Murex Chlamydia Verification Kit
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1 1F83-01 E C06DK05GB Murex Chlamydia Verification Kit DK05 For the confirmatory testing of clinical samples reactive in Murex Chlamydia (1F82-01) Note Changes Highlighted 2005 Abbott / Printed in the UK / Murex Chlamydia Verification Kit Key to symbols used: 8 C Lot: Store at 2 C to 8 C 2 C Use by For in vitro diagnostic use Consult instructions for use Legal Manufacturer Catalogue number See Reagents section for a full explanation of symbols used in component naming ABBOTT Diagnostics Division 1
2 MUREX CHLAMYDIA VERIFICATION KIT INTENDED USE The Murex Chlamydia Verification Kit is for use in conjunction with Murex Chlamydia (1F82), to confirm the presence of genus-specific chlamydial lipopolysaccharide (LPS) antigen in human urethral and endocervical swabs and male urines. The kit can be used to verify (the specificity of) results of specimens found to be reactive in Murex Chlamydia. SUMMARY AND EXPLANATION OF THE TEST Currently there are two main routine approaches used for the laboratory diagnosis of Chlamydia infections; direct detection of Chlamydia in clinical specimens or isolation of the organism from clinical specimens 1,2. Direct detection of Chlamydia in clinical samples can be performed using either an enzyme immunoassay 3,4,5 (e.g. Murex Chlamydia), or a fluorescent labelled monoclonal antibody 6. Isolation of Chlamydia trachomatis is dependent on the presence of viable organisms in the specimen. This can be affected by transportation and storage of specimens, sensitivity of cell lines and antimicrobial therapy 7,8, whereas antigen detection methods will detect both viable and non-viable organisms 9. In addition, enzyme immunoassays such as Murex Chlamydia will detect the presence of free chlamydial LPS as well as LPS present in elementary bodies. As a result some clinical specimens containing chlamydial LPS antigen will be reactive in enzyme immunoassays, but fail to react in other test systems (e.g. cell culture). Murex Chlamydia Verification Kit utilises a genus-specific murine monoclonal antibody which selectively binds to chlamydial LPS in a reactive specimen, thus reducing the binding of the chlamydial LPS to the capture antibody in Murex Chlamydia. PRINCIPLE OF THE TEST Murex Chlamydia Verification Kit consist of two reagents, a genus-specific murine monoclonal antibody (Verification Reagent) of different origin to that used in Murex Chlamydia and a murine monoclonal antibody with no anti-chlamydial activity (Control Reagent). The selective blocking test is performed on specimens found to be reactive in Murex Chlamydia. Each reactive specimen is re-tested using two wells in the Murex Chlamydia assay. The Verification Reagent is added to one of the test wells and Control Reagent to the other test well prior to the addition of the specimen or Positive and Negative Control. If chlamydial LPS antigen is present in the specimen, the verification antibody, but not the control antibody, will selectively inhibit the binding of chlamydial LPS to the capture antibody on the coated well. On completion of the Murex Chlamydia procedure, a significant reduction in the absorbance value obtained in wells containing the specimen or Positive Control and Verification Reagent, relative to the wells containing the specimen or Positive Control and Control Reagent, verifies the presence of chlamydial LPS antigen. Murex Chlamydia Positive and Negative Controls are included in each test run to quality control the test. REAGENTS DESCRIPTION, PREPARATION FOR USE AND RECOMMENDED STORAGE CONDITIONS WARNINGS AND PRECAUTIONS The reagents are for in vitro diagnostic use only. For professional use only. Please refer to the manufacturer s safety data sheet and the product labelling for information on potentially hazardous components. HEALTH AND SAFETY INFORMATION Verification Reagent and Control Reagent contain % sodium azide which is classified per applicable European Economic Community (EEC) Directives as harmful (Xn). The following are the appropriate Risk (R) and Safety (S) phrases. Xn R22 Harmful if swallowed. R32 Contact with acids liberates very toxic gas. S35 This material and its container must be disposed of in a safe way. S36 Wear suitable protective clothing. S46 If swallowed, seek medical advice immediately and show this container or label. Sodium azide can react with copper and lead, used in some plumbing systems, to form explosive salts. The quantities used in this kit are small, nevertheless when disposing of azide-containing materials they should be flushed away with relatively large quantities of water. Information for European customers: For product not classified as dangerous per European Directive 1999/45/EC - Safety Data Sheet available for professional user on request. 