Prolisa™ C. difficile GDH EIA Kit Instructions for Use - pro
Contents
1. 11 Do not reuse microwells 12 Exposing unused microwells to air for extended periods of time may com promise test results It is important to protect strips from moisture during storage by replacing unused stripwells in the provided pouch 13 Do not use a transfer pipette for more than one specimen 14 When dispensing samples into a microwell avoid splashing by placing the transfer pipette tip about halfway into the well and trickling solution slowly down the side of well 15 Stripwells should be washed precisely as directed in the assay procedure Inadequate washing can elevate background readings and lead to false positive results 16 Any deviation from set incubation times can affect test performance All parameters for this test have been optimized and any deviation from test protocol could affect results 17 Product contains material of animal origin and should be handled as a potential carrier and transmitter of disease 18 Only faecal specimens without added preservatives may be used in the test 19 Avoid scratching the microwells when handling as scratches could affect absorbance readings 20 It has been observed that strong positive samples may contaminate adja cent microwells leading to false positive test results It is recommended that weakly positive samples be retested if they occur immediately adjacent to strongly positive samples E a aE OO N wp REAGENT PREPARATION 1 Prepare 1X Wash Buffer from 22. 2 Wilkins T D and Lyerly D M 2003 Clostridium difficile Testing after 20 Years Still Challenging J Clin Microbiol 41 531 534 3 Eastwood K et al 2009 Comparison of nine commercially available Clostridium difficile toxin detection assays a real time PCR assay for C difficile tcdB and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods J Clin Microbiol 47 3211 3217 4 Health Protection Agency 2014 Processing of faeces for Clostridium difficile UK Standards for Microbiology Investigations B 10 https www gov uk gov ernment publications smi b 10 processing of faeces for clostridium difficile VY IVD y Bn Use by Lot number Catalogue number Manufacturer Authorized Representative in the European Community Contains sufficient for lt n gt tests In vitro diagnostic medical device Temperature limitation Consult instructions for use Revision 2015 01
3. infection is cytotoxigenic culture a test in which C difficile isolates from selective differential agar are enriched in broth and then tested for elaboration of Toxin B by cytotoxicity assay on cultured cells 2 Rapid immunoassays have been developed for detection of Toxin A and or Toxin B in faecal specimens however these tests lack sensitivity 3 Immunoassays for GDH a protein shared by toxigenic and non toxigenic C difficile have been developed and incorporated into algorithms for identification of toxigenic C difficile It has been shown that toxigenic C difficile can be more efficiently and economically identified by first testing for GDH and then Toxin A and or Toxin B rather than by testing for the toxins alone 3 PRINCIPLE OF THE TEST The Prolisa C difficile GDH EIA is a sandwich immunoassay that uses specific antibodies that recognize C difficile GDH The stripwells contain immobilized mouse monoclonal antibody and the immunoconjugate contains rabbit poly clonal antibodies conjugated to horseradish peroxidase To perform the test a portion of a faecal specimen is first thoroughly suspended in diluent to create a sample suitable for testing A portion of this sample and the immunoconjugate are then incubated simultaneously in a well containing immobilized monoclo nal antibody If GDH is present in the sample an insoluble antibody enzyme complex that cannot be easily washed from the wells is formed After the wells are
4. methods Table 1 summarizes the number of subjects and faecal C difficile prevalence in the study A total of 985 specimens were tested Table 1 Distribution of Samples by Site Faecal Specimens on C difficile Culture Positive Study Site Table 2 shows the sensitivity specificity and percent agreement values of the Prolisa C difficile GDH EIA relative to recovery of C difficile from faecal speci mens by culture Table 2 Performance of the Prolisa C difficile GDH EIA Relative to Culture Sites Combined a e f re f o 92 8 87 1 96 5 Relative specificity 91 3 89 2 93 1 Positive percent agreement 63 4 56 3 70 0 Negative percent agreement 98 7 97 7 99 4 95 confidence interval Prolisa C difficile GDH EIA Result Relative sensitivity Interfering Substances Substances sometimes found in faeces of patients with diarrhoea including common intestinal medications barium sulfate and blood were not reactive and did not interfere with detection of GDH in the Prolisa C difficile GDH EIA A ssay Specificity Forty five different enteric micro organisms comprising 42 bacterial strains and 3 viruses were tested both as pure samples and spiked into stool to determine their reactivity with Prolisa All bacteria were tested at gt 1 x 108 cfu ml viruses at 1 x 106 pfu ml in culture media spiked into negative stool samples and spiked into negative stool sampl
