral gastritis, duodenal ulcer, gastric ulcer
disease, gastric carcinoma and mucosa associated lymphatic tissue neoplasia. H. pylori infection of the
mucosa leads to a pronounced local and systemic immune response in most patients. The immune
response against H. pylori at the mucosal level is predominantly of the IgA type. Circulating antibodies to
H. pylori are predominantly of the IgG class. A systemic immune response to H. pylori of the IgA class is
present in a significant number of cases (>60% positive). In a minority of H. pylori infected patients only
H. pylori specific IgA antibodies can be detected. IgM antibodies are rarely found and seem to be of
minor importance. Determination of IgG antibodies to H. pylori in serum can be used as a non-invasive
test for the presence of gastric or duodenal H. pylori infection. The level of IgG antibodies to H. pylori
correlates with the severity of gastritis of the antrum. In addition, the outcome of treatment can be
monitored by serological follow-up.


4. PRINCIPLE OF THE ASSAY
The method for quantitative determination of specific IgG to H.pylori is illustrated below.




1.       Well coated with purified H.pylori antigen is incubated with diluted human serum for 60 minutes at
         37°Celsius.
2.       After washing with washbuffer anti-IgG antibody conjugated to peroxidase (conjugate) is added. Incubation
         for 60 minutes at 37°Celsius.
3.       After washing with washbuffer, TMB is added. TMB acts as a substrate for the peroxidase.

4.       By incubating the wells in the dark for 30 minutes at room-temperature the colour of the substrate will turn
         to blue.
5.       The enzymatic conversion of TMB is stopped by adding sulphuric acid, 0.5 M. The optical density is
         measured with a spectrophotometric reading instrument at 450 nanometer

4.1 Assay principle
The Helicobacter pylori IgG EIA is an indirect IgG immunosorbent assay for the detection of H.pylori
specific IgG in human serum. H. pylori antigen is prepared by extraction and inactivation of clinical
isolates of H.pylori, propagated in vitro. This H.pylori antigen is coated to the solid phase of the micro-
titerplate wells. The antigen will bind H. pylori specific human IgG present in a serum. Anti-IgG labelled
covalently to peroxidase (i.e. the conjugate) will complex to H.pylori-specific IgG. The conjugate will act
RE58041_IFU_en_Helicobacter pylori
IgG_quantitative_3410_V09_042007.doc; April 2007
                                                                                                                  -3-
as the indicator for the immunological reaction between the H. pylori specific human IgG and the H.pylori
specific antigen coated on the wells of the microtiterplate. TMB that acts as a chromogen will induce
colour proportionally to the amount of H.pylori specific IgG bound. By making use of a set of 4 calibrators,
the results obtained with each sample can be expressed quantitatively in Arbitrary Units per mL (AU/mL).


5. REAGENTS AND ACCESSORIES

5.1 Reagents provided in the kit
The kit contains the following reagents. A distinction can be made between reagents that are specific for
the assay and universal reagents.
 SYMBOL                        CAT. CODE        DESCRIPTION                                   QUANTITY
                                  3410-21       Calibrator 300 AU/mL (RED, ready-to-use)          1.5 mL

                                  3410-16       Calibrator 75 AU/mL (ROSE, ready-to-use)          1.5 mL

                                  3410-14       Calibrator 15 AU/mL (GREEN, ready-to-use)         1.5 mL

                                  3410-11       Calibrator 0 AU/mL (YELLOW, ready-to-use)         1.5 mL
                                                PO-labelled anti-IgG conjugate
                                  9000-40                                                         0.25 mL
                                                (100 x concentrated)
                                                Microtiterplate coated with H.pylori antigen
                                  3410-08                                                         1 plate
                                                (96 wells)
                                  9000-19       TMB Substrate Solution (ready-to-use).            15 mL