1. Do not pipette by mouth. Wear disposable gloves and eye protection while handling specimens and performing the assay. Wash hands thoroughly when finished. 2. The Health and Safety Information given in Murex Chlamydia (1F82) Instructions for Use also applies when using Murex Chlamydia Verification Kit (1F83). ANALYTICAL PRECAUTIONS 1. The Precautions given in the Murex Chlamydia (1F82) Instructions for Use also apply to Murex Chlamydia Verification Kit (1F83). 2. Do not transpose the caps of the Verification Reagent and Control Reagent as this may cause cross-contamination of the reagents. 3. Ensure all reagents are at room temperature (15-30 C) before use. 4. Use separate disposable pipettes or pipette tips for each sample, control or reagent (if not using dropper bottles) in order to avoid cross contamination of either samples, controls or reagents which could cause erroneous results. 5. Avoid touching or contaminating the tops or rim of wells with specimen or reagents as this may adversly affect the performance of the test. Avoid touching the well contents with the pipette tip when dispensing conjugate as this may lead to crosscontamination. 6. Components must not be used after the expiry date. Do not mix or interchange different batches/lots of reagents. 7. Microwells can not be reused. All components should be stored at 2 to 8 C unless otherwise stated, under which condition they will retain activity until the expiry date of the kit. 1. Verification Reagent One amber bottle of buffer containing 1 ml of murine monoclonal antibody specific for chlamydial LPS antigen. Contains % sodium azide as preservative. 2. Control Reagent One clear bottle of buffer containing 1 ml of murine monoclonal antibody free of Chlamydia specific activity. Contains % sodium azide as preservative. 2
3 SPECIMEN COLLECTION, TRANSPORT AND STORAGE Specimens should be collected, transported and processed as outlined in the Murex Chlamydia Instructions for Use. Murex Chlamydia reactive specimens that require verification should be stored at 2 to 8 C and tested within 5 days. For longer term storage, up to 4 weeks, specimens should be stored at 15 to 25 C. When testing specimens that have been frozen allow to thaw and equilibrate to room temperature (15 to 30 C) then vortex vigorously for a minimum of 1 minute prior to testing. PROCEDURE MATERIALS REQUIRED BUT NOT PROVIDED 1. Murex Chlamydia (1F82) kit. 2. Micropipettes of appropriate volume (25 to 1000 µl). 3. Other materials and equipment as described in the Murex Chlamydia (1F82) Instructions for Use. TEST PROCEDURE Procedural Notes 1. Please read carefully the Test Procedure contained in the Murex Chlamydia (1F82) Instructions for Use before performing the test. 2. Murex Chlamydia Positive and Negative Controls must be tested with each batch of specimens tested. Murex Chlamydia Positive and Negative controls are provided ready to use. DO NOT RE- HEAT. (If required, an additional control which will not verify as positive using the Murex Chlamydia Verification Kit may be used to monitor the performance and specificity of the Verification reagent e.g. an anti-mouse antiserum control. Tests using this control will produce positive absorbance values in both the Control and Verification Reagent wells despite the absence of any Chlamydia antigen. Anti-mouse antibody will form a bridge between capture and conjugate antibodies causing non-specific positive absorbance which will not be blocked by the Chlamydiaspecific Verification Reagent). 3. Vortex samples and controls for a minimum of 1 minute prior to testing. 