SIGNIFICANCE AND BACKGROUND

Transcription

1 BORRELIA VlsE-1/pepC10 PLUS TEST SYSTEM A Multiplexed, Microparticle-Based Immunoassay for IgG Antibodies to VlsE-1 and IgM Antibodies to pepc10 Antigens Product Number: A90151 INTENDED USE The ZEUS Scientific AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System is a multiplexed sandwich immunoassay for the qualitative detection of IgG class antibody to recombinant VlsE-1 and the IgM class antibody to synthetic pepc10 in human serum. The AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System is intended for use with the Luminex xmap platform and the AtheNA Multi-Lyte data management package in testing serum samples from symptomatic patients or those with a history of Lyme Borreliosis. All positive specimens should be tested with a second-tier test such as Western Blot, which if positive, is supportive evidence of infection with Borrelia burgdorferi. Diagnosis of Lyme Borreliosis should be made based on the presence of B. burgdorferi antibodies, history, symptoms, and other laboratory data. Negative first or second tier results should not be used to exclude Borreliosis. This kit is for in vitro diagnostic use. SIGNIFICANCE AND BACKGROUND Borrelia burgdorferi 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. B. burgdorferi 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. burgdorferi and the tick bite often causes a characteristic rash called erythema migrans (EM). EM develops around the tick bite in 60% to 80% 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. 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 later stages. Patients with early infection produce IgM class antibodies during the first few weeks after onset of EM and produce IgG class antibodies more slowly (1). Although only IgM 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. burgdorferi from skin biopsy, blood, and spinal fluid has been reported (2). 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. burgdorferi include indirect fluorescent antibody (IFA) staining, immunoblotting, and enzyme immunoassay (ELISA). B. burgdorferi is antigenically complex with strains that vary considerably. Early antibody responses often are to flagellin that has cross-reactive components. Patients in early stages of infection may not produce detectable levels of antibody. In addition, early antibiotic therapy after EM may diminish or abrogate good antibody response. Some patients may never generate detectable antibody levels. Thus, serological tests for antibodies to B. burgdorferi are known to have low sensitivity and specificity and because of such inaccuracy, these tests cannot be relied upon for establishing a diagnosis of Lyme disease (3, 4). In 1994, the Second National Conference on Serological diagnosis of Lyme disease recommended a two-step testing system toward standardizing laboratory serological testing for B. burgdorferi. 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. burgdorferi. Western Blot assays for antibodies to B. burgdorferi 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. burgdorferi, which could support a clinical diagnosis of Lyme disease but should not be used as a sole criterion for diagnosis. Scientists tested various antigens in recent years to improve the serological detection of antibodies to B. burgdorferi. One such attempt has been the use of an algorithm to test for IgG antibodies towards VlsE-1 antigen and IgM antibodies for pepc10 antigen (5). The AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System was developed by ZEUS Scientific using the protocol that detects IgG antibodies to VlsE-1 and IgM antibodies to pepc10. Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

2 PRINCIPLE OF THE ASSAY The ZEUS Scientific AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System is designed to detect IgG class antibodies in human sera to VlsE-1 antigen and IgM class antibodies to pepc10 antigen. The test procedure involves four incubation steps: 1. Test sera (properly diluted) are incubated in a filter plate well containing a multiplexed mixture of Bead Suspension-1. The multiplexed Bead Suspension-1 contains a mixture of distinguishable sets of polystyrene microspheres; one of these bead sets is conjugated with the VlsE-1 antigen. The bead mix also contains one bead set designed to detect non-specific binding and four separate bead sets used for assay calibration. If present in patient sera, specific antibodies will bind to the immobilized antigen on one or more of the bead sets. The microspheres are rinsed to remove non-reactive serum proteins. 2. Conjugate-1 is added to the microtiter well and the plate is incubated. The conjugate will react with IgG antibody immobilized on the solid phase in step 1. The microspheres are rinsed to remove unbound conjugate. 3. Bead Suspension-2 is added to the wells. The bead set contains beads conjugated with pepc10 and VlsE-1 antigens. A second aliquot of test sera at the same dilution as in step 1 is added to the well and mixed. The bead and specimen suspension is incubated. Following incubation, the microspheres are rinsed to remove the non-reacting serum proteins. 