                                  9000-03       Dilution buffer (BLUE, ready-to-use)              120 mL

                                  9000-07       Wash buffer (20 x concentrated)                   60 mL

                                  9000-08       Stop solution (ready-to-use)                      20 mL

5.2 Materials provided with the kit
− Resealable bag, 2 x
− Instructions for use, 1 x
− Certificate of Analysis, 1x

5.3 Equipment and materials required, but not supplied
− Pipettes to deliver volumes between 10 µL and 1000 µL (trueness ± 2%, precision ± 1%)
− Volumetric laboratory glassware
− Deionised (or distilled) water
                                              C
− Incubator thermostatically controlled at 37° ± 1ºC
− Clean disposable tubes for diluting patients sera (capacity appr. 3 mL)
− Clean disposable tubes for diluting conjugate and TMB (capacity 12 mL)
− Automatic plate washer (optional) with dispense volume 300-350 µL, wash cycle = 5 times
− Microtiter plate reader, equipped for measuring absorbances at 450 nm (optionally equipped for dual
    wavelength measurement at 450 and 620 nm), absorbance range; 0.0 to 3.0 absorbance units
− Vortex tube mixer
− Timer


6. COMPOSITION AND HANDLING OF REAGENTS

6.1 Specific kit reagents

6.1.1 Microtiterplate
Microtiterplate (96 wells) with 8-well breakable strips coated with H.pylori specific antigens. The strips are
                                                                           RE58041_IFU_en_Helicobacter pylori
                                                             IgG_quantitative_3410_V09_042007.doc; April 2007
 -4-
                                          C.
ready to use and should be stored at 2-8 ° After opening replace any unused wells in the resealable
plastic bag and store in the kitbox between 2-8 ºC. Resealed strips expire after one month.

6.1.2 Calibrator 300 AU/mL
A vial containing 1.5 mL of human serum, highly reactive for IgG directed to H.pylori prediluted in PBS
buffer, BSA, preservatives and an inert red dye. The H.pylori-IgG reactivity is set such that this calibrator
contains 300 AU/mL.The reagent is ready to use. After use, close cap, replace in the kitbox and store
               C.
between 2-8 ° Handled in this way, the calibrator will expire as indicated on the vial label.

6.1.3 Calibrator 75 AU/mL
A vial containing 1.5 mL of buffered solution, reactive for IgG antibodies against H.pylori prediluted in
PBS buffer, BSA, preservatives and an inert light-red dye. The H.pylori-IgG reactivity is set such that this
calibrator contains 75 AU/mL The reagent is ready to use. After use, close cap, replace in the kitbox and
                     C.
store between 2-8 ° Handled in this way, the calibrator will expire as indicated on the vial label.

6.1.4 Calibrator 15 AU/mL
A vial containing 1.5 mL human serum, with a low reactivity for IgG to H.pylori prediluted in PBS buffer,
preservatives and an inert green dye. The reagent is ready to use. The H.pylori-IgG reactivity is set such
that this calibrator contains 15 AU/mL After use, close cap, replace in the kitbox and store between 2-8
 C.
° Handled in this way, the calibrator will expire as indicated on the vial label.

6.1.5 Calibrator 0 AU/mL
A vial containing 1.5 mL human serum, without reactivity for IgG to H.pylori prediluted in PBS buffer,
preservatives and an inert yellow dye. The reagent is ready to use. After use, close cap, replace in the
                               C.
kitbox and store between 2-8 ° Handled in this way, the calibrator will expire as indicated on the vial
label.

6.2 Universal reagents

6.2.1 Conjugate
A vial containing 0.25 mL antibody specific for IgG labelled with horseradish peroxidase, PBS buffer, BSA
and preservatives. Prepare only the amount of working strength conjugate needed for the assay-run and
                                         C.
keep the concentrated conjugate at 2-8 ° Handled in this way, the conjugate will expire as indicated on
the vial label.

Note: The working strength conjugate cannot be stored and should be used immediately after
      preparation.

6.2.2 Dilution buffer
The bottle contains 120 mL PBS buffer, proteins, preservatives and an inert blue dye. The reagent is
                                                                                 C.
ready to use. After use, close lid, replace in the kitbox and store between 2-8 ° Handled in this way, the
dilution buffer will expire as indicated on the bottle label.

6.2.3 Wash buffer
The bottle contains 60 mL PBS buffer, Tween® 20 and preservatives. The wash buffer is 20 times
concentration must be prepared according to protocol. After use, close lid, replace in the kitbox and store
              C.
between 2-8 ° Handled in this way, the wash buffer will expire as indicated on the bottle label. Stability
at working concentration is one week at room temperature or one month at 2-8°    C.