4. DO NOT re-boil specimens previously boiled and tested in Murex Chlamydia. These specimens should be vortexed vigorously for a minimum of 1 minute immediately prior to testing. 5. Two wells are required for each sample and Positive and Negative Control tested. One well for the Verification Reagent and one for the Control Reagent. 6. Three Negative Controls and one Positive Control should also be included in each batch run of Murex Chlamydia to quality control the Murex Chlamydia reagents. Step 1 Prepare the Wash Fluid. Step 2 Use only the number of wells required for the test. Step 3 Add 25 µl of Control Reagent or Verification 25 µl Reagent into appropriate wells (See Figure 1). Step 4 Add 200 µl of vortexed Murex Chlamydia kit Controls 200 µl and Specimens to the wells. A minimum of three Negative and one Positive Control should be tested. (See Figure 1). Step 5 Add 50 µl (2 drops) of Conjugate to each well. 50 µl Mix the contents of the wells by gently tapping the edge of the plate. Continue from and including Step 5 of the Murex Chlamydia (1F82) procedure. Figure 1 Configuration of Test RESULTS CALCULATION OF RESULTS Each plate must be considered separately when calculating and interpreting results of the assay, regardless of the number of plates processed at the same time. Negative Control Calculate the mean (A 490 ) absorbance of the three Negative Control wells containing Control Reagent. Example: Well 1 = 0.122, Well 2 = 0.155, Well 3 = Total = Mean = 0.420/3 = If one of the Negative Control wells has an A 490 more than 0.05 above or below the mean of all three, discard that value and calculate the new mean A 490 from the remaining two replicates. If two Negative Control absorbance values are unacceptable the test must be repeated. Cut-off Value Calculate the Cut-off value by adding 0.05 to the mean of the Negative Control replicates (see above). Example: Mean Negative Control = Cut-off value = = Percentage Inhibition (PI) Calculate the percentage reduction in absorbance (A 490 ) values between the Control Reagent well and the Verification Reagent well according to the following formula: PI = (A 490 Control Reagent Well A 490 Verification Reagent Well ) x 100 A 490 Control Reagent well where: Control Reagent Well = Well containing Control/Specimen + Control Reagent Verification Reagent Well= Well containing Control/Specimen + Verification Reagent For examples see below. Positive Control Calculate the Percentage Inhibition (PI) of the Positive Control. Example: A 490 in Control Reagent Well = A 490 in Verification Reagent Well = Inhibition by specific antibody = ( ) x PI = 86% 3
4 QUALITY CONTROL Results of an assay are valid if the following criteria for the controls are met: Negative Control All (six) individual Negative Control A 490 values are less than or equal to 0.3. Positive Control The well containing Positive Control and Control Reagent has an A 490 reading greater than 0.5, and the PI (see above for example of calculation) is greater than 40%. Assays which do not meet these criteria must be repeated. In the unlikely event that the results obtained do not meet either the Quality Control criteria or the expected performance of the test, please contact your representative. INTERPRETATION OF RESULTS Positive Result 1. A specimen with a Control Reagent well giving an A 490 that is greater than 0.1 above the Cut-off is verified positive if the PI (as calculated above) is greater than 40%. Example: A 490 Verification Reagent well = A 490 Control Reagent well = PI = ( ) x = 83.4% PI > 40% therefore sample is verified as positive. 2. A specimen with a Control Reagent well giving an A 490 between that of the Cut-off and 0.1 above the Cut-off (ie a weakly reactive specimen), is verified positive if: a) The A490 of the Verification Reagent well is below the Cut-off and b) The PI is greater than 25% Example: A 490 Verification Reagent well = A 490 Control Reagent well = PI = ( ) x = 27.9.