5. nm lt 0 100 Positive OD 450 630 nm 0 100 A positive result indicates the presence of GDH in the sample A negative result indicates the absence of GDH in the sample or a level of GDH below the level that can be detected by the test Samples containing high amounts of GDH can produce a visible black pre ci pitate upon addition of Stop Solution The presence of the precipitate will not affect interpretation of results LIMITATIONS OF THE PROCEDURE w P The Prolisa C difficile GDH EIA should not to be used alone to diagnose C difficile associated disease Diagnosis should consider the test results patient clinical history and the results of additional laboratory tests The Prolisa C difficile GDH EIA does not distinguish between toxigenic and non toxigenic C difficile Other tests are needed to confirm the presence of toxigenic C difficile False positive test results can be obtained if plates are not washed ade quately Contact Pro Lab Diagnostics Technical Service for assistance if false positive test results are suspected ERFORMANCE CHARACTERISTICS The clinical evaluation was performed at two external trial sites using faecal specimens submitted for routine testing for the presence of C difficile Samples were tested by the Prolisa C difficile GDH EIA according to the instructions p rovided with the kit Results from the test were compared to the results from C difficile culture
6. washed to remove unbound material the bound enzyme is detected through the use of a chromogenic substrate j Canada 800 268 2341 Fax 905 731 0206 20 Mural Street Unit 4 Richmond Hill ON L4B 1K3 Prolisa C difficile GDH EIA for in vitro diagnostic use MATERIALS PROVIDED Coated and PL 2115 1 Mouse monoclonal Stabilized Plate plate antibody to GDH pouch coated onto strip wells PL 2113 2x A protein free 30 ml solution with preservative PL 2112 1x Recombinant GDH 2 5ml ina buffered pro tein solution with preservative P Immunoconjugate L 2114 1x Rabbit polyclonal 7 ml anti GDH antibody conjugated to horseradish per oxidase Each pouch contains 1 plate with a sealer and 2 desiccants White bottle Dropper bottle with blue cap Sample Diluent Positive Control Dropper bottle with red cap 20X Wash Buffer PL 2110 2x Concentrated White bottle 25ml buffer containing detergent and 0 1 thimerosal w v Substrate Solution PL 2104 x 3 3 5 5 tetrameth Amber bottle 4ml ylbenzidine ina mildly acidic buffer Stop Solution PL 2103 x 0 2 N sulfuric acid Dropper bottle 4ml with yellow cap Plate Sealer Transfer pipette Instructions for N A Use MATERIALS REQUIRED BUT NOT PROVIDED Wooden applicator sticks or loop Timer Pipette capable of delivering 50 ul to 1000 ul Pipette tips Test tubes 12 X 75 mm or other suitable size for sample dilution Distilled or deion
7. 0X Wash Buffer by mixing the supplied con centrate with 950 ml of distilled or deionized water 2 Bring the entire kit including plate pouch to room temperature before use SPECIMEN STORAGE Faecal specimens tested within 2 hours of collection do not require refrig eration Specimens not tested within 2 hours of collection should be stored at 2 8 C and tested within 24 to 48 hours of collection if possible If specimens cannot be tested within 48 hours of collection they should be stored frozen at U K 0151 353 1613 Fax 0151 353 1614 3 Bassendale Road Bromborough Wirral Merseyside CH62 3QL EC REP lt 20 C 4 Avoid repeatedly freezing and thawing specimens as this may lead to erroneous test results SAMPLE PREPARATION FOR MANUAL USE Add 300 ul of Sample Diluent to a sample tube Transfer 100 ul of unformed faeces or approximately 20 ul of solid faeces equivalent to a spherical mass with a diameter of approximately 4 mm to the sample tube Vortex the sample tube for 10 seconds to thoroughly emulsify the specimen in Sample Diluent Test sample immediately after preparation If the plate is to be washed with an automated plate washer centrifuge the samples at 5000 x g for 10 minutes or until the particulate matter forms pellets before adding the sample supernatant to the test wells Large par ticulate matter in samples may interfere with automated plate washing TEST PROTOCOL FOR MANUA
8. 2 ODIL sects INTENDED USE The Prolisa C difficile GDH EIA is a microwell assay for the qualitative detection of Clostridium difficile glutamate dehydrogenase GDH in faecal specimens The Prolisa C difficile GDH EIA is intended for use as an aid in the diagnosis of C difficile infections This test detects GDH and will not differentiate between toxigenic and non toxigenic strains of C difficile Like alternative C difficile tests results should be considered in conjunction with patient history and additional laboratory investigations SUMMARY AND EXPLANATION Mechanism of Disease Clostridium difficile is an anaerobic spore forming bacillus that produces two clinically important toxins called Toxin A and Toxin B that act in the gut to produce local tissue damage that can progress to pseudomembranous colitis Toxigenic C difficile can be carried asymptomatically however serious sequelae sometimes follow overgrowth of C difficile resulting from antimicrobial therapy Institutional outbreaks of C difficile associated disease are frequently caused by ingestion of acid resistant spores present in the environment Clostridium difficile strains that do not produce Toxin A and Toxin B are considered non pathogenic 1 Diagnosis of Disease Clostridium difficile associated disease is diagnosed by a combination of clinical and microbiological findings The gold standard for microbiological identifica tion of toxigenic C difficile