6.2.4 TMB substrate (chromogen)
The bottle contains 15 mL TetraMethylBenzidine (TMB) chromogen/substrate solution and is presented
in an amber bottle. The TMB solution is at working strength and is ready to use. Take out only the
amount of TMB needed for the assay-run. After opening take out the volume needed and immediately re-
close the bottle. The TMB should at all times kept away from direct light, as this can induce the auto-
coloration. Always check the colour of the TMB prior to use. The TMB should be clear, colourless or with
                                                                  C.
a very faint blue tinge. The TMB solution should be stored at 2-8° Handled in this way, the TMB will
expire as indicated on the bottle label.

Note: Do not pour back any unused TMB substrate solution!

RE58041_IFU_en_Helicobacter pylori
IgG_quantitative_3410_V09_042007.doc; April 2007
                                                                                                           -5-
6.2.5 Stop Solution
The bottle contains 20 mL 0.5 M sulphuric acid solution. The reagent is ready to use. After use, close cap
and replace in the kitbox. Handled in this way, the Stop Solution will expire as indicated on the vial label.


7. COLLECTION, HANDLING AND STORAGE OF SERUM SPECIMENS
Either human serum or plasma may be used. Samples must not be haemolized, nor contain particulate
material. To obtain sera for the detection of H.pylori IgG antibodies, patient blood should be drawn and
allowed to clot at roomtemperature. Centrifuge within one day, transfer the serum into a vial. Sera may be
            C                                                                 C        C.
stored at 4° for up to 7 days. If storage time exceeds 7 days, store at -20° to -70° Avoid repeated
freeze-thaw cycles.


8. PROCEDURE

8.1 Washing procedure
Efficient washing is a fundamental requirement of EIA's. It is essential that each washing procedure is
carried out with care to obtain reproducible inter- and intra- assay results.

Prepare the washbuffer: mix per 8-well strip 1.5 mL wash buffer (20x) with 28.5 mL distilled water.
Alternatively, mix the total volume (60 mL) of the wash buffer (20x) with 1140 mL distilled water.

Both manual washing and washing with an automatic plate washer can be done:

8.1.1 Manual washing
1.   Empty the contents of each well by turning the strips in the holder upside down followed by a firm
     short vertical movement. Keep the strips tightened by pressing the sides of the strip holder.
2.   Fill all the wells to the rims (300-350 µL) with rinsing buffer, for instance with a 8-channel pipet. Be
     aware of carry-over.
3.   Turn the strips upside down and empty the wells by a firm short vertical movement.
4.   This washing cylce (2 and 3) should be carried out 5 (five) times.
5.   Place the inverted plate on absorbent paper towels and tap the plate firmly to remove residual
     washing solution in the wells.
6.   Take care that none of the wells dries out before the next reagent is dispensed. Therefore, proceed
     immediately with the next step.

8.1.2 Washing with automatic microtiterplate wash equipment
When using automatic plate wash equipment, check that all wells can be aspirated completely, that the
washing buffer is accurately dispensed reaching the rim of each well during each washing cycle. The
washer should be programmed to execute 5 (five) washing cycles. After the last cycle, remove the
washing buffer from the wells by tapping firmly the plate on absorbant towels.

8.2 Assay and reagent preparation procedure

                                                    C)
Note: Bring all reagents to room temperature (18-23° before assaying. Perform all assay steps
      in the order given and without any appreciable delays between the steps. Check the expiry
      date before use.

1.     Dilute patient sera (1+100): mix 1.0 mL dilution buffer (BLUE) with 10 µL patient serum. After
       dilution thoroughly mix with a vortex to ensure adequate mixing. The calibrators are ready to use
       and need no further dilution.

2.     Leave as many wells as needed in the holder. Label appropriately.

Note: Bring the coated strips to room temperature before opening the pouch to avoid
      development of condensed water in the wells. Place unused strips in the pouch, securely
                               C
      reseal and store at 2-8 ° in the kitbox.

3.     Dispense per well 100 µL of the calibrators in duplicate (see scheme). Use 8 wells for calibrators:
       300 AU/mL (RED), 75 AU/mL (ROSE), 15 AU/mL (GREEN) and 0 AU/mL (YELLOW). Dispense
                                                                           RE58041_IFU_en_Helicobacter pylori
                                                             IgG_quantitative_3410_V09_042007.doc; April 2007
 -6-
          100 µL of each diluted patient sample (BLUE) into a well.

