The presence of anti-vca IgM together with anti-ebna and anti-ea-r are associated with reactivation of the latent viral carrier state (13, 14).

Transcription

1 . Sumaya CV: Endogenous reactivation of Epstein-Barr virus infections. J. Infect. Dis. :7, 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., th Ed., U.S. Department of Labor, Occupational Safety and Health Administration; Occupational Exposure to Bloodborne Pathogens, Final Rule. Fed.Register :7-8, Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids and Tissues; Approved Guideline. NCCLS Document M9, Vol.7(), CLSI. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline. CLSI document EP-A. CLSI, 9 West Valley Road, Suite, Wayne, Pennsylvania USA,. 9. CLSI. How to define, determine, and utilize reference intervals in the clinical laboratory; approved guideline. CLSI Document C8-A. CLSI, 9 West Valley Road, Suite, Wayne, Pennsylvania USA;99. EBV VCA IgM Plus 8 Test System A Multiplexed, Microparticle-Based Immunoassay for IgM Antibodies to Epstein Barr Virus (EBV), Viral Capsid Antigen (VCA) Product Number: A9MB Marketed exclusively in the USA by: Research Way Princeton, NJ 8 For Customer Service (USA): enter # For Technical Service (USA): enter # Fax: Manufactured by: AtheNA Multi-Lyte EBV VCA IgM Plus 8/Issue Date: 8-9-/K9/R7PEB/FCD- Zeus Scientific, Inc. Printed in USA Zeus Scientific, Inc. P.O. Box 8, Raritan, New Jersey 889, USA For Technical Service call Toll Free: 8-8- Phone: * FAX: info@zeusscientific.com Zeus Authorized Representative: Emergo Europe, Molenstraat, BH, The Hague, The Netherlands INTENDED USE The Zeus Scientific, Inc. AtheNA Multi-Lyte EBV VCA IgM Plus 8 Test System is a microparticle-based immunoassay intended for the qualitative detection of IgM class antibody to the Epstein-Barr virus, viral capsid antigen in human serum using the AtheNA Multi-Lyte System. The test system is intended to be used for the laboratory diagnosis of EBV-associated infectious mononucleosis and provides epidemiological information on the diseases caused by Epstein-Barr virus. Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients, cord blood, neonatal specimens, or infants. Assay performance characteristics have not been established for the diagnosis of nasopharyngeal carcinoma, Burkitt's lymphoma, and other EBV-associated lymphomas. SUMMARY AND EXPLANATION OF THE TEST Epstein-Barr Virus (EBV) is a ubiquitous human virus which causes infectious mononucleosis (IM), a self limiting lymphoproliferative disease (). By late adulthood virtually everyone has been infected with the virus. In underdeveloped countries, seroconversion to the virus takes place in early childhood and is usually asymptomatic (). In more affluent countries, primary EBV infections are often delayed until adolescence or later, and manifests as IM in about % of this age group (-). Following seroconversion, whether symptomatic or not, EBV establishes a chronic, latent infection in B lymphocytes which probably lasts for life (). EBV replicates in oropharyngeal epithelial cells and is present in the saliva of most patients with IM (7). Also, -% of healthy persons who are EBV antibody positive shed the virus in their oral secretions (,7,8). Reactivation of the latent viral carrier state, as evidenced by increased rates of virus shedding, is enhanced by immunosuppression, pregnancy, malnutrition, or disease (8, 9). Chronic EBV infections, whether latent or active, are rarely associated with disease. The Paul-Bunnell-Davidsohn test for heterophile antibody is highly specific for IM (). However, -% of adults and higher percentages of children and infants with primary EBV infections do not develop heterophile antibodies (). In these situations EBV-specific serological tests are needed to differentiate primary EBV infections that are heterophile negative from mononucleosis-like illnesses caused by other agents such as cytomegalovirus, adenovirus, and Toxoplasma gondii (). The presence of antibody to specific EBV antigens correlates with different stages of IM (, -). Both IgM and IgG antibodies to the viral capsid antigen (VCA) peak to weeks after primary EBV infection. IgM anti-vca decline rapidly and is usually undetectable after weeks. The presence of anti-vca IgM together with anti-ebna and anti-ea-r are associated with reactivation of the latent viral carrier state (, ). PRINCIPLE OF THE AtheNA Multi-Lyte EBV VCA IgM PLUS 8 ASSAY The Zeus Scientific, Inc. AtheNA Multi-Lyte EBV VCA IgM Plus 8 Test System is designed to detect IgM class antibodies in human sera to EBV VCA antigen. The test procedure involves two incubation steps:. Diluted test sera are incubated in a vessel containing a multiplexed mixture of the bead suspension. The multiplexed bead suspension contains a mixture of distinguishable sets of polystyrene microspheres. Conjugated to the primary set of microspheres is the EBV viral capsid antigen. The bead mix also contains one bead set designed to detect non-specific and/or RF IgM antibodies in the patient sample (if present) and four separate bead sets used for assay calibration. If present in patient sera, antibodies to the EBV viral capsid antigen will bind to the immobilized antigen on the primary bead set. The microspheres are rinsed to remove non-reactive serum proteins.. Phycoerythrin-conjugated goat anti-human IgM (µ chain specific) is added to the vessel and the plate is incubated. The conjugate will react with IgM antibody immobilized on the solid phase in step. The bead suspension is then analyzed by the AtheNA Multi-Lyte instrument. 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 establish the assay s cutoff. KIT COMPONENTS Note: 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 expose these reagents to strong sources of visible light unnecessarily.

