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1. Genarrayt Microarray 200 Food IgG 16pt G E N E S I S Quantitative assay for investigation of lgG mediated food sensitivity Product code GD201 4 16 patients For in vitro diagnostic use CE 266 201pt16 01 1 Intended use The Genarrayt Microarray 200 Food IgG kit is a rapid colorimetric microarray based ELISA for the measurement of IgG antibodies to 221 foods in human whole blood serum or plasma 2 Explanation of the Test Many people exhibit chronic food sensitivity reactions to specific food antigens Unlike the immediate effects of IgE mediated allergy IgG mediated food sensitivity reactions may take several days to appear Controlled removal of the problem foods from the patient s diet will in many cases rapidly improve the patient s condition General lethargy weight gain dermatitis arthritis and tiredness are associated with food allergies Irritable bowel syndrome may also be linked to food sensitivity 3 Principle of the test 221 food extracts have been microarrayed onto nitrocellulose pads on a glass microscope slide Food extracts are incubated with either diluted patient whole blood serum or plasma in sample diluent After washing away unbound proteins anti human IgG conjugated to horseradish peroxidase is added to the pads and this binds to food extract bound antibodies in the primary incubation Unbound conjugate is removed by washing and a solution containing 3 3 5 5 tetramethylb2. 6 Precautions 6 1 Safety Precautions 1 Only experienced laboratory personnel should use this test The test protocol must be followed strictly 2 Human lgG used in the preparation of the Standard and the Positive Control for this product has been tested and found negative for antibodies to HIV HBsAg and HCV No test method however can offer complete assurance that infectious agents are absent Therefore all reagents containing human material should be handled as if potentially infectious Operators should wear powder free examination gloves and protective clothing when handling any patient sera or serum based products and throughout the assay procedure 3 The Sample Diluent contains 0 09 sodium azide Avoid contact with the skin and eyes Rinse immediately with plenty of water if any contact occurs Flush sinks with copious amounts of water after discarding these reagents 4 Diluted samples and any liquid that has been brought into contact with potentially infectious material has to be discarded in a container with a disinfectant Disposal must be performed in accordance with local legislation 5 The slide frame holder should be disinfected after use by immersing it in ethanol or iso propyl alcohol for no longer than ten minutes 6 2 Technical Precautions 1 The microarrays should not be used if the foil bag is damaged and solutions should not be used if liquids have leaked Allow all reagents and the microarrays to reach ro
3. enzidine TMB and enzyme substrate is added to trace specific antibody binding After washing with distilled water the slides are dried by centrifugation prior to scanning The optical densities of the standards positive and negative controls and samples are measured using a high resolution flat bed scanner with associated software 4 Materials included in the kit e 4x 4 Pad Food microarrays on modified glass slides in a slide carrier sealed in a foil bag with desiccant Each microarray is comprised of 221 food extracts e Sample Diluent 10mM Tris buffered saline pH 7 2 with 0 09 sodium azide and proteins 10ml ready to use blue cap e Wash Buffer 10mM Tris buffered saline pH 7 2 with detergent 100ml ready to use e Conjugate Goat anti human IgG conjugated to a horseradish peroxidase 10ml ready to use red cap e TMB Substrate aqueous solution of TMB and mild oxidising agent 10ml ready to use e 1x 96 well microtitre plate e Instructions for Use e Genarrayt Reporting Software 5 Other equipment required e 8 channel pipette and tips to deliver 100uI and 120ul Single channel pipettes and tips to deliver 5ul to 250ul e distilled or deionised water e powder free examination gloves e 4 reagent reservoirs slide frame holder e slide centrifuge wash station high resolution flat bed scanner with associated computer and spot finding software e high speed 14 000 x g centrifuge Ethanol or iso propyl alcohol
4. lasma samples should be gently thawed if necessary mixed well DO NOT VORTEX and centrifuged at 14 000g for 10 minutes If the sample is not used immediately repeat the mix and centrifugation step prior to testing Dilute samples 1 49 in sample diluent by adding 5ul serum plasma to 245ul Sample Diluent For whole blood dilute samples 1 24 by adding 10ul whole blood to 240ul Sample Diluent Samples can be diluted directly into the 96 well microtitre plate or into microtubes and then transferred to the 96 well microtitre plate if required When transferring samples to the microtitre plate record the well position for each sample as this will dictate the position of the sample on the microarrayed slides For example well A1 corresponds to Slide 1 Pad 1 Pad 4 10 Assay Procedure 1 Load slides into the slide frame holder with the membrane side facing up The X or dot on the slide should be on the bottom left 2 Using a single or 8 channel pipette add 100ul of diluted patient sample in Sample Diluent to the pad Agitate cover and incubate for 30 minutes 3 Vigorously flick out the slide contents add 120ul of wash buffer and agitate by tapping the side of the frame Repeat step 3 once more 4 Vigorously flick out the slide contents add 120ul of wash buffer and agitate by tapping the side of the frame Cover and incubate for 5 minutes Repeat step 4 once more Flick off the wash buffer Do not allow the slide
