Captia Borrelia burgdorferi IgG/IgM

Transcription

1 wells turn yellow. The color, which is indicative of the concentration of antibody in the serum, can be read on a suitable spectrophotometer or ELISA microwell plate reader. 8,9,1,11,12,13,14 Captia Borrelia IgG/IgM Pour d'autres langues Für andere Sprachen Para otras lenguas Per le altre lingue Dla innych języków Tests Tests INTENDED USE The Trinity Biotech Captia Borrelia (B. ) IgG/IgM Enzyme-Linked Immunosorbent Assay (ELISA) is intended for the qualitative presumptive (first-step) detection of total (IgG/IgM) antibodies to Borrelia in human serum. This ELISA should only be used for patients with signs and symptoms that are consistent with Lyme disease. Equivocal or positive results must be supplemented by testing with a standardized Western-blot (second-step) procedure. Positive supplemental (second- step) results are supportive evidence of exposure to B. and can be used to support a clinical diagnosis of Lyme disease. The diagnosis of Lyme disease must be made based on history, signs (such as erythema migrans), symptoms, and other laboratory data, in addition to the presence of antibodies to B.. Negative results (either first- or second step) should not be used to exclude Lyme disease. For in vitro diagnostic use. High Complexity test. INTRODUCTION Borrelia is a spirochete that causes Lyme disease. The organism is transmitted by ticks of the genus Ixodes. In endemic areas, these ticks are commonly found on vegetation and animals such as deer, mice, dogs, horses, and birds. 1 B. infection shares features with other spirochetal infections (diseases caused by three genera in humans: Treponema, Borrelia, and Leptospira). Skin is the portal of entry for B. and a characteristic rash called erythema migrans (EM) develops around the tick bite in 6% to 8% of patients. Spirochetemia occurs early with wide spread dissemination through tissue and body fluids. Lyme disease occurs in stages, often with intervening latent periods and with different clinical manifestations. 2,3 In Lyme disease there are generally three stages of disease often with overlapping symptoms. Symptoms vary according to the sites affected by the infection such as joints, skin, central nervous system, heart, eye, bone, spleen, and kidney. Late disease is most often associated with arthritis or CNS syndromes. Asymptomatic subclinical infection is possible and infection may not become clinically evident until the later stages. Patients with early infection produce IgM antibodies during the first few weeks after onset of EM and produce IgG antibodies more slowly. 4 Although IgM only may be detected during the first month after onset of illness, the majority of patients develop IgG antibodies within one month. Both IgG and IgM antibodies can remain detectable for years. Isolation of B. from skin biopsy, blood, and spinal fluid has been reported. 5 However, these direct culture detection methods may not be practical in the large scale diagnosis of Lyme borreliosis. Serological testing methods for antibodies to B. include Indirect Fluorescent Antibody (IFA) staining, Immunoblotting, and Enzyme Immunoassay (EIA). B. is antigenically complex with strains that vary considerably. Early antibody responses often are to flagellin, which has cross-reactive components. Patients in early stages of infection may not produce detectable levels of antibody. Also, early antibiotic therapy after EM may diminish or abrogate good antibody response. Thus, serological tests for antibodies to B. are known to have low sensitivity and specificity, and because of such inaccuracy, these test cannot be relied upon for establishing a diagnosis of Lyme disease. 2,6 In 1994, the Second National Conference on Serological Diagnosis of Lyme disease recommended a two-step testing system toward standardizing laboratory serologic testing for B.. 7 Because ELISA and IFA methods were not sufficiently specific to support clinical diagnosis, it was recommended that positive or equivocal results from a sensitive ELISA or IFA (first step) should be further tested, or supplemented, by using a standardized Western-blot method (second step) for detecting antibodies to B. (Western-blot assays for antibodies to B. are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM). Two-step positive results provide supportive evidence of exposure to