4.        Incubate the wells in a second resealable bag or in 100% moist atmosphere for 60 minutes ± 5
                        C
          minutes at 37° ± 1ºC.




                1        2       3       4       5       6        7       8       9       10      11     12

      A        300      S1

      B        300      S2

      C         75      S3

      D         75      S4

      E         15      S5

      F         15      S6

      G         0       S7

      H         0       S8

300 = Calibrator 300AU/mL 15 = Calibrator 15AU/mL S1 = Diluted Sample S3 = Diluted Sample
75 = Calibrator 75AU/mL   0 = Calibrator 0 AU/mL  S2 = Diluted Sample S4 = Diluted Sample

4.        Prepare working strength anti-IgG-PO conjugate: mix per 8-well strip 1.0 mL dilution buffer (BLUE)
          with 10 µL anti-IgG-PO conjugate (100x). See scheme below.

                        Dilution scheme for preparation of anti-IgG-PO conjugate
                                              Dilution buffer                  Anti-IgG-PO
     Number of 8-well strips in use
                                                  (BLUE)                     conjugate (100x)
                   1                              1.0 mL                           10 µL
                   2                              2.0 mL                           20 µL
                   6                              6.0 mL                           60 µL
                  12                             12.0 mL                          120 µL

5.        When incubation has completed, aspirate the liquid and wash the 8-well strips 5 (five) times with
          wash buffer according to the washing protocol (see 8.1).

7.        Dispense 100 µL per well working strength anti-IgG-PO conjugate (BLUE).

8.        Incubate the strips in the resealable bag or in a 100% moist atmosphere for 60 minutes ± 5
                        C
          minutes at 37° ± 1ºC.

Note: In case the incubations can not be performed in a 100% moist atmosphere, the background
     OD levels may rise. In that case it is advised to incubate with 150 µl conjugate per well.

9.        Wash the strips 5 (five) times with washbuffer according to the washing protocol (see 8.1).

10.       Dispense 100 µL TMB solution ready-to-use per well.

Note: Use only clean disposable containers.

11.                                                                     C),
          Incubate for 30 minutes ± 2 minutes at roomtemperature (18-23° away from direct or intense
          light.

RE58041_IFU_en_Helicobacter pylori
IgG_quantitative_3410_V09_042007.doc; April 2007
                                                                                                          -7-
12.    Add 100 µL stop solution per well (colour shift: blue ⇒ yellow) in the same order and the same rate
       as for TMB-substrate

13.    Measure the absorbance of specimens with a spectrophotometer at 450 nm (optionally with a 620
       nm reference filter) within 10 minutes of adding the stop solution.



9. CALCULATION OF RESULTS
9.1 Calculations
Calculate the mean absorbance value of the calibrators. The concentration of H.pylori specific IgG in a
patient sample is determined by comparing the absorbance values of patient samples with that of the
calibrators. The calibrators have fixed values expressed in AU/mL which represent the reactivity of the
sera. Plot the validated mean absorbances of the calibrators against the fixed AU/mL values into a curve
as indicated in the example standard curve (figure below). The concentration expressed in Arbitrary Units
per mL of individual serum sample can now be read by interpolation from this calibration curve. Similarly,
a curve fitting program such as spline analysis can also be used to calculate the AU/ml values.
                                         E x a m p l e H . p y lo r i Ig G C a l ib r a t i o n C u r v e


                                      1600

                                      1400
                                      1200
                    Absorbance (OD)




                                      1000
                                       800

                                       600
                                       400

                                       200
                                         0
                                             0      50       100          150      200       250    300
                                                          A r b it r a r y U n its p e r m L



Note: Do not use this example calibration curve to read your absorbance results.
      In each run and each microtiterplate a calibration curve should be produced!

9.2 Validation of test
The following criteria must be met to validate each run. Validation should be based on mean values,
calculated for each calibrator.
       1. Calibrator 0 AU/mL:         OD<0.7 times OD of the 15 AU/mL Calibrator
       2. Calibrator 15 AU/mL:        0.150 < OD < 0.500
       3. Calibrator 75 AU/mL:        Ratio OD75AU/mL / Ratio OD15AU/mL: range 2.5 – 5.0
       5. Calibrator 300 AU/mL:       Ratio OD300AU/mL / Ratio OD15AU/mL: range: 5.0 – 10.0

Note: If these criteria are not met, the run should be considered invalid and must be repeated.