% Cut-off = Thus since, A490 of Verification Reagent well is less than the Cut-off and the PI is greater than 25% the sample is verified as positive. Non-specific Result If the absorbance values in the Verification Reagent well and the Control Reagent well for a specimen are both above the Cut-off value and the PI is less than 40% then the result must be considered to be nonspecific. In such cases it is advisable to obtain another specimen from the patient for testing to exclude the possibility of technical or sampling errors. To ensure that the results of some specimens, which are strongly reactive in Murex Chlamydia, when retested with Murex Chlamydia Verification Kit are not misinterpreted as non-specific reactions, further dilutions of such specimens is recommended (see Limitations of the Procedure). Strongly Positive Samples Specimens with a high A 490 value (e.g. 2.0 or greater in the Control Reagent well and greater than 1.2 in the Verification Reagent well) may contain a high concentration of chlamydial antigen and may not be fully blocked by the Verification Reagent. Such specimens may not show the reduction in absorbance value of greater than 40% required to verify the original result. These specimens should be diluted 1 in 10 with working strength Murex Chlamydia Extraction Buffer and re-tested using Murex Chlamydia Verification Kit. Unverified Result If the absorbance value for a specimen previously weakly reactive in Murex Chlamydia falls below the Cut-off value in the Control Reagent well when retested using the Murex Chlamydia Verification Kit, then the result should be considered inconclusive. In such cases it is advisable to obtain another specimen from the patient for retesting using the Murex Chlamydia kit. SUMMARY OF INTERPRETATION OF RESULTS AND RECOMMENDED REPORTING A 490 in Control % Reduction between Result Reporting Reagent Well Control Reagent Well and Recommendation Verification Reagent Well > (CO+0.1) >40% Positive Chlamydial antigen present (verified by blocking) <40% Negative Non-specific reactivity. Obtain new specimen. Between (CO-0.015) >25% Positive Chlamydial antigen and (CO + 0.1) present (verified by blocking) <25% Inconclusive Inconclusive. Obtain new specimen < (CO ) Inconclusive Inconclusive. Obtain new specimen. SPECIFIC PERFORMANCE CHARACTERISTICS CLINICAL STUDIES Murex Chlamydia Verification Kit was evaluated in clinical studies performed at three centres. Clinical studies were conducted on specimens that were reactive or non-reactive on initial testing with Murex Chlamydia. A total of 141 specimens were tested. (85 endocervical swabs, 46 male urines, 9 male urethral swabs and 1 high vaginal swab.) In addition, Murex Chlamydia Verification Kit was evaluated using antigen preparations derived from cultures of each serovar of C. trachomatis, two strains of C. pneumoniae and three strains of C. psittaci. CLINICAL PERFORMANCE The results of the clinical studies are presented in Table specimens initially reactive in Murex Chlamydia were retested using Murex Chlamydia Verification Kit. 107 of these specimens gave absorbance values above the Cut-off value in the well containing Control Reagent and absorbance values at least 40% lower in the well containing the Chlamydia specific Verification Reagent thus verifying the specificity of the original reactive Murex Chlamydia result. Two of the specimens which were initially weakly reactive in Murex Chlamydia were negative on testing by direct immunofluorescence (DIF) and using Murex Chlamydia Verification Kit (below the Cut-off in both Verification and Control wells). Of the remaining two Murex Chlamydia reactive specimens not verified as positive using Murex Chlamydia Verification Kit, one had absorbance values in both the Control and the Verification Reagent wells below the Cut-off value and was positive by DIF and the other specimen (positive by DIF) was reactive in both the Control and the Verification Reagent wells. Overall 98.2% (107/109) of specimens that were reactive by Murex Chlamydia and positive by cell culture or DIF were verified as positive by re-testing using Murex Chlamydia Verification Kit. All of the antigen preparations derived from each C. trachomatis serovar and the strains of C. pneumoniae and C. psittaci gave positive results in Murex Chlamydia. All results verified as positive when retested using Murex Chlamydia Verification Kit. Table 1 Verification of Reactive Specimens using Murex Chlamydia Verification Kit Specimen No. of No. of No. of No. of Type SpecimensSpecimensSpecimensSpecimens Murex Chlamydia initially Murex Chlamydia verified reactive negative and Murex Chlamydia reactive and using Murex Culture or DIF reactive Culture or DIF Chlamydia negative positive Verification kit Cervical/High Vaginal Swab Male Swabs/ Urines 4