B., which could support a clinical diagnosis of Lyme disease but should not be used as a criterion for diagnosis. Antibody detection methods do not provide definitive results for establishing or ruling out a diagnosis of Lyme disease in that the predictive value of positive results will be low when specimens are tested from patients with diagnoses that are not supported by history of exposure, symptoms, signs, and laboratory data. PRINCIPLE OF THE ASSAY Para outras línguas Για τις άλλες λώσσες För andra språk For andre språk Enzyme-Linked Immunosorbent Assays (ELISA) rely on the ability of biological materials, (i.e., antigens) to adsorb to plastic surfaces such as polystyrene (solid phase). When antigens bound to the solid phase are brought into contact with a patient's serum, antigen specific antibody, if present, will bind to the antigen on the solid phase forming antigen-antibody complexes. Excess antibody is removed by washing. This is followed by the addition of goat anti-human IgG/IgM conjugated with horseradish peroxidase which then binds to the antibody-antigen complexes. The excess conjugate is removed by washing, followed by the addition of Chromogen/Substrate Tetramethylbenzidine (TMB). If specific antibody to the antigen is present in the patient's serum, a blue color develops. When the enzymatic reaction is stopped with 1N H2SO4, the contents of the MATERIALS SUPPLIED KIT PRESENTATION Each kit contains the following components in sufficient quantities to perform the number of tests indicated on the package label. 1. Purified B. antigen, strain B-31 (passed less than 15 times, washed, concentrated and detergent treated in glycine buffer) coated microassay plate: 96 wells, configured in twelve 1x8 strips, stored in a foil pouch with desiccant. (96T: one plate; 48T: five plates) 2. B. Serum Diluent Type V: Ready for use. Contains gentamycin and sodium azide (<.1%) as preservatives. (96T: one bottle, 3 ml; 48T: three bottles, 5 ml each) 3. Cutoff ibrator (ibrator): human serum or defibrinated plasma. Sodium azide (<.1%) and pen/strep (.1%) added as preservatives, with kit specific factor printed on vial label. The Cutoff ibrator is used to calibrate the assay to account for day-to-day fluctuations in temperature and other testing conditions. (96T: one vial,.4 ml; 48T: one vial,.8 ml) * 4. High Positive Control: human serum or defibrinated plasma. Sodium azide (<.1%) and pen/strep (.1%) added as preservatives, with established range printed on vial label. The High Positive Control is utilized to indicate the upper limit of dynamic range of the assay. (96T: one vial,.4 ml; 48T: one vial,.8 ml) * 5. Low Positive Control: human serum or defibrinated plasma. Sodium azide (<.1%) and pen/strep (.1%) added as preservatives, with established range printed on vial label. The Low Positive Control is utilized to control the assay near the cutoff of the assay. (96T: one vial,.4 ml; 48T: one vial,.8 ml) * 6. Negative Control: human serum or defibrinated plasma. Sodium azide (<.1%) and pen/strep (.1%) added as preservatives, with established range printed on vial label. The Negative Control is utilized to control the negative range of the assay. (96T: one vial,.4 ml; 48T: one vial,.8 ml) * 7. Horseradish-peroxidase (HRP) Conjugate: Ready for use. Goat anti-human IgG/IgM, containing Proclin 3 (.1%) as a preservative. (96T: one bottle, 16 ml; 48T: five bottles, 16 ml each) 8. Chromogen/Substrate Solution Type I: Tetramethylbenzidine (TMB), ready to use. (96T: one bottle, 16 ml; 48T: five bottles, 16 ml each) 9. Wash Buffer Type I (2X concentrate): dilute 1 part concentrate + 19 parts deionized or distilled water. Contains TBS, Tween-2 and Proclin 3 (.1%) as a preservative. (96T: one bottle, 5 ml; 48T: one bottle, ml) 1. Stop Solution: Contains a H2SO4 solution, ready to use. (96T: one bottle, 15 ml; 48T: five bottles, 15 ml each) * Note: serum vials may contain excess volume. The following components are not Kit Lot # dependent and may be used interchangeably with the Trinity Biotech ELISA IgG/IgM assays: Chromogen/Substrate Solution Type I, Wash Buffer Type I, and Stop Solution. The Serum Diluent Type V is specific for the B. kits. Do not interchange the Serum Diluent with other kits. ADDITIONAL REQUIREMENTS Cylinder (1 ml) Flask (1 L) Timer: to 6 minutes Micropipettes capable of accurately delivering 1-2 µl volumes (less than 3% CV) Deionized or distilled water Paper towels Wash bottle, semi-automated or automated wash equipment. Single or dual wavelength microplate reader with 45 nm filter. If dual wavelength is used, set the reference filter to 6-65 nm. Read the Operator s Manual or contact the instrumentation manufacturer to establish linearity performance specifications of the reader. Test tubes for serum dilution Disposal basin and disinfectant (e.g.,.5% sodium hypochlorite). Note: Use only clean, dry glassware. STORAGE AND STABILITY 1. Store unopened kit between 2 and 8 C. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Unopened microassay plates must be stored between 2 and 8 C. Unused strips must be immediately resealed in a sealable bag with desiccant and returned to storage between 2 and 8 C. 3. Store HRP conjugate between 2 and 8 C. 4. Store the Cutoff ibrator, High Positive Control, Low Positive Control, and Negative Control between 2 and 8 C. 5. Store Serum Diluent and 2X Wash Buffer between 2 and 8 C. 6. Store the Chromogen/Substrate Solution between 2 and 8 C. 7. Store 1X (diluted) Wash Buffer at room temperature (21 to C) for up to 5 days, or 1 week between 2 and 8 C. Note: If constant storage temperature is maintained, reagents and substrate will be stable for the dating period of the kit. Refer to package label for expiration date. Precautions were taken in the manufacture of this product to protect the reagents from contamination and bacteriostatic agents have been added to the liquid reagents. Care should be exercised to protect the reagents in this kit from contamination. PRECAUTIONS 1. For in vitro diagnostic use only. 2. The human serum components used in the preparation of the Controls and Cutoff ibrators in this kit have been tested by an FDA approved method for the presence of antibodies to Human Immunodeficiency Virus 1 & 2 (HIV 1&2) and Hepatitis C (HCV) as well as hepatitis B surface antigen and found negative. Because no test methods can offer complete assurance that HIV, HCV, Hepatitis B virus, or other infectious agents are absent, specimens and human-based reagents should be handled as if capable of transmitting infectious agents. 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2 3. The Centers for Disease Control and Prevention, and the National Institutes of Health recommend that potentially infectious agents be handled at the Biosafety Level The components in this kit have been quality control tested as a Master Lot unit. Do not mix components from different lot numbers except Chromogen/Substrate Solution, Stop Solution and Wash Buffer. Serum Diluent supplied with B. kits can be used only with other B. kits. Do not mix with components from other manufacturers. 5. Do not use reagents beyond the stated expiration date marked on the package label. 6. All reagents must be at room temperature (21 - C) before running assay. Remove only the volume of reagents that is needed. Do not pour reagents back into vials as reagent contamination may occur. 7. Before opening Control and Cutoff ibrator vials, tap firmly on the benchtop to ensure that all liquid is at the bottom of the vial. 8. Use only distilled or deionized water and clean glassware. 9. Do not let wells dry during assay; add reagents immediately after completing wash steps. 1. Avoid cross-contamination of reagents. Wash hands before and after handling reagents. Cross-contamination of reagents and/or samples could cause false results. 11. If washing steps are performed manually, wells are to be washed three times. Up to five wash cycles may be necessary if a washing manifold or automated equipment is used. 12. Sodium azide inhibits Conjugate activity. Clean pipette tips must be used for the Conjugate addition so that sodium azide is not carried over from other reagents. 13. It has been reported that sodium azide may react with lead and copper in plumbing to form explosive compounds. When disposing, flush drains with water to minimize build-up of metal azide compounds. 14. Never pipette by mouth or allow reagents or patient sample to come into contact with skin. Reagents containing proclin, sodium azide, and TMB may be irritating. Avoid contact with skin and eyes. In case of contact, flush with plenty of water. 15. If a sodium hypochlorite (bleach) solution is being used as a disinfectant, do not expose to work area during actual test procedure because of potential interference with enzyme activity. 