4. Conjugate 2 is added to the microtiter well and the plate is incubated. The conjugate will react with IgM antibody immobilized on the solid phase in step 1 and step The AtheNA Multi-Lyte instrument then analyzes the entire bead suspension. The bead set(s) are sorted (identified) and the amount of reporter molecule (PE conjugate) is determined for each bead set. Using the Intra-Well Calibration Technology, internal calibration bead sets are used to convert raw fluorescence into outcome (the Bioinformatics Score or BIS, patent pending). DETECTION OF ANTIGEN SPECIFIC IgG AND IgM ANTIBODIES: After the bead set mix is allowed to react with human serum as described in steps 1 through 4, human antibodies if present, will bind to the bead sets and then will be analyzed. The amount of reporter is a direct measurement of IgM, IgG or both IgM and IgG if a patient has both IgG and IgM to the same antigen. The amount of IgG in such an instance can be determined by a mathematical formula of like-coated bead set results using a sequence of measurements by ZEUS Scientific, Inc (patent pending). KIT COMPONENTS REACTIVE REAGENTS: All reactive reagents contain sodium azide as a preservative at a concentration of <0.1% (w/v). Multiplexed Bead Suspension 1: Ready to use, 5.5mL bottle. The Suspension contains separate BEAD distinguishable 5.6-micron polystyrene beads that are conjugated with VlsE-1 antigen. The bead mix also contains one bead set designed to detect non-specific antibodies in the patient sample (if present) and four separate bead sets used for assay calibration Multiplexed Bead Suspension 2: Ready to use, 5.5mL bottle. The Suspension contains separate BEAD distinguishable 5.6-micron polystyrene beads that are conjugated with the following antigens: VlsE-1 and pepc10. CONJ Conjugate 1: Phycoerythrin conjugated goat anti-human IgG (γ chain specific). Ready to use, 15mL amber bottle. Conjugate 2: Phycoerythrin conjugated goat anti-human IgM (µ chain specific). Ready to use, 15mL amber bottle. Note: The Bead Suspension and Conjugate are light sensitive reagents. CONJ Both have been packaged in light protective containers. Normal exposure experienced during the course of performing the assay will not affect assay performance. Do not unenecessarily expose these reagents to strong sources of visible light. CONTROL + Human Positive serum Controls. Two, 0.2mL vials, one specific for VlsE-1 and one specific for pepc10. CONTROL - Human VlsE-1 and pepc10 Negative serum Control. One, 0.2mL vial. DIL SPE SAVe Diluent. One 50mL bottle containing phosphate-buffered-saline. Ready to use. Note: The SAVe Diluent will change color in the presence of serum. WASHBUF 10X Wash Buffer Concentrate: One 60mL bottle containing 10 X concentrate of phosphate buffered saline.. Dilute 1 part concentrate + 9 parts deionized or distilled water. NON-REACTIVE REAGENTS: 1. One, dilution plate. 2. One, 96-well filtration plate for rinsing the microspheres. 3. Data Labels: One label is adhered to the inside lid of the kit box and a second label is inside the kit box. 4. Package Insert providing instructions for use. 5. Calibration CD: A compact disc that includes all lot-specific kit calibration values required for specimen analysis and assay quality control. 2 Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088

3 PRECAUTIONS 1. Dilution or adulteration of these reagents may generate erroneous results. 2. Do not use reagents from other sources or manufacturers. 3. The Bead Suspension and Conjugate are light sensitive reagents. Both have been packaged in light protective containers. Normal exposures experienced during the course of performing the assay will not affect assay performance. Do not unnecessarily expose these reagents to strong sources of visible light. 4. To optimize read times the Bead Suspension must be thoroughly mixed just prior to use. The most effective means to resuspend the Beads is to first vortex the Bead Suspension for approximately 30 seconds followed by sonication of the bead suspension for 30 seconds in a small bath sonicator. 5. Never pipette by mouth. Avoid contact of reagents and patient specimens with skin and mucous membranes. 6. Avoid microbial contamination of reagents. Incorrect results may occur. 7. Cross contamination of reagents and/or samples could cause erroneous results. 8. Strict adherence to the specified time and temperature of incubations is essential for accurate results. Allow all reagents to reach room temperature (20-25 C) before starting the assay. Return unused reagents to refrigerated temperature immediately after use. 9. Avoid splashing or generation of aerosols. 10. Treat the waste solution with 10% household bleach (0.5% sodium hypochlorite). Avoid exposure of reagents to bleach fumes. 11. Do not expose any of the reactive reagents to bleach-containing solutions or to any strong odors from bleach-containing solutions. Trace amounts of bleach (sodium hypochlorite) may destroy the biological activity of many of the reactive reagents within this kit. WARNINGS 1. For in vitro diagnostic use only. 2. CAUTION! POTENTIAL BIOHAZARD: The controls contain human source material. Using FDA-approved test methods, findings show that derivative source materials were negative for HIV-1 antigen, HBs antigens, and for antibodies against HCV and HIV. However, since no test method can offer complete assurance that infectious agents are absent, these products should be handled at the Biosafety Level 2. Follow recommendations for any potentially infectious human serum or blood specimen found in the Centers for Disease Control/National Institutes of Health manual Biosafety in Microbiological and Biomedical Laboratories : current edition (6); and OSHA s Standard for Bloodborne Pathogens (7). The AtheNA Multi-Lyte Plus Test System conjugated microspheres do not contain viable organisms. However, the reagent should be considered a POTENTIAL BIOHAZARD and handled accordingly. 