9.3 Interpretation of results

Detection of serological evidence of H.pylori infection
Serological evidence of a H.pylori infection can be found by interpretation of the H.pylori IgG
concentration in a sample expressed in AU/mL as follows:

RESULTS                        INTERPRETATION
                               A serum should be considered positive for H.pylori specific IgG antibodies when the
POSITIVE                       concentration is > 20 AU/mL. Interpretation needs to be done with care as indicated in
                               section 13, Limitations of the assay.
                               A serum should be considered negative for H.pylori specific IgG antibodies when the
NEGATIVE                       concentration is <15 AU/mL. Interpretation needs to be done with care as indicated in
                               section 13, Limitations of the assay.

                                                                                                                 RE58041_IFU_en_Helicobacter pylori
                                                                                                   IgG_quantitative_3410_V09_042007.doc; April 2007
 -8-
                    A serum may be considered equivocal, if the H.pylori IgG concentration is between 15
EQUIVOCAL           and 20 AU/mL. In such case it is advised to confirm the results by testing that serum
                    again in duplicate. In the case the repeated result is again equivocal, a second serum
                    should be tested and judged for a change in result.

NOTE: The results must be interpreted in relation with clinical data available. Specially, the
history of antobiotic therapy is of importance: After effective therapy H.pylori specific IgG can
remain present over a prolonged period and may lead to misinterpretation of a positive result.



9.4 Detection of effect of therapy of H. pylori infection by serology.
To estimate the effect of therapy of H.pylori infections by serology it is advised to test serum pairs. The
second serum of a pair can be drawn 3-6 months after the first serum is obtained. Each serum pair
should be tested at the same day in the same test to allow interprepation of significant antibody level
decrease. If one or both sera have an absorbance value higher than that of the 300 AU/ml positive
control, retest both sera in a 1:1000 serum dilution. To obtain the concentration in AU/ml of a serum
measured with 1:1000 dilution, multiply by 10:

         Concentration(1:100)= Concentration(1:1000) x 10 AU/ml.

A significant difference in antibody level between 2 sera is found when,
(1) the second serum is negative and the first serum has a concentration of > 20 AU/ml, or,
(2) the second serum has a twofold lower concentration than the first serum.

NOTE:    If the results of a serum pair do not meet the criteria for a significant antibody level
         difference, it is advised to draw a third sample 3 months later and retest the third sample
         together with the first.

10. CHANGES IN PROCEDURE AND PERFORMANCE
IFU 3410 V09: Instructions for Use are changed as follows: Front page: address and Logo are updated.
All pages: reference to Meddens is made.

11. SPECIFIC PERFORMANCE CHARACTERISTICS
When the kit is employed according to the instructions given, and the appropriate equipment is used in
optimal conditions, the following performances could be reached.

11.1 Assay precision
Different samples containing different concentrations of anti-Helicobacter pylori IgG determined, were
assayed to assess repeatability and reproducibility of the assay (within- and between-run variability). The
assay precision computed on these samples gives coefficient of variation values lower then 7.8% (within-
run variability) and 10.0% (between-run variability). The calibrators used in the kit are calibrated an
internal standard preparation. An internal study is performed to investigate the uncertainty of the values
assigned to the calibrators and the total uncertainty of the assay. A total of eight runs with four kits of the
same lot have been tested to assess the intra-run variation and inter-run variation. Traceability of the
values of the kit calibrators is reached by testing the internal reference standard preparation in different
concentrations together with the kit-calibrators in different runs. The uncertainty of the reference material
(standard deviation) is 1%. The uncertainty of the test-procedure at calibrator 15 AU/mL is 6%, the bias
when compared with the internal reference standard preparation standard is 1,8 AU/mL.
11.2 Analytical specificity
Analytical specificity may be defined as the ability of the assay to detect specific analyte in presence of
potentially interfering factors in the sample matrix (e.g. anticoagulants, haemolysis, effects of sample
treatment). Controlled studies of potentially interfering substances or conditions showed that the assay
performance was not significantly affected by either fat, slight heamolysis or freezing. The results showed
an increase in absorbance values obtained in the lower region of the calibration curve with plasma
samples containing EDTA when compared with serum samples.