16. Avoid contact of sulfuric acid with skin or eyes. If contact occurs, immediately flush area with water. 17. Caution: Liquid waste at acid ph must be neutralized prior to adding sodium hypochlorite solutions (bleach) to avoid formation of poison gas. Recommend disposing of reacted, stopped plates in biohazard bags. See Precaution The concentrations of anti-b. in a given specimen determined with assays from different manufacturers can vary due to differences in assay methods and reagent specificity. The safety data sheet is available upon request. Serum Diluent, Conjugate, and Wash Buffer contain.1% ProClin 3, a biocidal preservative that may cause sensitization by skin contact; prolonged or repeated exposure may cause allergic reaction in certain sensitive individuals. H317: May cause an allergic skin reaction. P28: Wear protective gloves / protective clothing / eye protection / face protection. P32 + P352: IF ON SKIN: Wash with plenty of soap and water. P333 + P313: If skin irritation or rash occurs: Get medical advice/ attention. P51: Dispose of contents and container in accordance to local, regional, national and international regulations. Serum Diluent and Controls contain <.1% sodium azide. H32: Harmful if swallowed P264: Wash thoroughly with plenty of soap and water after handling P27: Do no eat, drink or smoke when using this product P31+P312: IF SWALLOWED: l a POISON CENTER or doctor/physician if you feel unwell P33: If swallowed, rinse mouth P51: Dispose of contents/container to in accordance to local, regional, national and international regulations. SPECIMEN COLLECTION AND STORAGE 1. Handle all blood and serum as if capable of transmitting infectious agents. 2. Optimal performance of the kit depends upon the use of fresh serum samples (clear, nonhemolyzed, non-lipemic, non-icteric). A minimum volume of 5 µl is recommended, in case repeat testing is required. Specimens should be collected aseptically by venipuncture. 16 Early separation from the clot prevents hemolysis of serum. 3. The CLSI (Approved Standard - Procedures for the Handling and Processing of Blood Specimens, H18-A4. 21) 16 provides the following general recommendations for storing blood specimens, recognizing that there can be several exceptions: Separated serum should remain at room temperature for no longer than eight hours. If assays will not be completed within eight hours, serum should be refrigerated (2 to 8 C). If assays are not completed within 48 hours, or the separated serum will be stored beyond 48 hours, serum should be frozen at or below -2 C. 4. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory Do not use a frost-free freezer because it may allow the specimens to go through freeze-thaw cycles and degrade antibody. Samples that are improperly stored or are subjected to multiple freeze-thaw cycles may yield spurious results. 16 METHODS FOR USE PREPARATION FOR THE ASSAY 1. All reagents must be removed from refrigeration and allowed to come to room temperature before use (21 to C). Return all reagents to refrigerator promptly after use. 2. All samples and controls should be vortexed before use. 3. Dilute 5 ml of the 2X Wash Buffer Type I to 1 L with distilled and/or deionized H2O. Mix well. ASSAY PROCEDURE 1. Place the desired number of strips into a microwell frame. Allow six (6) Control/Cutoff ibrator determinations (one Negative Control, three Cutoff ibrators, one High Positive Control, and one Low Positive Control) per run. A reagent blank (RB) should be run on each assay. Check software and reader requirements for the correct Control/ibrator configuration. Return unused strips to the sealable bag with desiccant, seal and immediately refrigerate. Example Configuration: Plate Location 1A 1B 1C 1D 1E 1F 1G 1H Sample Description RB NC HPC LPC Patient #1 Plate Location 2A 2B 2C 2D 2E 2F 2G 2H Sample Description Patient #2 Patient #3 Patient #4 Patient #5 Patient #6 Patient #7 Patient #8 Patient #9 RB = Reagent Blank - Well without serum addition run with all reagents. Utilized to blank reader. NC = Negative Control = ibrator HPC = High Positive Control LPC = Low Positive Control 2. Dilute test sera, Cutoff ibrator, High, Low and Negative Control sera 1:21 (e.g., 1 µl + 2 µl) in Serum Diluent. (For manual dilutions, it is suggested to dispense the Serum Diluent into the test tube first and then add the patient serum). 