3. Caution: Sodium azide can react with copper and lead plumbing to form explosive metal azides. On disposal, flush reagents with a large volume of water to prevent the buildup of azides if disposal into a drain is in compliance with Federal, State and local requirements. 4. Normal precautions exercised in handling laboratory reagents should be followed when performing the AtheNA Multi-Lyte Plus Test System. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, gloves, and eye/face protection. Never pipette by mouth. Avoid contact of reagents and patient specimens with skin and mucous membranes. Do not breathe vapor. Dispose of hazardous or biologically contaminated materials according to the practices of your institution. Discard all materials in a safe and acceptable manner, and in compliance with all Federal, State, and local requirements. MATERIALS REQUIRED BUT NOT PROVIDED 1. AtheNA Multi-Lyte System (Luminex 100 or 200IS instrument). 2. Pipettes capable of accurately delivering 10 to 200µL. 3. Multichannel pipette capable of accurately delivering (10 to 200µL). 4. Reagent reservoirs for multichannel pipettes. 5. Disposable pipette tips. 6. Laboratory timer to monitor incubation steps. 7. Small bath sonicator. 8. Plate shaker capable of shaking at 800 RPM (optional for mixing). 9. Vacuum aspirator and vacuum manifold for washing the microspheres. SPECIMEN COLLECTION It is recommended that specimen collection be carried out in accordance with NCCLS/CLSI document M29: Protection of Laboratory Workers from Infectious Disease (8). No known test method can offer complete assurance that human blood samples will not transmit infection. Therefore, consider all blood derivatives potentially infectious. Only freshly drawn and properly stored sera obtained by approved aseptic venipuncture procedures should be used in this assay. Do not use sera containing anticoagulants or preservatives. Do not use hemolyzed, icteric, lipemic, or bacterially contaminated sera. Store sample at room temperature for no longer than 8 hours. If performance of testing will not occur within 8 hours, sera may be stored at 2-8 C, but for no longer than 48 hours. If anticipating a longer delay in testing, store test sera at -20 C or lower. Avoid multiple freeze/thaw cycles, which may cause loss of antibody activity and give erroneous results. Seal specimen containers tightly before storage. For freezing, the use of self-sealing, air tight tubes is recommended. Whenever possible, avoid the use of self-defrosting freezers because of the danger of desiccation of specimens. Refer to CLSI H18-A2: Procedures in for the Handling and Processing of Blood Specimens (9). Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

4 ASSAY PROCEDURE: Set-up of the Assay: Remove the individual components from storage and allow them to warm to room temperature (20-25 C). Determine the total number of controls and samples to be tested. It is necessary to include the Negative Control and the Positive Controls with each run. The Negative Control should be tested in well A1. The Positive Controls should be tested in wells B1and C1. Each control and sample requires one microwell for processing. Note 1: To optimize read times the Bead Suspension must be thoroughly mixed just prior to use. The most effective means to resuspend the beads is to first vortex the Bead Suspension for approximately 30 seconds and then sonicate for approximately 30 seconds in a small bath sonicator. Note 2: For proper performance, it is important that the contents of the assay are thoroughly mixed. Suitable means of mixing include mixing the plate on a plate shaker for approximately 30 seconds at approximately 800 RPM or to set a pipettor to roughly ½ of the volume in the plate and repeatedly aspirate and expel (pump up and down) the contents of the well for a minimum of 5 cycles. STORAGE CONDITIONS 1. Store the unopened kit at 2-8 C. Kit reagents are stable through the end of the month indicated by the label expiration date. Do not use reagents beyond the expiration date. 2. Multiplex Bead Suspension: Store at 2-8 C. After re-suspending the beads, remove only the required amount of solution to analyze the specimens to be tested and return the unused portion to storage at 2-8 C. 3. Phycoerythrin conjugated goat anti-human IgG antibody: Store at 2-8 C. 4. Phycoerythrin conjugated goat anti-human IgM antibody: Store at 2-8 C. 5. Human Controls: Store at 2-8 C. 6. SAVe Diluent : Store at 2-8 C. 7. Wash Buffer Concentrate (10X). Store at 2-25 o C. Diluted Wash Buffer (1X) is stable at room temperature (20-25 o C) for up to 7 days or for 30 days at 2-8 o C. ASSAY PROCEDURE SERUM INCUBATION: 1. Prepare a 1:21 dilution of the Negative Control, the Positive Controls and each of the patient sera. (Example: Combine 10µL of serum with 200µL of SAVe Diluent). The Diluent will undergo a color change confirming that the specimen has been combined with the diluent. For proper performance, it is important that the sample dilutions be thoroughly mixed. Mix according to Note 2 above. 