5 LIMITATIONS OF THE PROCEDURE 1. Verification testing using Murex Chlamydia Verification Kit is subject to the performance limitations outlined in the Murex Chlamydia Instructions for Use. Results of antigen detection tests for Chlamydia should be interpreted in conjunction with clinical and patient information and/or other laboratory tests. All enzyme immunoassay systems may produce false positives due to nonspecific reactions. Analytical studies show that very high concentrations of some strains of Staphylococcus aureus (>3 x 10 7 CFU/ml) can cause non-specific reactivity in Murex Chlamydia. Murex Chlamydia Verification Kit should be used to verify Murex Chlamydia reactive results when determined necessary by the user. Antigen detection systems such as Murex Chlamydia should not be used to provide information for medico-legal cases. Only standard Chlamydia cultures, using Chlamydia species specific monoclonal antibody immunofluorescence reagents, should be used in the evaluation of suspected abuse or other situations in which the possibility of a false positive result with antigen detection systems is unacceptable. 2. Murex Chlamydia Verification Kit can only be used for verification of positive results obtained in Murex Chlamydia. For more definitive confirmation of Chlamydia infection an alternative test system based on detection of another marker of Chlamydia should be used Specimens giving very low reactive absorbance values in Murex Chlamydia (within 0.1 absorbance units of the Cut-off) may not repeat positive using Murex Chlamydia Verification Kit due to specimen variation associated with the uneven distribution of aggregated chlamydial LPS in specimens. Under these circumstances it is recommended that a further specimen is taken from the patient. Some patient specimens will contain Chlamydia organisms at levels below the detection limit of Murex Chlamydia and will therefore give negative results. 4. Specimens containing very high concentrations of chlamydial LPS may not be fully blocked with the Chlamydia Verification Reagent and therefore may not show a reduction in absorbance units of greater than 40%. These specimens should be further diluted 1/10 in the Murex Chlamydia Extraction Buffer and retested using Murex Chlamydia Verification Kit. BIBLIOGRAPHY 1 Darougar, S. (Ed) (1983). Chlamydia Disease. Bri. Med. Bull. 39, Caul, E.O. (1990). Laboratory Diagnosis of C. trachomatis infections. PHLS Microbiology Digest 7(3), Pugh S.F., et al. (1985). Enzyme amplified immunoassay: A novel technique applied to direct detection of C. trachomatis in clinical specimens. J. Clin. Path. 38, Wiesmeier E., et al. (1988). Enzyme linked immunoabsorbent Assays in the Detection of C. trachomatis: How valid are they? Diag. Micro. Infect. Dis. 9, Lebar W.D., et al. (1990). Comparison of IDEIA III and cell culture for the detection of C. trachomatis in Endocervical specimens. J. Clin. Micro. 28(6), Alexander I., et al. (1985). Evaluation of a genus reactive monoclonal antibody in rapid identification of C. trachomatis by direct immunofluorescence. Genitourin. Med. 61, Mohanty K.C., et al. (1986). Comparison of enzyme immunoassays and cell culture for detecting C. trachomatis. Genitourin. Med. 62, Ridgway G.L., et al. (1991). Current problems in microbiology: 1. Chlamydia infections : Which laboratory tests? J. Clin. Path. 448, 1. 9 Morgan-Capner P., et al. (1986). Detection of C. trachomatis by enzyme immunoassays, immunofluorescence and cell culture. J. Clin. Path. 39, Recommendations for the prevention and management of C. trachomatis Infections. (1993). MMWR 1993 August 6: Volume 42, No. RR-12. Manufacturer: Murex Biotech Limited Central Road, Temple Hill, Dartford DA1 5LR UK Distributed in Europe by: ABBOTT Max-Planck-Ring Wiesbaden Germany C06DK05GB August
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