3. To individual wells, add 1 µl of the appropriate diluted Cutoff ibrator, Controls and patient sera. Add 1 µl of Serum Diluent to reagent blank well. Check software and reader requirements for the correct reagent blank well configuration. 4. Incubate each well at room temperature (21 - C) for minutes +/- 5 minutes. 5. Aspirate or shake out liquid from all wells. If using semi-automated or automated washing equipment, add -3 µl of diluted Wash Buffer to each well. Aspirate or shake out to remove all liquid. Repeat the wash procedure two times (for a total of three (3) washes) for manual or semi-automated equipment or four (4) times (for a total of five (5) washes) for automated equipment. After the final wash, blot the plate on paper toweling to remove all liquid from the wells. **IMPORTANT NOTE: Regarding steps 5 and 8 -Insufficient or excessive washing will result in assay variation and will affect validity of results. Therefore, for best results, the use of semiautomated or automated equipment set to deliver a volume to completely fill each well (- 3 µl) is recommended. A total of up to five (5) washes may be necessary with automated equipment. Please contact Trinity Biotech with any questions regarding appropriate wash equipment. Complete removal of the Wash Buffer after the last wash is critical for the accurate performance of the test. Also, visually ensure that no bubbles are remaining in the wells. 6. Add 1 µl Conjugate to each well, including reagent blank well. Avoid bubbles upon addition as they may yield spurious results. 7. Incubate each well minutes +/- 5 minutes at room temperature (21 - C). 8. Repeat Wash as described in Step Add 1 µl Chromogen/Substrate Solution to each well, including reagent blank well, maintaining a constant rate of addition across the plate. 1. Incubate each well 1-15 minutes at room temperature (21 to C). 11. Stop reaction by addition of 1 µl of Stop Solution (1N H2SO4) following the same order of Chromogen/Substrate Solution addition, including reagent blank well. Tap the plate gently along the outsides, to mix contents of the wells. The plate may be held up to 1 hour after addition of the Stop Solution before reading. 12. The developed color should be read on an ELISA plate reader equipped with a 45 nm filter. If dual wavelength is used, set the reference filter to 6-65 nm. The instrument should be blanked on air. The reagent blank must be less than.15 Absorbance at 45 nm. If the reagent blank is >.15 the run must be repeated. Blank the reader on the reagent blank well and then continue to read the entire plate. Dispose of used plates after readings have been obtained. QUALITY CONTROL For the assay to be considered valid the following conditions must be met: 1. ibrator and Controls must be run with each test run. 2. Reagent blank (when read against air blank) must be <.15 Absorbance (A) at 45 nm. 3. Negative Control must be <. A at 45 nm (when read against reagent blank). 4. Each Cutoff ibrator must be >. A at 45 nm (when read against reagent blank). 5. High Positive Control must be >.5 A at 45 nm (when read against reagent blank). 6. The ISR (Presumptive Immunoglobulin Status Ratio) Values for the High, Low, and Negative Controls should be in their respective ranges printed on the vials. If the Control values are not within their respective ranges, the test should be considered invalid and the test should be repeated. 7. Additional controls may be tested according to guideline, or requirements of local, state, and/or federal regulations or accrediting organizations. 8. Refer to NCCLS C24-A for guidance on appropriate Quality Control practices. 9. If above criteria are not met on repeat, contact Trinity Biotech Technical Service. INTERPRETATION 1. Cutoff ibrator Value - culate the mean value for the Cutoff ibrator from the three Cutoff ibrator determinations. If any of the three Cutoff ibrator values differ by more than 15% from the mean, discard that value and calculate the average of the two remaining values. 2. Correction Factor - To account for day-to-day fluctuations in assay activity due to room temperature and timing, a Correction Factor is determined for each lot of kits. The Correction Factor is printed on the Cutoff ibrator vial. 3. Cutoff O.D. Value - The Cutoff O.D. Value for each assay is determined by multiplying the Correction Factor by the mean Cutoff ibrator Value determined in step 1. Page 2 of 5 EN Rev K