11.3 Diagnostic specificity
Diagnostic specificity is the probability of the assay procedure of scoring negative in non infected
samples:

RE58041_IFU_en_Helicobacter pylori
IgG_quantitative_3410_V09_042007.doc; April 2007
                                                                                                              -9-
                                                                       TN
                                          Diagnostic specificity =
                                                                     TN + FP
TN = True Negatives, FP = False Positives

Out of the total of 102 samples 41 samples are expected to be negative, the kit proved to be negative in
39 cases, giving a diagnostic specificity of 95% in this group

11.4 Diagnostic sensitivity
Diagnostic sensitivity is the probability of the assay procedure of scoring positive in infected samples:

                                                                       TP
                                          Diagnostic sensitivity =
                                                                     TP + FN
TP = True Positives, FN = False Negatives

Out of the total of 102 samples a total of 61 serum samples were considered positive (infection with H.
pylori). The Meddens test proved to give a positive result in 60 cases giving it a diagnostic sensitivity of
98%. The accuracy obtained was 98%.

11.5 Limit of Detection
Three replicates of twelve sera tested negative within an independent assay for the presence of
Helicobacter pylori IgG were tested during an internal evaluation. The limit of detection was determined at
6,3 AU/mL.

12. TRACEABILITY OF CALIBRATORS
The level of the calibrators as presented in this kit, represents the level as used in the clinical trials as
shown above. This is organised such that a manufacturer’s working reference is maintained to which
manufacturer’s product reference is calibrated. This manufacturer’s product reference is used for
validating kit performance. In this way the sensitivity and specificity of each lot represents that as shown
above.

13. LIMITATIONS OF THE ASSAY
−      Appropriate medical decisions are only possible if the medical traceability is ensured. The product is
       intended for professional use as an aid in the diagnosis of active H. pylori infections.
−      Bacterial contamination or repeated freeze-thaw cycles of the specimens may affect the absorbance
       values of the samples with consequent alterations of antibody to H.pylori levels.
−      Diagnosis of an infectious disease should not be established on the basis of a single test result. A
       precise diagnosis, in fact, should take into consideration clinical history, symptomatology, as well as
       serological data. Serological data, however, have restricted value in immunosuppressed patients.
−      It should be noted that results must be interpreted in relation with clinical data available. Specially, the
       history of antibiotic therapy is of importance: after effective therapy H.pylori specific IgG can remain
       present over a prolonged period and may lead to misinterpretation of a positive result.
−      The performance characteristics mentioned in section 11 are acquired with the utmost care.
       However, a negative result does not totally exclude an active H.pylori infection. Therefore results
       need to be interpreted with caution.

14. WARNINGS AND PRECAUTIONS
−      All reagents supplied are for in vitro use only.
−      All calibrators provided in this H. pylori-IgG testkit contain serum or plasma from human origin. They
       have been assayed for Hepatitis B antigen, anti-HCV and anti-HIV antibodies and found negative.
       However, these sera must be considered as potentially infectious, and sera should be handled by
       appropriate procedures.
−      The reagents included in the kit have been formulated with materials of animal origin. These materials are
       sourced where possible from countries that have no current status of outbreaks of TSE’s or other transmittable
       infectious agents within cattle, or are treated during the manufacturing process in such a way as to protect
       personnel and preserve the performance of the device. However, the reagents must be considered as
       potentially infectious, and should be handled by appropriate procedures.
−      Avoid contact of substrate, sulfuric acid, washing and dilution buffer with skin and mucous membranes. If these
       reagents come into contact with skin or mucous membranes, wash with abundant tap water.
−      Each well is ultimately used as an optical cuvette. Therefore, do not touch the undersurface of the strips,