2. After determining the total number of wells to process, use a multichannel or a repeating pipette to dispense 50µL of the Bead Suspension 1 into each of the wells of the filtration plate (re-suspend beads according to Note 1 above). 3. Transfer 10µL of each diluted sample (1:21) and control from the dilution plate to the filtration plate. For proper performance, it is important that the sample dilution and Bead Suspension be thoroughly mixed. Mix according to Note 2 above. 4. Incubate the plate at room temperature (20-25 C) for 30 ± 10 minutes. 5. After the incubation, wash the beads by vacuum filtration: a. Place the filtration plate on the vacuum manifold and remove the solution, leaving the beads behind. b. Turn off the vacuum and add 200µL of Diluted Wash Buffer (1X). c. Apply the vacuum and remove the solution. d. Repeat steps 5.b and 5.c for a total of three rinses with the Diluted Wash Buffer (1X). 6. Following the final wash gently blot the bottom of the filter plate and allow the filter plate dry for 3-5 minutes before proceeding to the next step. 7. Add 150µL of Conjugate 1 to each well at the same rate and in the same order as the specimens were added. For proper performance, it is important that the conjugate solution and Bead Suspension be thoroughly mixed. Mix according to Note 2 above. 8. Incubate the plate at room temperature (20-25 C) for 30 ±10 minutes. 9. After the incubation, wash the Beads by vacuum filtration according to step 5 and 6 above. 10. After the washes add 50µL of the Bead Suspension 2 into each of the wells of the filtration plate. Re-suspend Beads according to Note Transfer 10 µl of each diluted sample (1:21) and control prepared in step 1 from the dilution plate to the filtration plate. For proper performance, it is important that the sample dilution and Bead Suspension be thoroughly mixed. Mix according to Note 2 above. 12. Incubate the plate at room temperature (20-25 C) for 30 ±10 minutes. 13. After the incubation, wash the Beads by vacuum filtration according to step 5 and 6 above. 14. Add 150µL of the Conjugate 2 to each well at the same rate and in the same order as the specimens were added. For proper performance, it is important that the conjugate solution and Bead Suspension be thoroughly mixed. Mix according to Note 2 above. 15. Incubate the plate at room temperature (20-25 C) for 30 ±10 minutes. 16. After the incubation, wash the Beads by vacuum filtration according to step 5 and 6 above. 17. After the final wash step re-suspend the microspheres in 150µL of Diluted Wash Buffer (1X). Mix the Beads according to Note 2 above. 4 Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088

5 ABBREVIATED ASSAY PROTOCOL: Step Procedure 1. Dilute specimens 1:21 (10µl specimen plus 200µl diluent) in SAVe Diluent. Mix well. 2. Dispense 50µL of Bead Suspension 1 into each of the wells of the empty filter plate. Transfer 10µL of each diluted specimen from the dilution plate to the filter plate containing the Bead Mix. For proper performance, it is important that the sample dilution and Bead Suspension be thoroughly mixed. 3. Incubate the plate at room temperature for 30 ±10 minutes 4. Wash the microspheres 3X with 200µl Diluted Wash Buffer (1X) each rinse. 5. Add 150µL of Conjugate 1 to each well. Mix well. 6. Incubate at room temperature for 30 ±10 minutes. 7. Wash the microspheres 3X with 200µl Diluted Wash Buffer (1X). 8. Add 50µL of bead suspension 2 to the wells of the filter plate. Then add 10µL of diluted specimen to the wells containing Bead Suspension B. Mix well. 9. Incubate at room temperature for 30 ±10 minutes. 10. Wash the microspheres 3X with 200µl Diluted Wash Buffer (1X). 11. Add 150µL of Conjugate 2 to each well. Mix well. 12. Incubate at room temperature for 30 ±10 minutes. 13. Wash the microspheres 3X with 200µl Diluted Wash buffer (1X). 14. Re-suspend the microspheres in 150µl of Diluted Wash Buffer (1X) and read the results within 60 minutes. SPECIMEN ANALYSIS: 1. Note: For proper specimen analysis, it is important that the instrument is set-up, calibrated and maintained according to the manufacturer s instructions. Please review the instrument manual for instrument preparation prior to reading the assay results. 2. Set the AtheNA Multi-Lyte instrument to analyze the reactions by selecting the ZEUS ScientificAtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System (VlsE-1 + pepc10) template. Refer to the operator s manual for details regarding the operation of the AtheNA Multi-Lyte instrument. 