3 4. ISR Value - culate a Presumptive Immunoglobulin Status Ratio (ISR) for each specimen by dividing the specimen O.D. value by the Cutoff O.D. Value determined in step 3. Example: ODs obtained for Cutoff ibrator =.38,.4,.42 Mean O.D. for Cutoff ibrator =.4 Correction Factor =.5 Cutoff O.D. Value =.5x.4 =.2 O.D. obtained for patient sera =.6 Presumptive Immunoglobulin Status Ratio =.6/.2 = 3. ANALYSIS 1. The patients ISR (Presumptive Immunoglobulin Status Ratio) is interpreted and reported as follows: ISR Value Results Interpretation.9 Negative by Trinity Biotech B Equivocal by Trinity Biotech B. 1.1 Positive by Trinity Biotech B. No detectable antibody; result does not exclude B. infection. An additional sample should be tested within 4-6 weeks if early infection suspected. 17 Equivocal results should only be interpreted as initial evidence for detection of antibodies to B., with the recommendation for second-step testing to be performed before reporting results to a clinician. Current recommendations 7 state that equivocal results should be followed by supplemental (second-step) Western Blot testing. (Western Blot assays for antibodies to B. are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM). This equivocal result should be reported with results from Wester Blot testing. Results should not be reported until the supplemental testing is completed. Antibody to B. presumptively detected. Positive results should only be interpreted as initial evidence for detection of antibodies to B., with the recommendation for second-step testing to be performed before reporting results to a clinician. Per current recommendations, 7 the result cannot be further interpreted without supplemental (second-step) Western-Blot testing. (Western Blot assays for antibodies to B. are supplemental rather than confirmatory because their specificity is less than optimal, particularly for detecting IgM). Results should not be reported until the supplemental testing is completed. 2. There is a low predictive value of a negative result from the assay when used early in infection, and a low predictive value of a positive result when exposure, history, symptoms, and clinical findings are not consistent with Lyme disease. A positive result from a first-step serologic test is presumptive for the presence of antibodies to B. and may indicate infection with B.. Positive or equivocal results should not be reported until second-step testing of the specimen is done using a method that is more specific for antibodies to B. (currently limited to IgG and IgM Western Blot assays). Reporting positive or equivocal first-step results before, or in the absence of, second-step testing may increase the likelihood of misdiagnosis of Lyme disease, and potentially expose the patient to inappropriate and toxic therapy. 3. To determine the cutoff of the assay, one hundred ninety-four normal sera were assayed by the Trinity Biotech B. test. Ninety-six sera were from an endemic area and ninety-eight sera were from a non-endemic area. The mean and standard deviation of the optical density readings for the sera were.9 and.57, respectively. The positive threshold for the assay was determined by adding the mean and three standard deviations ( (.57) =.26). A positive serum was titrated to give a constant ratio of the threshold value to obtain a Cutoff ibrator serum. On all subsequent assays this serum was run and the assay was calibrated by multiplying the O.D. value for the Cutoff ibrator by the ratio to the cutoff to obtain the Cutoff O.D. This value was then divided into the O.D. for the patient sera to obtain a Presumptive Immunoglobulin Status Ratio (ISR). By definition the cutoff ISR is equal to 1.. To account for inherent variation in immunoassay, values of are considered equivocal. Therefore, values <.9 are considered negative and the values >1.1 are considered positive. 4. The magnitude of the ISR above the cut-off is not indicative of the amount of B. antibody present. LIMITATIONS OF USE 1. False positive results may occur with sera from patients with relapsing fever, other spirochetal diseases, Rocky Mountain Spotted Fever, autoimmune diseases, EBV and CMV infections. Patients with syphilis may have antibodies which will cross-react with the B. antigen in the Trinity Biotech B. kit. 18 The reactivity of the assay with sera containing antibodies to H. pylori has not been determined. 