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Hp culture for collecting multiple strains and antigen preparation according to the mentioned protocols in culture section, identity confirmation of grown bacteria by gram staining, urease, catalase and motility test.
2.2 Antigen preparation
Phosphate Buffered Saline (PBS, pH 7.2): Weight 8 g NaCl, 0.2 g KCl, 1.1 g Na2HPO4, 0.2 g KH2PO4 and transfer to an appropriate container. Add deionized water to a volume of 800 mL. Mix and adjust the pH with HCl. Make up to 1 L with water and store at 4°C (See Note 1 for adding NaN3).
Protease inhibitor solution: A solution of 1 tablet complete one, Mini (Roche, Germany), dissolved in 10 mL solution (see Note 2 For application of protease inhibitor)
0.01% Sodium azide solution (NaN3)
Disposal items (Maxisorb ELISA plates; 0.8µm, 0.45µm and 0.2µm syringe-end filters)
Equipments (Pepetman, sonicator, centrifuge, spectrophotometer, -20°C freezer)
All reagents should be removed from refrigerator and allowed to come to room temperature before use. Return all reagents to the refrigerator immediately after use.
Coating buffer (pH 9.6): Weight 1.59 g Na2CO3, 2.93 g NaHCO3 and transfer to an appropriate container. Add deionized water to a volume of 800 mL. Mix and adjust the pH. Make up to 1 L with dH2O and store at 4°C (See Note 1 for adding NaN3).
Blocking buffer: 1% (w/v) bovine serum albumin (BSA) in sterile PBS.
Washing buffer: 0.1% Tween 20 in sterile PBS.
Primary and secondary antibody diluent buffer (pH 7.2): Sterile PBS supplemented with 1% BSA and 0.1% Tween 20. Mix gently till BSA powder particles become resolved in the buffer (See Note 3 for Tween 20 and Note 4 for resolving BSA).
Standard sera: Standard A: Negative control; Standard B: Cut off control equal to 10U/mL anti Hp specific IgG; Standard C: Weakly positive control equal to 25U/mL anti Hp specific IgG; Standard D: Strongly positive control equal to 150U/mL anti Hp specific IgG.
Dilute serum samples in a final 1:100 dilution rate (e.g. 2 µl + 200 µl) in the diluent buffer mentioned above.
Diluted secondary antibody: Polyclonal Rabbit Anti Human IgG conjugated with Horse Radish Peroxidase (HRP) (Dako, Denmark) diluted 1:10,000 in antibody diluent buffer mentioned above.
Reagents (Tween 20, ready to use TMB solution)
Stopping solution (2M H2SO4)
Equipments (Microtiter plate reader capable of reading absorbance at 450nm, multichannel pipetman, 37°C incubator)
3.1 Hp culture for collecting multiple strains and antigen preparation (according to the mentioned protocols in culture section, identity confirmation of grown bacteria by gram staining, urease, catalase and motility test)
3.2 Antigen preparation
Harvest Hp colonies from the surface of 3-5 days incubated agar plates with sufficient volume of sterile PBS (pH 7.2) after identity confirmation of Hp.
Centrifuge the bacterial suspension at 2,300 rcf at 4°C for 10 minutes. Remove the supernatant and resuspend the pellet in the same volume of PBS and repeat the centrifugation procedure.
Resuspend the pellet in sterile PBS at a final concentration of 0.5 g/mL.
Sonicate the bacterial suspension at 20,000 Hz on ice 5 x 45 sec with 45 sec intervals.
Centrifuge at 7,200 rcf for 1 hour. Collect the supernatant (See note 4)
Filter the supernatant through 0.8, 0.45 and 0.22 µm filters.
Measure the protein concentration at 260 and 280 nm by spectrophotometer.
Add protease inhibitor solution to the prepared antigen and aliquot it into small volumes. Store aliquoted samples at -20°C for further use.
Place desire number of strips into an ELISA microwell frame.
Dilute antigen solution to a final concentration of 5 μg/mL, in the coating buffer (carbonate-bicarbonate buffer), add 100μl to each well. Incubate the plate at 4°C over night.
Wash coated wells with sterile PBS three times (300 μl/ well) (See Note 5).
Block coated wells with 100 μl blocking buffer for 1 hour at room temperature.
Remove the blocking solution and add 100 μl diluted sera to blocked wells and incubate at RT for 1 hour.
Remove serum samples and wash the wells with 300 μl washing buffer (5times).
Add 100 μl diluted secondary antibody to each well and incubate at RT for 1 hour.
Repeat washing procedure as described in step 5.
Add 100 μl ready to use substrate solution (TMB) to each well and keep the plate in dark for 10 minutes (See Note 6).
Add 100 μl stop solution (2M H2SO4) to each well in the same order of substrate addition and mix well the contents of each well (See Note 6).
Read the plate at 450 nm by an ELISA reader. If a dual filter is used, set the second filter to 630 nm.
Dispose the plate after reading.
Calculate the estimated IgG concentration of each serum sample based on standards. The cut off value is given by the optical density measured for Standard B sample. Samples in the range of 20% around the cut off value have to be considered as borderline.
For quantitative evaluation of results, draw the standard curve using measured optical densities of standard serum samples (on y axis, linear) against their IgG international units (on x axis, logarithmic) on a semi-logarithmic graph paper. The concentration of anti Hp IgG in each sample can be observed from the standard curve.
Sodium azide (NaN3) serves as a preservative. In case of contact with eyes or skin, wash immediately with water.
Protease inhibitor cocktail is used for the inhibition of serine, cysteine and metalloproteases in bacterial, mammalian, yeast and plant cell extracts. One tablet is enough for the inhibition of the proteolytic activity in 10 mL extraction solution.
Tween 20 is a non ionic detergent which its action is based on its binding to water insoluble components rendering them hydrophilic.
Avoid harsh shaking to prevent BSA proteolysis.
It is important to prevent pellet material since it will block the filters.
Insufficient washing will result in assay variations affecting the validity of test. Thus, the perfect volume of washing buffer should be delivered to each well is 250-300 µl. Remove wash buffer completely after the last wash. Make sure that no bubbles remain in the wells.
The colorless substrate solution becomes blue during incubation in dark while after adding the stop solution it turns to yellow.