1.     Antigen strips      Avoid unnecessary exposure of antigen strips to light. 2.     Reagents      Store all reagents at 2-8°C.      For best results, dispense all reagents while cold and return to 2-8°C storage as soon as      possible. CAUTION : Avoid unnecessary exposure of substrate to light.

 

Rocker platform Pipettors and tips Aspirator with sodium hypochlorite trap 56°C water bath (optional)

Patients’ sera can be inactivated but this is not a requirement for optimal test performance. Inactivate as follows: Loosen caps of serum containers. Heat sera to 56°C for 30 minutes in water bath. Allow sera to cool before retightening caps. Sera can be stored frozen until analysis. We recommend that the patients' sera should not undergo repeated freeze-thaw cycles prior to testing.

1.     DILUTED WASH BUFFER a)    Dilute 1 volume of WASH BUFFER CONCENTRATE (20X) with 19 volumes of reagent grade         water. Mix well. 2.     BLOTTING BUFFER a)    BLOTTING BUFFER should be prepared fresh prior to use. b)     Dilute 1 volume of STOCK BUFFER CONCENTRATE (10X) with 9 volumes of reagent grade         water. Mix well. c)     Add 1g of BLOTTING POWDER to every 20 ml of DILUTED STOCK BUFFER prepared in step         2(b) above. Mix well. 2.     WORKING CONJUGATE SOLUTION a)    Prepare WORKING CONJUGATE SOLUTION by diluting CONJUGATE 1:1000 in BLOTTING         BUFFER, for example, 10 ul Conjugate to 10 ml blotting buffer. b)     WORKING CONJUGATE SOLUTION should be prepared fresh prior to use. 3.     SUBSTRATE SOLUTION (Ready to use) a)    Dispense directly the required volumes from the bottle. Use a clean pipette. Cap         tightly after use.

NOTE:      Aspirate all used chemicals and reagents into a trap containing sodium                 hypochlorite. Using forceps, carefully remove the required number of STRIPS from the tube and place numbered side up into each well. Include strips for one reactive and one non-reactive control. Add 2 ml of DILUTED WASH BUFFER to each well. Incubate the strips for at least 5 minutes at room temperature (25±3°C) on a rocking platform. Remove buffer by aspirations. Add 2 ml of BLOTTING BUFFER to each well followed by 20 ul each of patients’ sera or controls to appropriate wells. Cover the tray with the cover provided and incubate for 1 hour at room temperature (25±3°C) on a rocking platform. Carefully uncover the tray to avoid splashing or mixing of samples. Aspirate the mixtures from the wells. Change aspirator tips between the samples to avoid cross contamination. Wash each strip 3 times with 2 ml of DILUTED WASH BUFFER, allowing 5 minutes soak on the rocking platform between each wash. Add 2 ml of WORKING CONJUGATE SOLUTION to each well. Cover tray and incubate for 1 hour at room temperature (25±3°C) on a rocking platform. Remove Conjugate from the wells. Wash as in step 7. Add 2 ml of SUBSTRATE SOLUTION to each well. Cover tray and incubate for 15 minutes on the rocking platform. Remove the substrate and rinse the strips several times with reagent grade water to stop the reaction. Using forceps, gently remove strips onto the paper towels. Cover with paper towels and dry. Mount strips on worksheet (non-absorbent white paper) for visual reading or on appropriate template for automated reading (i.e. AutoScan). For storage, keep the strips in the dark. Do not apply adhesive tape over the developed bands.

The Non-Reactive and Reactive controls must be run with every assay. In order for the results obtained from any assay to be considered valid, the following conditions must be met. 1.     NON-REACTIVE CONTROL The non-reactive control must not react with any proteins used in the criteria for interpretation. There may be bands or a broad band appearing in the 60kD region, but reactivity with proteins in this region alone is not specific for H.pylori (see Figure 1). 2.     REACTIVE CONTROL All relevant molecular weight bands must be evident. Figure 1 provides a guide to the relative positioning of bands visualized on the Genelabs Diagnostics HELICO BLOT 2.1. The bands are 116kD(CagA), 89kD(VacA), 37kD, 35kD, 30kD(UreA) and 19.5kD. The current infection marker band (below the serum control band) must be evident. 3.      The serum addition control band must be present on all strips. This band serves as an internal          control for sample and reagent additions.