3. The plate should be read within 60 minutes after the completion of the conjugate incubation. One may decide to shake the plate for approximately 15 seconds prior to reading. This optional step may reduce the amount of time required to read the plate. CONVERSION OF FLUORESCENCE TO AtheNA SCORE: 1. Assay Calibration The AtheNA Multi-Lyte Plus Test System utilizes ZEUS Scientific s patented Intra-Well Calibration Technology. Intra-Well Calibration Technology includes a multi-point standard curve within the bead suspension. With Intra-Well Calibration Technology, each well of the assay is calibrated internally without any user intervention. The standard curve is designed to self-adjust based upon the unique characteristics of the patient or control serum. Calibrator values are assigned to the internal standards by the manufacturer. These values are lot specific and are encoded within the lot specific Calibration CD included in the kit box. 2. Analyte Measurement Each analyte of the AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Test System is measured individually (producing an intermediate value for each), and the results of the two assays are converted into a single Bioinformatics Score (BIS). Assay calibrator values are lot specific and are determined by the manufacturer for each kit lot. These values are encoded within the lot specific Calibration CD included in the kit box. 3. Calculations Through Intra-Well Calibration Technology, all calculations are performed automatically when using the AtheNA Multi-Lyte system. Intra-Well Calibration Technology performs a regression analysis of the internal standards and then adjusts the calculated unit values based upon an additional standard and the characteristics of the serum sample. Finally, the individual VlsE1 and pepc10 results are combined into a single BIS. QUALITY CONTROL 1. Each time the assay is run, it is necessary to include the Negative Control (in well A1) and the Positive Controls (in well B1 and C1). The Positive and Negative Controls are intended to monitor for substantial reagent failure. 2. Run validity is determined through the performance of the positive and negative controls. These criteria are analyzed automatically through Intra-Well Calibration Technology. a. The Negative Control and the Positive Control must all be negative on the non-specific or control antigen bead. b. The Negative Control must be negative for every analyte included in the multiplexed bead suspension. c. The Positive Controls must be positive for one of the two analytes included in the multiplexed bead suspension. These ranges are lot specific and are encoded within the Calibration CD. PC ranges may be viewed by clicking on the Control Graphs button of the AtheNA Multi-Lyte software and then clicking Control Upper/Lower Limits. d. If any of the above criteria are not met, do not report the patient results. The entire run will be considered invalid and should be repeated. Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

6 3. Specimen validity is based upon the characteristics of the calibration beads and their interactions with the patient sera. There are various parameters monitored automatically through Intra-Well Calibration Technology. If any of the criteria are found to be out of specification, the patient s results are considered invalid and should be repeated. Should this occur, the data report would indicate the particular specimen that has been invalidated, as well as a trouble shooting code. If a specimen is repeatedly invalid, it must be tested using an alternate methodology since it is incompatible with the AtheNA Multi-Lyte Plus Test System. 4. Additional controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. External controls must be representative of normal human serum since AtheNA Multi-Lyte s calibration system is partially based upon the characteristics of the serum sample. If the specimen formulation is artificial (not human serum), erroneous results may occur. INTERPRETATION OF RESULTS Cut off Determination: The cut off for this assay was established using 25 negative control specimens as well as 5 clinically characterized specimens for each antigen. The mean and standard deviation was established for the negative population. Using a mathematical calculation involving this data, a theoretical cut-off is established and validated with the characterized specimens. Based upon the results of this testing, the manufacturer has established the following guidelines for interpretation of patient samples. Bioinformatics Score Result Interpretation (BIS) <1 Negative An AtheNA Multi-Lyte BIS of less than 1 indicates no significant amount of antibodies to VlsE1 and pepc10 detected. If exposure to B. burgdorferi is suspected, a second sample should be collected and tested two to four weeks later. 1 Positive An AtheNA Multi-Lyte BIS of greater than or equal to 1 indicates that antibodies specific to B.burgdorferi were detected. This indicates presumptive evidence of probable exposure. Note: If there is too much activity on the NSC (non-specific control) bead, Intra-Well Calibration Technology will invalidate that particular specimen. Invalid specimens should be re-tested. Repeatedly invalid specimens should be re-tested using an alternate procedure. LIMITATION OF THE ASSAY 1. Interpret test results in conjunction with the clinical evaluation and the results of other diagnostic procedures. 2. Do not perform as a screening procedure for the general population. The predictive value of a positive or negative result depends on the prevalence of analyte (antibodies present to VlsE-1 and pepc10 antigens) in a given patient population. Test only when clinical evidence suggests the diagnosis of Borrelia infection or related etiological conditions observed by the physicians. 3. Hemolytic, icteric, or lipemic samples and specimens with abnormal IgG and RF antibody concentrations may interfere with the outcome of this assay. Avoid the use of these types of specimens. 4. Interpret test results of specimens from immunosuppressed patients with caution. 