                                                                                 RE58041_IFU_en_Helicobacter pylori
                                                                   IgG_quantitative_3410_V09_042007.doc; April 2007
    - 10 -
     prevent damage and dirt.
−    Use only components that are provided in this kit: intermixing between kits may cause interpretation problems.
−    The reagents supplied should be used only as indicated in this instruction manual.
−    Do not eat, drink, smoke or apply cosmetics in the assay laboratory.
−    Do not pipette solutions by mouth.
−    Avoid direct contact with all potentially infectious materials by using protective clothing such as lab coats,
     protective glasses and disposable gloves. Wash hands thoroughly at the and of each assay.
−    Avoid splashing or forming an aerosol. Any reagent spill should be washed with 3% sodium hypochlorite
     solution and disposed of as though potentially infectious.
−    All samples, biological reagents and materials used in the assay must be considered potentially able to transmit
     infectious agents. Disposal should be according to local, state or national legislation. Dispose of through
     authority facilities or pass to chemical disposal company. Disposable ignitable materials must be incinerated;
     liquid waste and non-ignitable materials must be decontaminated with sodium hypochlorite at a final
     concentration of 3 % for at least half an hour. Liquid waste containing acid must be neutralized before
     treatment. Any materials to be reused must be autoclaved using an overkill approach. A minimum of one hour
              C
     at 121 ° is usually considered adequate, though the users must check the effectiveness of their
     decontamination cycle by initially validating it and routinely using biological indicators.
−    Thiomersal is included as a preservative in some of the components included in the kit. Thiomersal has been
     reported to form mercury build-up in the laboratory plumbing. To prevent build-up, flush plumbing with a large
     volume of water while disposing of these solutions in the sink.
−    TMB substrate solution is a stabilized chromogenic substrate for use with horse radish peroxidase
     immunoassays. It contains both 3,3’, 5,5’ tetramethylbenzidine and hydrogen peroxide (H2O2) in low
     concentrations. This formulation contains no DMF or DMSO. TMB is known to be sensitizing to the skin when
     exposed to high concentrations. TMB substrate is very light sensitive and direct exposure to sunlight should be
     avoided.To avoid contamination of the entire bottle of substrate, never pour back unused substrate solution.
     Always pour necessary volume of substrate into a separate container for use.
−    Stop Solution (Stopping reagent, H2SO4, 0.5 M):
     R36/38 - irritating to eyes and skin,
     S26        - In case of contact with eyes, rinse immediately with plenty of water, and seek medical advice.

15. LITERATURE
1.    Van Zwet AA, Thijs JC, Diagnostic and therapeutic studies in Helicobacter pylori infection. 1995. Thesis, Free
      University Amsterdam, Chapter 5, 39-56.
2.    Veenendaal RA, Immunological and serological aspects of Helicobacter pylori infection. Thesis Leiden, ISBN 90-
      9007016-8.
3.    Kreuning J, Lindeman J, Biemond I, Lamers CBHW, Relation between IgG and IgA antibody titres against
      Helicobacter pylori in serum and severety of gastritis in asymptomatic subjects. J Clin Pathol 1994; 47: 227-223.
4.    Peña AS, Endtz HPh, Offerhaus GJA, Hoogenboom-Verdegaal A, van Duijn W, de Vargus N, den Hartog G,
      Kreuning J, van der Reyden J, Mouton RP, Lamers CBHW. Value of serology (ELISA and immunoblotting) for the
      diagnosis of Campilobacter pylori infection. Digestion 1989, 44: 131-141.
5.    Kosunen TU, Seppälä K, Sarna S, Sipponen P. Diagnostic value of decreasing IgG, IgA and IgM antibody titres
      after eradication of Helicobacter pylori.Lancet 1992, 339: 893-895.
6.    Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, Jellum E, Orentreich N, Vogelman J H, Friedman GD.
      Helicobacter pylori infection and gastric lymphoma. N Engl J Med 1994, 330: 1267-1271.
7.    Parsonnet J, Friedman GD, Vandersteen MS, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter
      pylori infection and the risk of carcinoma. N Engl J Med 1991, 325:1127-1131.
8.    Strauss RM, Wang TC, Kelsay PB, Compton CC, Ferraro M-J, Perez-Perez G, Parsonnet J, Blaser J. Association
      of Helicobacter pylori infection with dyspeptic symptoms in patients undergoing gastroduodenoscopy. Am J Med
      1990, 89:646-469.
9.    Newell DG, Rathbone BJ. The serodiagnosis of Campilobacter pylori infection. Review article. Serodiagn
      Immunother Inf Dis 1989, 3: 1-6.