Record sample's identity number, and other appropriate information onto the provided report sheet. A NON-REACTIVE control and a REACTIVE control must be run with each assay. The serum control band serves as a check for serum and reagent (conjugate and substrate) additions in the assay. Absence of this band on a strip would indicate that no test serum or conjugate or substrate has been added, or other operational errors. EACH strip is compared to the strips used with the NON-REACTIVE and the REACTIVE control for the assay. Use the REACTIVE control strip to identify bands on the patients' strips. Record the appropriate bands on the patients’ strips onto the provided report sheet. RECOMMENDED CRITERIA: The recommended criteria for Genelabs HELICO BLOT 2.1 has been designed such that all of the bands which are used have high specificity and are easy to locate within the pattern. The recommended criteria for determining a sample as H. pylori seropositive is any one of the following conditions: 1.     116kD (CagA) positive, where CagA has to be present with one or more of the following bands:         89kD (VacA), 37kD, 35kD, 30kD (UreA) and 19.5kD together, OR with current infection         marker. 2.     Presence of any one band at 89kD, 37kD or 35kd, with or without current infection marker. 3.    Presence of both 30kD and 19.5kD with or without current infection marker. In general, individuals which have infections with H. pylori would have reactivity to several of these and other bacterial proteins on the blot. In addition, individuals who have current infections with H. pylori will most likely have reactivity to the current infection marker band. In several populations tested, the positive predictive value* of the current infection marker band compared against urea breath test (UBT) or other invasive tests (histology, culture, urease test) is 85-94%. The high predictive value for the current infection marker therefore serves as a quick reference for the current infection status. Samples which meet the above criteria for positive should be reported as POSITIVE. Samples which have no reactivity to any bands except serum control band or reactivity which does not meet the criteria for positive should be reported as NEGATIVE. Positive predictive value = TP / (TP+FP) where TP=number of true positives, FP=number of false positives.

Optimal assay performance requires strict adherence to the recommended assay procedure described. Deviation from the recommended procedure may lead to aberrant results. A NEGATIVE result does not exclude the possibility of exposure to or infection with H. pylori. A NEGATIVE result for the current infection marker does not exclude the possibility that one is currently infected due to variations in the prevalence of antibody response to the current infection marker in different geographical populations. There is much heterogeneity among various isolates of H. pylori for levels of protein expression of the various antigens, and for sequence homology as well. The antibody response of various individuals can be quite diverse. There is very good sensitivity and specificity for the 7 bacterial proteins used in the criteria for interpretation with infection, regardless of clinical status and the strain of H. pylori which has been used in the blot is useful for diagnosis of cases worldwide.

Patients were tested using the HELICO BLOT 2.1 test kit. Results were compared against gold standard tests for active infection (histology, culture, rapid urease test or urea breath test [UBT]). For gold standard positives to be considered positive, at least 2 out of the 3 tests for the above have to be positive. For gold standard negatives, all of the performed test, if performed, should be negative. Combined total number of positive subjects = 48 Combined total number of negative subjects = 56 Compared against histology, culture, rapid urease test and/or urea breath test(UBT): Sensitivity = 96% Specificity = 95% Positive predictive value for current infection marker: Compared against gold standard active infection tests = 94% Compared against UBT only = 91%

Should there be a technical inquiry, please do the following: 1.     Note the kit lot number, strip lot number and their expiry dates. 2.     Retain the kit and the results that were obtained. 3.     Contact the nearest Genelabs office or your local distributor.

The manufacturer makes no warranty other than that the test kit will function as a research use only assay within the specifications and limitations described in the Product Instruction Manual when used in accordance with the instructions contained therein. The manufacturer disclaims any warranty, expressed or implied, including such expressed or implied warranty with respect to merchantability, fitness for use or implied utility for any purpose. The manufacturer is limited to either replacement of the product or refund of the purchase price of the product. The manufacturer shall not be liable to the purchaser or third parties for any damage, injury or economic loss howsoever caused by the product in the use or in the application thereof.