5. Performance characteristics of this device have not been established for matrices other than serum. 6. Performance characteristics of this device have not been established with specimens containing heterophile antibodies, which are known to cause false positive results in various immunoassays. 7. Specimens known to contain potentially cross reactive antibodies to B.burgdorferi with infections to tick-borne relapsing fever, rickettsial diseases, ehrlichiosis, babesiosis, and leptospirosis have not been tested, therefore the performance of this device is unknown if there is any cross-reactivity with these antibodies. 8. This assay is platform dependant and used in conjunction with the Luminex 100 or 200 IS system and the AtheNA Multi- Lyte Test System Data Analysis Package. EXPECTED RESULTS Demographics and Age Distribution in a Prospective Population: Internal and external investigators assessed the device s performance with 756 masked samples prospectively collected from patients between the ages of 1 and 94 that were submitted for Borreila antibody testing. Site 1, a hospital laboratory located in the northeast tested 107 samples. Site 2, a hospital laboratory in the northeast tested 103 samples. The third clinical site was a state Department of Health located in the northeast. This facility tested 446 samples collected in the northeast. Demographics for 346 of the 756 samples were unavailable. Site 4, the manufacturer s research facility tested 100 samples collected in Connecticut. The available patient demographics and age distribution for 410 of the 756 samples are summarized below followed by expected results in the prospective population in Table 1. 6 Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088

7 Summary of Patient Demographics (Prospective Samples): Distribution of Patient Age and Sex Frequency < Age Group Total Males Females Table 1: AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Results from the Prospective Study Age Specimen Group Positive Negative 1-9 Males 7 14 Females Males 1 13 Females Males 13 Females Males 1 19 Females Males 3 25 Females Males 3 41 Females Males 4 28 Females Males 7 15 Females 1 27 Age / Sex Unknown 1 15 Total: Prospective Males Prospective Females Female age Unknown 1 Age / Sex Unknown Total Grand Total 756 Other Studies: Internal and external investigators assessed the device s performance with varying populations. The available patient demographics, quantity of samples tested and the number of samples which tested positive for each population are summarized in Table 2. Table 2: AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Results from Other Populations Populations Number Tested Gender Male Female Age Range Positive/Tested Characterized 229 *NA NA NA 171/229 Retrospective /242 Endemic Controls 300 NA NA NA 38/300 Non-Endemic Controls 400 NA NA NA 39/400 *Not available Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

8 PERFORMANCE CHARACTERISTICS CLINICAL STUDIES AND METHOD COMPARISON WITH A COMMERCIALLY MARKETED ELISA PREDICATE DEVICE: The clinical studies consisted of 1,967 serum samples evaluated at four sites located in the United States. All serum samples evaluated for concordance were tested with the ELISA (IgG/IgM) reference assay. The following populations were tested at a total of four clinical sites: Sites 1 and 2 were hospital laboratories located in the northeast, site 3 was a state Department of Health Laboratory located in the northeast, and site 4 was the manufacturer s research facility. 1. Characterized Samples 2. Prospective Population 3. Retrospective Samples 4. CDC Lyme Panel 5. Endemic and Non-Endemic Control Samples 6. Precision and Reproducibility STUDY 1: CHARACTERIZED SAMPLES Two hundred and twenty-nine characterized serum samples were acquired and tested at a northeastern state Department of Health Laboratory. Twenty-one samples were acute patients with a history of Borreliosis. Fifty samples were from convalescent patients with a history of Borreliosis; 14 of these patients presented with neurological, 2 with cardiac and 34 with arthritic symptoms. Seventy-nine samples were paired acute (culture proven, early acute Lyme disease) and early convalescent sera from these same patients. Table 3: Characterized Samples - Summary of Comparative Testing Results AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Predicate ELISA(IgG/IgM) Western Blot(IgG and/or IgM) Clinical Diagnosis Pos Neg Total % agreement with clinical diagnosis Pos Neg or Eqv Total % agreement with clinical diagnosis Pos Neg Total % agreement with clinical diagnosis Acute % (21/21) % (21/21) % (20/21) 95% CI 86.7%-100% 86.7%-100% 76.2%-99.9% Convalescent % (47/50) % (50/50) % (43/50) 95% CI 83.5%-98.8% 94.2%-100% 73.3%-94.2% Culture (+) early acute % (41/79) * 47.4% (37/78) * 58.5% (31/53) 95% CI 40.4%-63.3% 36.0%-59.1% 44.1%-71.9% Early Convalescent % (62/79) * 93.6 (73/78) * 82.7% (62/75) 95% CI 67.8%-86.9% 85.7%-97.9% 72.2%-90.4% Total % (171/229) % (181/227) % (156/199) 95% CI 68.5%-80.2% 73.9%-84.8% 72.0%-83.9% *one sample invalid *blot results unavailable for all 79 samples STUDY 2: PROSPECTIVE POPULATION A total of 756 unselected samples from patients with an order for a Borrelia antibody test were included in the study. The samples submitted for Borrelia antibody testing were sequentially numbered, de-identified and archived. After the collection, 103 samples were tested at a hospital laboratory located in the Mid-Atlantic, 100 samples were tested at a hospital laboratory in upper Connecticut, 107 samples were tested at a hospital laboratory in lower Connecticut and 446 samples were tested at a state Department of Health Lab, also located in the northeast. Table 4: Prospective Samples - Summary of Comparative Testing Results. Predicate ELISA (IgG/IgM) Positive Equivocal Negative Site Total PPA NPA 95% CI AtheNA Multi- Lyte Borrelia VlsE-1/ pepc10 Positive %(162/199) Equivocal Negative %(509/557) Site Total If available, western blot testing was performed on the discrepant results. 