RE58041_IFU_en_Helicobacter pylori
IgG_quantitative_3410_V09_042007.doc; April 2007
                                                                                                                  - 11 -
16. QUICK REFERENCE PROTOCOL

                     QUICK REFERENCE PROTOCOL FOR H.pylori IgG EIA*
          PREPARATION OF REAGENTS                                   TEST PROCEDURE

    A. Dilute patient test serum
       mix 1.0 mL dilution buffer (BLUE) +
       10 µL patient test serum.

                                                      1. Dispense 100 µL per well of each calibrator in
                                                         duplicate: 300 AU/mL (RED), 75 AU/mL
                                                         (ROSE), 15 AU/mL (YELLOW) and 0 AU/mL
                                                         (GREEN), diluted patient test sera (BLUE), and
                                                         incubate 60 ± 5 minutes at 37 ± 1ºC in a
                                                         resealable bag or in a 100% moist chamber.

    B. Prepare diluted conjugate:
       mix per 8-well strip: 1.0 mL dilution buffer
       (BLUE) +10 µL anti-IgG-PO conjugate
       (100x).


    C. Prepare wash buffer: mix per 8-well strip
       28.5 mL distilled water + 1.5 mL wash buffer
       (20x).

                                                      2. Wash 5 times, dispense per well 100 µL
                                                         working strength conjugate (BLUE) and
                                                         incubate 60 ± 5 minutes at 37 ± 1ºC in a
                                                         resealable bag or in a 100% moist chamber.


                                                      3. Wash 5 times, dispense per well 100 µL TMB
                                                         substrate solution and incubate in the dark 30
                                                         ± 2 minutes at room temperature.


                                                      4. Add per well 100 µL stop solution and read
                                                         absorbance at 450 nm (optionally with 620 nm
                                                         reference).




          Read the entire protocol before starting the assay




INSTRUCTIONS FOR USE: IFU3410-V09, 2 April 2007

                                                                       RE58041_IFU_en_Helicobacter pylori
                                                         IgG_quantitative_3410_V09_042007.doc; April 2007
 - 12 -
         Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύµβολα


   REF         Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–Cat.: / Αριθµός-Κατ.:
    LOT        Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθµός -Παραγωγή:
               Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: /
               Χρησιµοποιείται από:
               No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: /
               Αριθµός εξετάσεων:
  CONC         Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συµπύκνωµα
   LYO         Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασµένο
               In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In
    IVD        Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In
               Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ∆ιάγνωση.
               Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos
               para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης.
               Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. /
               Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni
               prima dell’uso. / ∆ιαβάστε τις οδηγίες πριν την χρήση.
               Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. /
               Garder à l’abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la
               luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να
               φυλάσσεται µακριά από θερµότητα και άµεση επαφή µε το φως του ηλίου.
               Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση
               στους:
               Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός:
               Caution! / Vorsicht! / Attention! / ¡Precaución! / Cuidado! / Attenzione! / Προσοχή!
                          Symbols of the kit components see MATERIALS SUPPLIED.
            Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben.
                       Voir MATERIEL FOURNI pour les symbôles des composants du kit.
           Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS.
                     Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS.
                       Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT.
               Για τα σύµβολα των συστατικών του κιτ συµβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ.




                                               IBL AFFILIATES WORLDWIDE
                                                                                          Tel.:          + 49 (0) 40 532891 -0 Fax: -11
           IBL International GmbH
                                                                                          E-MAIL:        IBL@IBL-International.com
           Flughafenstr. 52A, D-22335 Hamburg, Germany
                                                                                          WEB:           http://www.IBL-International.com
                                                                                          Tel.:          + 31 570-66 15 15 Fax: -60 73 86
           IBL Deventer B.V.
                                                                                          E-MAIL:        IBL@IBL-International.com
           Zutphenseweg 55, NL-7418 AH Deventer, The Netherlands
                                                                                          WEB:           http://www.IBL-International.com
                                                                                          Toll free:     +1 (866) 645 -6755
           IBL - Transatlantic Corp.                                                      Tel.:          +1 (416) 645 -1703 Fax: -1704
           288 Wildcat Road, Toronto, Ontario M3J 2N5                                     E-MAIL:        IBL@IBL-Transatlantic.com
                                                                                          WEB:           http://www.IBL-Transatlantic.com
LIABILITY: Complaints will only be accepted in written and if all details of the test performance and results are included (complaint form available
from IBL or supplier). Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results.
These cases invalidate any claim for replacement. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the
test kit. Any damage caused to the kit during transportation is not subject to the liability of the manufacturer.

Symbols Version 3.5 / 2008-10-01

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:20
posted:7/28/2011
language:English
pages:12