9. L USE N ee wn oO N Cut the re sealable foil pouch and carefully remove the assay plate from the pouch Remove the sealing tape from the stripwells Return any extra wells to the pouch re seal the pouch and return it to storage at 2 8 C Add 1 drop 50 ul of the Immunoconjugate to the wells Use a transfer pipette to transfer 100 ul equivalent to the first calibration point of the pipette of diluted specimen to the wells and add 100 ul of Positive Control and 100 ul of Sample Diluent negative control to the appropriate wells Incubate the plate for 60 minutes at room temperature without shaking Alternatively incubate the plate for 20 minutes at room temperature with 1000 rpm shaking Discard the samples controls from the strip s and wash the wells 5 7 times with 1X Wash Buffer Option 1 Discard plate contents in an appropriate biohazard container Strike the inverted plate firmly on a clean stack of paper towels Completely fill all wells with 1X Wash Buffer using a wash bottle Repeat washing cycle discard strike and fill 4 6 additional times After the last refill discard contents and strike the plates firmly on fresh paper towels to remove any excess wash buffer Option 2 Wash plate with an automated plate washer 5 7 times by filling the wells with 300 ul of 1X Wash Buffer Add 100 ul of the Substrate Solution to each well tap the plate holder gen tly and incubate for 10 minut
10. es at room temperature Add 3 drops 100 ul of the Stop Solution to the wells and tap the plate holder gently to ensure that the contents are mixed properly Read the test results within 10 minutes after completion of Step 8 Ensure that bottom of wells are clean and dry Use a lint free towel to wipe the underside of wells when necessary 10 Measure OD450 630 nm in a microplate reader SAMPLE PREPARATION FOR AUTOMATION USE 4 Add 600 ul Sample Diluent to a sample tube provided by an automated EIA system or equivalent tube Transfer 200 ul of unformed faeces or approximately 40 ul of solid faeces to the sample tube Cover the sample vials and vortex the sample tube for 10 seconds to thor oughly emulsify the specimen in the Sample Diluent Centrifuge the samples at least 5000 x g for 10 minutes at room tempera ture Note If 5000 x g in a centrifuge for a specific sample vial is not available a longer 5 centrifugation time should be applied e g 3000 x g for 20 minutes Do not disturb the sample tubes and place the sample tube in an appropri ate position in the automated EIA system TEST PROTOCOL FOR AUTOMATION USE Cut the re sealable foil pouch and carefully remove the assay plate from the pouch Remove the sealing tape from the stripwells Return any extra wells to the pouch re seal the pouch and return it to storage at 2 8 C Place the required strips with the holder in an appropriate p
11. es containing GDH contrived positive sample to evaluate cross reactivity and test interference Table 3 lists the organisms that were tested and were non reactive in the assay Test reactivity was seen with Clostridium sporogenes which is not part of the normal intestinal flora None of the remaining organisms were reactive in the GDH test nor did they interfere with test results Prolisa was also tested for reactivity with 18 C difficile strains The test reacted with all of the tested strains including strains producing Toxin A and Toxin B strains producing Toxin B only and strains producing neither Toxin A or Toxin B Table 3 Non Reactive Microorganisms in Prolisa C difficile GDH EIA Adenovirus serotype 1 Aeromonas hydrophila Arcobacter butzleri Bacillus cereus Bacillus subtilis Campylobacter coli Campylobacter fetus Campylobacter jejuni Citrobacter freundii Citrobacter braakii freundii Clostridium butyricum Clostridium histolyticum Clostridium novyi Clostridium perfringens Clostridium septicum Clostridium sordellii Cocksackievirus Cytomegalovirus Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis van B Enterococcus faecium van A Escherichia coli 0157 H7 Escherichia hermanii Gardnerella vaginalis Klebsiella pneumonia Listeria innocua Listeria monocytogenes Peptostreptococcus anaerobius Prevotella melaninogenica Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimu
12. ized water Wash bottle or a plate washer or an automated EIA system Graduated cylinder EIA plate reader with 450 630 nm absorbance reading capability or an auto mated EIA system 10 Vortex Mixer 11 Centrifuge 0000 OY SOI OO ND U S A 800 522 7740 Fax 800 332 0450 21 Cypress Blvd Suite 1070 Round Rock Texas 78665 1034 PRODUCT CODE PL 2002 Z 96 STABILITY AND STORAGE The expiration date is indicated on the kit label Store the kit at 2 8 C 20X wash buffer may be stored at room temperature Return the kit promptly to 2 8 C storage after each use 1X Wash Buffer may be stored at room temperature for up to 1 month PRECAUTIONS For in vitro diagnostic use only Specimens may contain infectious agents and should be handled at Biosafety Level 2 All reagents should be mixed gently before use Stored wash buffer may separate into layers Shake well before use Do not interchange reagents from different kit lot numbers The substrate is sensitive to light do not expose it to light Reagent vials should be held vertically at a suitable distance above the well to insure proper drop size and delivery Do not use kit components beyond the expiry date on the label Dispose of used wash buffer and all test materials in a manner appropriate for potentially biohazardous materials 10 Avoid skin contact with Stop Solution it contains 0 2 N sulfuric acid Flush with water immediately if solution contacts skin or eyes
13. nd operation manual of the automated EIA system Conduct a test of the Prolisa C difficile GDH EIA kit in the automated EIA sys tem and analyze the data in the system Note Ifa wavelengh 630 nm filter is not available in the automated EIA system set up dual wavelength at 450 nm 620 nm Itis recommended that the Positive Control and the Negative Control be added in well A1 B1 and C1 D1 in duplicate The mean of OD readings of the Positive Control must be greater than 0 800 and the mean of OD readings of the Negative Control must be less than 0 100 It is recommended that five 5 cycle washings with 300 ul 1X Wash Buffer in each well be included in the running program for an automated EIA system However more washing cycles gt 5 may be required in different automated systems QUALITY CONTROL PROCEDURES The Positive and Negative Controls must be used with each assay run to assure q 1 uality of the reagents and test procedure The Positive Control should read gt 0 800 at 450 630 nm 2 The Negative Control should read lt 0 100 but greater than 0 000 at 450 630 nm If the Negative Control is lt 0 000 re blank the plate reader to air and re read the plate A well that is not visually positive yellow but has yielded a positive result should be wiped on the underside repositioned and reread INTERPRETATION OF THE RESULTS Spectrophotometric Dual Wavelength 450 630nm Negative OD 450 630
14. osition in the automated system Prepare adequate volume of 1X Wash Buffer by diluting 20X Wash Buffer in distilled or deionized water Transfer the 1X Wash Buffer to an appropriate container in the automated system Carefully read the User Manual of the automated EIA system Set up a program for running the Prolisa C difficile GDH EIA in the automated EIA system a ccording to the following policies step 4 11 Contact Pro Lab Diagnostics Technical Service for questions related to the set up of a program in an auto mated EIA system 4 Transfer adequate volumes of the Immunoconjugate the Substrate Solution wn and the Stop Solution to containers provided with the automated system and place them in the appropriate positions in the system Transfer 50 ul of the Immunoconjugate to each well 6 Transfer 100 ul of Positive Control and 100 ul of Sample Diluent negative control to the appropriate wells Transfer 100 ul of diluted specimen to the wells 7 Incubate the plate for 60 minutes at room temperature without shaking 8 Aspirate the samples controls from the strip s and wash the wells 5 times with 1X Wash Buffer 9 Transfer 100 ul of the Substrate Solution to each well and incubate for 10 1 1 minutes at room temperature 0 Transfer 100 ul of the Stop Solution to the wells and shake the plate briefly 1 Measure OD450 630nm in the automated system within 10 minutes after Step 10 Follow the maintenance a
15. rium Serratia liquefaciens Shigella boydii Shigella dysenteriae Shigella flexneri Shigella sonnei Staphylococcus aureus Staphylococcus epidermidis Streptococcus agalactiae Vibrio parahaemolyticus Yersinia enterocolitica z LOT REF EC REP Analytical Sensitivity The detection limit of the Prolisa C difficile GDH EIA for recombinant C difficile GDH is approximately 3 5 ng ml in faecal sample Assay Precision The interassay variability of the Prolisa C difficile GDH EIA was evaluated with a panel of stool samples spiked with different amounts of GDH The panel comprised negative high negative low positive and moderate positive samples Testing was done by two operators at each of three sites on five different days Between run variability of the positive samples ranged from 3 1 12 2 and of negative samples ranged from 20 4 50 6 Intra assay variability was 1 1 29 5 on positive samples and 1 2 39 4 on negative samples Overall agreement between test result and expected sample result with negative samples was 100 Overall agreement between test result and expected sample result with positive samples was 97 The low positive sample was set at the limit of detection and thus it was anticipated that up to 5 could be negative due to assay variability REFERENCES 1 Bartlett J G 1990 Clostridium difficile Clinical Considerations Rev Infect Diseases 12 Supplement 2 243 S251
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