5. om temperature before use Do not touch the microarrays Strictly observe the indicated incubation times Ensure that no cross contamination occurs between pads Never pour unused reagents back into the original bottles Do not allow the microarrays to dry between incubation steps Strictly follow the described wash procedure Insufficient washing may cause high background signal 9 Avoid direct sunlight and exposure to heat sources during all incubation steps 10 The use of lint free wipes is essential in reducing potential dust contamination of the arrays 11 Replace colour coded caps on their correct vials to avoid cross contamination 12 Wear powder free nitrile gloves when handling the microarrayed slides and when handling patient samples N eo 7 Shelf life and storage conditions On arrival store the kit at 2 8 C Do not use kits beyond their expiry date Do not freeze any kit component Once opened use the kit within 3 months 8 Specimen collection and storage Serum plasma or whole blood samples may be used Serum and plasma should be stored at 20 C for long term storage Repeated freezing and thawing can affect results Addition of preservatives to any sample type may adversely affect the results Microbially contaminated heat treated or specimens containing particulate matter should not be used Grossly haemolysed icteric or lipaemic specimens should be avoided 9 Preparation of samples Serum or p
6. ry Genesis units Response Negative lt 24 24 30 These are suggested ranges based on in house studies at Genesis Diagnostics Ltd Users of the kit should verify these ranges in their own laboratory under local conditions and adjust as required 13 Limitations of the Procedure 1 Results must always be correlated to the clinical condition of the patient since a raised food IgG level need not manifest as specific symptoms 2 It should be noted that results from this kit give no information about IgE mediated allergy 14 Assay characteristics Within assay imprecision lt 20 Between assay imprecision lt 22 15 221 Food IgG Food Antigen Layout See the food reporting software provided with the kit Method Summary Pipette diluted sample onto the microarray Cover and incubate for 30 minutes at room temperature Wash the microarrays twice with120ul wash buffer Flick off buffer and add 120ul wash buffer and incubate for 5 minutes Repeat once Flick off Dispense 100ul of conjugate onto each microarray Incubate at room temperature for 30 minutes Wash the microarrays twice with120ul wash buffer Flick off buffer and add 120ul wash buffer and incubate for 5 minutes Repeat once Flick off Incubate with 100ul TMB substrate for 10 minutes Load slides into a wash station containing water and incubate for 2 minutes Centrifuge the slides in a centrifuge for 30 seconds Remove from centrifuge and leave for 30 minute
7. s before scanning Scan the microarray using a high resolution flat bed scanner and apply associated spot finding software Process the data using the Genarrayt reporting software following the user manual provided Further reading Atkinson et al IgG antibodies in IBS Gut 2004 53 1459 1464 James M Toward an understanding of allergy and in vitro testing Nat Med Journal 1999 2 4 7 15 Gaby AR The role of hidden food allergy intolerance in chronic disease Alt Med Review 1998 3 2 90 100 Hofman T IgE and IgG antibodies in children with food allergy Rocz Akad Med Bialmyst 1995 40 3 468 473 Sampson HA Metcalfe DD Food allergies JAMA 1992 268 20 2840 2844 El Rafei A et al Diagnostic value of IgG4 measurement in patients with food allergy Ann Allergy 1989 62 94 99
8. to dry 5 Using a single or 8 channel pipette add 100ul of conjugate to each pad Agitate cover and incubate for 30 minutes 6 Vigorously flick out the slide contents add 120ul of wash buffer and agitate by tapping the side of the frame Repeat step 6 once more 7 Vigorously flick out the slide contents add 120ul of wash buffer and agitate by tapping the side of the frame Cover and incubate for 5 minutes Repeat step 7 once more Flick off the wash buffer Do not allow the slide to dry 8 Using a single or 8 channel pipette add 100ul of TMB substrate to each pad Agitate cover and incubate for 10 minutes 9 Carefully flick off the slide contents and gently remove slides from the slide frame holder and carefully place into the wash station containing 400ml distilled de ionised water for 2 minutes DO NOT AGITATE 10 Carefully remove slides and centrifuge in the slide centrifuge for 30 seconds Remove from centrifuge and leave for at least 30 minutes before scanning gt 30 minutes if gt 60 RH 11 Scan using the high resolution flat bed scanner 12 Process the data using the Genarrayt reporting software following the user manual provided 11 Quality control The microarrays include positive and negative controls which are intended to monitor for substantial reagent failure 12 Interpretation of Results Results are derived from internal IgG standards included in the array Range U ml Units are arbitra
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