2. Testing of potentially cross-reacting specimens with Rocky Mountain Spotted Fever and elevated ESR has not been performed. 3. Screening of the general population should not be performed. The positive predictive value depends on the likelihood of Lyme disease being present. Testing should only be performed when clinical symptoms are present or exposure is suspected. 4. This assay detects IgG and IgM immunoglobulins to B., whereas other available tests may detect total immunoglobulins (IgG/M/A) or only IgM or IgG. The analytical sensitivity for each immunoglobulin class has not been defined for this product. 5. The affinity and/or avidity of anti-b. IgG and IgM for the B. antigen used have not been determined for this assay; samples with IgM only may not be detected. 6. The assay performance characteristics have not been established for matrices other than sera. 7. Icteric, lipemic, hemolyzed or heat inactivated sera may cause erroneous results and should be avoided. 8. Kit procedures or practices outside those in this package insert may yield questionable results. EXPECTED VALUES A population of 4 serum samples collected from a healthy population with random gender, age (18 5), race and geographic location were tested on the Trinity Biotech Borrelia assay. The Trinity Biotech Borrelia yielded a 4.75 % (19/4) positivity rate and 5. % (21/4) equivocal rate for this group of samples. The distribution of ISR values from this study is presented in the following chart. This prevalence may vary according to age and geographical area of the test population. Frequency Chart 1 Distribution of ISR Values in a Normal Population (n=4) # of Specimens ISR Value PERFORMANCE CHARACTERISTICS Evaluation of Trinity Biotech Borrelia Percent Agreement Positive and Percent Agreement Negative Relative to Western Blot The Trinity Biotech Borrelia was evaluated by masked testing of 474 sera versus MarDx Western Blot for both IgG and IgM. Any sera with 1-step positive or equivocal test results were further analyzed with supplemental (second step) Western Blot testing. The results are illustrated in Table 1. Table 2 illustrates the percent agreement positive and percent agreement negative for all 474 sera tested relative to the MarDx Western Blot. Trinity B. IgG/IgM + eq. Type of Result Table 1 MarDx IgG and IgM Western Blot Trinity (95% CI) 1-step (ELISA) Pos. or Eq % (11.2 % %) (68/474) 1-step (ELISA) Pos. or Eq. & 2-step (WB) pos. 2-step (WB) Pos. Among 1-step (ELISA) Pos. or Eq. CI = Confidence Interval 3.16 % (1.78 % %) (15/474) 22.6 % (12.9 % %) (15/68) 28 The 95% confidence intervals were calculated using the normal method. Table 2 Trinity Biotech Borrelia % Agreement Positive and % Agreement Negative Relative to MarDx Western Blot Trinity Biotech Borrelia MarDx Western Blot + - Total eq % Agreement Positive = 15 / 16 = 93.75% 95% Confidence Interval = 69.8 % to 99.8 % % Agreement Negative = 45 / 433 = 93.53% 95% Confidence Interval = 91.2 % to 95.8 % % Agreement = 42 / 449 = 93.54% 95% Confidence Interval = 91.3 % to 95.8 % Equivocals were not included in the above calculations. The 95% confidence intervals were calculated using the normal method. Total PRECISION The precision of the Trinity Biotech Borrelia was determined by testing six different sera ten times each on three separate days. The mean coefficients of variation from the intra- and inter- assays are presented in Table 3. With appropriate technique the user should obtain precision of < 15% CV Table 3 Trinity Biotech Borrelia Precision Assay 1 (n=1) Assay 2 (n=1) Assay 3 (n=1) Inter-Assay (n=3) X SD CV X SD CV X SD CV X SD CV X = Mean SD =Standard Deviation CV = Coefficient of Variation = SD/X x 1 The methods in NCCLS EP5 were utilized for precision parameters. Page 3 of 5 EN Rev K