12/31 samples were negative by blot and 9/31 samples were positive by blot for sera which tested negative on the AtheNA Multi-Lyte test system and positive by ELISA (10/31) samples had no blot data provided). The 3 equivocal samples by the predicate would be considered for second step Western blot testing along with positives. 7/45 samples tested positive and 5/45 samples tested negative by blot for the discrepant samples that were positive on the AtheNA Multi-Lyte test system and negative by ELISA (33 samples had no blot data available). 8 Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088

9 STUDY 3: RETROSPECTIVE SAMPLES 242 samples believed to have screened positive for Borrelia burgdorferi antibodies were tested at two external sites. 124 samples were tested in a hospital facility in Connecticut and 118 samples were tested in a Pennsylvania hospital laboratory. Table 5: Retrospective Samples - Summary of Comparative Testing Results. Predicate ELISA (IgG/IgM) Positive Equivocal Negative Site Total PPA NPA 95% CI AtheNA Multi- Lyte Borrelia VlsE-1/ pepc10 Positive % (180/225) Equivocal Negative %(6/17)* Site Total Western blot testing was done on discrepant results. 2/39 samples were negative by blot and 37/39 samples were positive by blot for sera which tested negative on the AtheNA Multi-Lyte test system and positive by ELISA. 4/4 samples tested positive by blot for the discrepant samples that were positive on the AtheNA Multi-Lyte test system and negative by ELISA. *Statistical significance evaluation can not be made on limited number of samples. STUDY 4: CDC CHARACTERIZED LYME PANEL 40 samples of various reactivity were acquired from the CDC and evaluated internally at the manufacturer s site. 5 samples were from normal blood donors. 35 samples were from patients diagnosed with Borreliosis. The results of the testing are presented here as a means of conveying further information on the performance of this assay with a characterized serum panel. This does not imply an endorsement of the assay by the CDC. Table 6: CDC Characterized Lyme Panel - Summary of Comparative Testing Results AtheNA Multi-Lyte Borrelia Predicate ELISA(IgG/IgM) VlsE-1/pepC10 Time % agreement Neg % agreement From Pos Neg Total with clinical Pos or Total with clinical Onset diagnosis Eqv diagnosis Western Blot (IgG and/or IgM) Pos Neg Total % agreement with clinical diagnosis Normals % (5/5) % (5/5) % (5/5) 0-1 month % (3/3) % (3/3) % (3/3) 1-2 months % (5/9) % (8/9) % (6/9) 81.3% 3-12 months % (11/16) (13/16) % (11/16) >12 months % (6/7) % (7/7) (6/7) Total % (25/40) % (31/40) % (26/40) STUDY 5: ANALYTICAL SPECIFICITY Testing of normal population was done on 300 samples acquired from blood donors in the New England endemic area and 400 samples acquired from blood donors and individuals undergoing routine testing not infectious in nature in the New Mexico nonendemic area. Table 7: Analytical Specificity Sample Type Number Negative Positive % Positivity* Endemic % Non-endemic % *% positivity with the predicate was found to be: endemic =14.3%; non-endemic-= 6.5%. Reproducibility: Two separate studies were done to assess reproducibility; one was a five day, three site reproducibility study and the second was a 12 day single site repeatability study. Both studies evaluated the measurement of the individual VlsE-1 and pepc10 intermediate values (AU/mL) and not the BIS. Assay reproducibility was evaluated at three external clinical sites. The study was conducted as follows: Five samples were identified and/or prepared (by ZEUS Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte test system. Selected samples were negative, near cut-off, low positive, and moderate and high positive. To assess reproducibility, on each day of testing, each sample was diluted twice and then each dilution was run in triplicate. This was done twice per day by two different technicians, and was repeated for five days. Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