4 CROSS-REACTIVITY The following potentially cross-reactive sera were run on the Trinity Biotech Borrelia assay to assess cross-reactivity with the assay: lipemic, bilirubinemic, RPR+, dsdna+, RF+, EBV+, CMV+, and CRP. The data in Table 4 illustrates the amount of reactivity with the sera. Table 4 Cross-Reactivity Laboratory result (Titer) # of samples # of positives Lipemic (+ + +) 5 1 Bilirubinemic ( ) 5 RPR + (1:2-1:64) 29 3 Rheumatoid Factor + (O.D ) 7 Epstein Barr Virus Antibody + (O.D ) 8 1 Cytomegalovirus Antibody + (O.D ) 7 1 CRP + ( mg/dl) 5 dsdna + ( IU) 5 BORRELIA BURGDORFERI IGG/IGM SUMMARY OF ASSAY DILUTE SERUM ADD TO MICROWELLS 1 µl WASH 1 21 Minutes at Room REFERENCES 1. Bates, H.M Lyme Disease. Lab Mgmt. 22: Steere, A.C., et al The Spirochetal Etiology of Lyme Disease. N Engl J Med. 38: Reik, L. et al Neurological abnormalities of Lyme Disease. Medicine. 58: Steere, A.C. et. al J. Infect. Dis. 154: Rosenfeld, M.E.A., et.al Serodiagnosis of Lyme Disease. J. Clin Microbiol. 31: Bakken, L.L., S.M. lister, P.J. Wand, and R.F. Schell Interlaboratory Comparison of Test Results for Detection of Lyme Disese by 516 Patients in the Wisconsin State Laboratory of Hygiene/College of American Pathologist Proficiency Testing Program. J. Clin Microbiol. 35: Centers for Disease Control and Prevention Recommendation for Test Performance and Interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. Morbid. Mortal. Weekly Rep. 44: Engvall, E., K. Jonsson, and P. Perlman Enzyme-Linked Immunosorbent Assay, (ELISA) Quantitative Assay of Immunoglobulin G. Immunochemistry. 8: Engvall, E. and P. Perlman Enzyme-Linked Immunosorbent Assay, ELISA. In: Protides of the Biological Fluids, H. Peeters, Ed., Proceedings of the Nineteenth Colloquium, Brugge Oxford. Pergamon Press. p Engvall, E., K. Jonsson, and P. Perlman Enzyme-Linked Immunosorbent Assay. II. Quantitative Assay of Protein Antigen, Immunoglobulin-G, By Means of Enzyme-Labeled Antigen and Antibody-Coated Tubes. Biochem. Biophys. Acta., 1: Van Weeman, B.K.and A.H.W.M. Schuurs Immunoassay Using Antigen-Enzyme Conjugates. FEBS Letter. 15: Bakerman, S Enzyme Immunoassays. Lab. Mgmt. August, p Voller, A., and D. E. Bidwell Brit. J. Exp. Pathology. 56: Engvall, E. and P. Perlman Enzyme-Linked Immunosorbent Assay, ELISA. III. Quantitation of Anti-Immunoglobulins in Antigen-Coated Tubes. J. Immunol. 19: CDC/NIH Guidelines Biosafety in Microbiological and Biomedical Laboratories. 3rd Edition. pp U.S. Dept. of Health and Human Services, Public Health Service. 16. National Committee for Clinical Laboratory Standards Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture Approved Standard. NCCLS Publication H18-A. 17. Barbour, A Laboratory Aspects of Lyme borreliosis. Clin. Micro. Rev.1: Magnarelli, L., et al Cross-Reactivity in Serologic Tests for Lyme Disease and Other Spirochetal Infections. J. Infec. Dis. 156: ADD CONJUGATE 1 µl WASH ADD TMB SOLUTION 1 µl ADD STOP 1 µl 1N H2SO4 READ AT 45 NM The safety data sheet is available upon request. Minutes at Room 1-15 Minutes at Room Serum Diluent, Conjugate, and Wash Buffer contain.1% ProClin 3, a biocidal preservative that may cause sensitization by skin contact; prolonged or repeated exposure may cause allergic reaction in certain sensitive individuals. H317: May cause an allergic skin reaction. P28: Wear protective gloves / protective clothing / eye protection / face protection. P32 + P352: IF ON SKIN: Wash with plenty of soap and water. P333 + P313: If skin irritation or rash occurs: Get medical advice/ attention. P51: Dispose of contents and container in accordance to local, regional, national and international regulations. Serum Diluent and Controls contain <.1% sodium azide. H32: Harmful if swallowed P264: Wash thoroughly with plenty of soap and water after handling P27: Do no eat, drink or smoke when using this product P31+P312: IF SWALLOWED: l a POISON CENTER or doctor/physician if you feel unwell P33: If swallowed, rinse mouth P51: Dispose of contents/container to in accordance to local, regional, national and international regulations. KIT Catalog No ORDERING INFORMATION Captia Borrelia IgG/IgM Test Kit Item Quantity Captia Borrelia IgG/IgM Test Kit 96 Tests Captia Borrelia IgG/IgM Test Kit 48 Tests Page 4 of 5 EN Rev K

5 Manufactured CONTROL + + High Pos or Positive Control Authorized Representative CONTROL + Low Pos or Cut-Off Control Consult accompanying documents CONTROL - Negative Control REF Product Number CAL ibrator LOT Lot C.F. Coefficient Factor Use by RNG Range Caution, consult accompanying documents STD Standard Store at 2-8ºC IVD For In Vitro Diagnostic use Store at 2-3ºC or Hazard Trinity Biotech USA Jamestown, NY 1471 Tel Fax: Trinity Biotech plc Bray Co. Wicklow, Ireland Tel Fax Rev K 7/215 Page 5 of 5 EN Rev K