10 Table 8:Summary Of Reproducibility Panel Member Sample Mean Within-Run Within -Day Between-Run/Day Between-Site Total N AU/mL SD %CV SD %CV SD %CV SD %CV SD %CV VlsE-1 Negative VlsE-1 Near Cut-off VlsE-1 Low Positive VlsE-1 Moderate Positive VlsE-1 High Positive pepc10 Negative pepc10 Near Cut-off pepc10 Low Positive pepc10 Moderate Positive pepc10 High Positive PRECISION: Assay repeatability was evaluated at the manufacturer site. The study was conducted as follows: six samples were identified and/or prepared (by ZEUS Scientific, Inc.) for use in the study based upon their activity on the AtheNA Multi-Lyte test system. Selected samples were negative, high negative, near cut-off, low positive, and moderate and high positive. On each day of testing, the samples were diluted twice and tested. This was repeated in a second run on the same day by a different technologist for a total of twelve days. Table 9.Summary of Repeatability Panel Member Sample N Mean AU/mL Within Run Within Day Total SD %CV SD %CV SD %CV VlsE-1Negative VlsE-1 high negative near cut-off low pos mod pos high pos pepc10 Negative pepc10high neg pepc10 near cut-off pepc10 low pos pepc10 mod pos pepc10 high pos CROSS REACTIVITY: ZEUS Scientific, Inc. conducted a study to assess cross reactivity with the AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus test system using sera that were sero-positive to EBV VCA IgG, RF, ANA, T.pallidum, CMV IgG, CMV IgM, Rubella, VZV IgM and Toxoplasma. ELISA, IFA and micro-particle immunoassay test systems manufactured by various companies for commercial distribution were used to determine the sero-positivity of the samples. Ten samples for each possible cross-reactant were tested. The cross reactivity data has been summarized in table 12. In total, 90 samples were tested for possible cross reactivity with 9 analytes. None of the ninety samples showed cross-reactivity with any of the nine analytes tested. Table 10: Cross Reactivity Summary AtheNA Multi-Lyte Borrelia VlsE-1/pepC10 Plus Cross Reactivity Study PossibleCross-Reactants Positive Results/Number Tested EBV VCA IgG 0 / 10 ANA 0 / 10 T. pallidum 0 / 10 CMV IgG 0 / 10 CMV IgM 0 / 10 Rubella IgG 0 / 10 Toxo IgG 0 / 10 VZV IgM 0 / 10 RF 0 / Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088

11 INTERFERING SUBSTANCES: The effect of potential interfering substances on sample results generated using the AtheNA Multi-Lyte VlsE-1/pepC10 Plus test system was evaluated with the following possible interfering substances: albumin, bilirubin, cholesterol, hemoglobin, triglycerides and intralipids. The quantity of analyte in each interfering substance is as follows: Bilirubin: 1mg/dL (low), 15 mg/dl (high) Albumin: 3.5 g/dl (low), 5 g/dl (high) Cholesterol: 150 mg/dl (low), 250 mg/dl (high) Triglycerides: 150 mg/dl (low), 500 mg/dl (high) Hemoglobin: 20 g/dl (low), 20 g/dl (high) Intralipid: 300 mg/dl (low), 750 mg/dl (high) Three samples each for VlsE-1 and pepc10 were chosen based on their performance on the AtheNA Multi-Lyte Borrelia VlsE- 1/pepC10 Plus test system: (strongly reactive, weakly reactive and negative). The samples were exposed to the possible interfering substance, tested in duplicate and mean was determined. All samples showed less than a 20% change in signal in the VlsE-1 study with the exception of the borderline VlsE-1 sample which exhibited an increase in signal of 32% with the high spike of bilirubin and an increase in signal of 27% with the high spike of cholesterol. The negative VlsE-1 sample showed a reduction in signal of 36% with the high spike of hemoglobin, a change in signal of 23% with the low spike of bilirubin and 27% with the high spike of bilirubin, a change in signal of 25% with the low spike of cholesterol and 45% with the high spike of cholesterol and a change in signal of 45% with both the low and high spikes of triglycerides. The change of signal in these negative samples did not change the qualitative outcome (or BIS), the results remained negative. All samples showed less than a 20% change in signal in the pepc10 study with the exception of the borderline pepc10 sample which exhibited a reduction in signal of 24% with the high spike of hemoglobin and an increase in signal of 28% with the low spike of triglyceride. REFERENCES 1. Steere AC, et al: J. Infect. Dis. 154: , Rosenfeld MEA:Serodiagnosis of Lyme disease. J. Clin. Microbiol. 31: , Steere AC, et al:the Spirochetal Etiology of Lyme Disease. N. Engl. J. Med. 308: , Bakken LL, Callister SM, Wand PJ, and Schell RF:Interlaboratory Comparison of Test Results for Detection of Lyme Disease by 516 Patients in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Proficiency Testing Program. J. Clin. Microbiol. 35: , Bacon R M, et al: J. Infect. Dis. 187: , U.S. Department of Health and Human Services. Public Health Service. Centers for Disease Control and Prevention and National Institutes of Health. U.S. Government Printing Office, Washington D.C., 4th Ed., U.S. Department of Labor, Occupational Safety and Health Administration; Occupational Exposure to Bloodborne Pathogens, Final Rule. Fed.Register 56: , Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids and Tissues; Approved Guideline. NCCLS/CLSI Document M29, Vol.17 (12), Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. NCCLS/CLSI H18-A2, Vol.19 (21), Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR

12 Marketed exclusively in the USA by: Manufacturer: 2 Research Way ZEUS Scientific, Inc. Princeton, NJ Evans Way, Branchburg, New Jersey, 08876, USA Send correspondence to: For Technical Service: P.O. Box 38, Raritan, New Jersey 08869, USA Telephone: For Technical Service call Toll Free: EXT-2 Fax: Phone: * FAX: US.TechSupport@alere.com info@zeusscientific.com website: For Customer Service: Telephone: ZEUS Authorized Representative: inverness medical is a registered trademark of the Inverness Medical family of products. All rights reserved. ZEUS Scientific, Inc. - Printed in U.S.A. 12 Borrelia VlsE-1/pepC10 Plus/ISSUE DATE: /R2755/DMR-00088