2 Reactive Reagents: (All reactive reagents contain sodium azide as a preservative at a concentration of.% w/v).. Multiplexed bead suspension. Ready to use,. ml bottles. The suspension contains separate distinguishable. micron polystyrene beads that are conjugated with EBV VCA gp antigen (affinity purified from EBV infected cell lines). The bead mix also contains one bead set designed to detect non-specific and/or RF IgM antibodies in the patient sample (if present) and four separate bead sets used for assay calibration.. Phycoerythrin conjugated goat anti-human IgM (µ chain specific). Ready to use, ml amber bottles.. Human positive serum control. Three,.mL vials.. Human negative serum control. Three,.mL vials.. SAVe Diluent. Three ml bottles containing phosphate-buffered-saline. Ready to use. NOTE, the sample diluent will change color in the presence of serum.. Wash Buffer Concentrate: Dilute part concentrate + 9 parts deionized or distilled water. Five ml bottles containing X concentrate of phosphate buffered saline. Non-reactive Components:. Five, 9-well filtration plates for rinsing the microspheres. Data Labels: One label is adhered to the inside lid of the kit box and a second label is inside the kit box.. Package Insert providing instructions for use. Calibration CD. A compact disc that includes all lot-specific kit calibration values required for specimen analysis and assay quality control WARNINGS. For In Vitro Diagnostic Use. CAUTION! POTENTIAL BIOHAZARD: The controls contain human source material. Source materials from which these products were derived were found negative for HIV- antigen, HBsAg and for antibodies against HCV and HIV by FDA-approved test methods. However, since no test method can offer complete assurance that infectious agents are absent, these products should be handled at the Biosafety Level as recommended for any potentially infectious human serum or blood specimen in the Centers for Disease Control/National Institutes of Health manual Biosafety in Microbiological and Biomedical Laboratories : current edition (); and OSHA s Standard for Bloodborne Pathogens (). 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.. 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.. Normal precautions exercised in handling laboratory reagents should be followed when performing the AtheNA Multi-Lyte Plus assay. 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. Results of the Interference Study: Sample Sample Sample Percent Positive Percent Positive Percent Positive Spiked Level: VCA M Strong Pos Signal Recovered VCA M Strong Pos Signal Recovered VCA M Weak Pos Signal Recovered Control N/A Bilirubin Low 88.% 7 99.% 8 8.% Bilirubin High 87.% 7.% 7 7.% Albumin Low 7 9.9% % 8.9% Albumin High 7 9.% 78 9.% 77 7.% IgG Low 79 9.% 79 8.%.9% IgG High 8.% 7.% 89.% Cholesterol Low % 78 8.% 8 7.% Cholesterol High 87.% 8 9.% 8.% Triglycerides Low 87 9.% 8.9% 8 7.% Triglycerides High 88.% 77.%.% Hemoglobin Low 7 9.8% 7 8.% 7 7.% Hemoglobin High 8.% 8 9.% 8.% Intralipid Low 78.% 78 8.% 8.8% Intralipid High 78.% 7 9.7% 87.8% All substances tested showed some level of interference with detection of low positive EBV-VCA IgM specimens using the AtheNA Multi-Lyte Plus assay. Recovery of positive signal for low positive Sample ranged from.% to 87.8%, depending on the interferant identity and level tested (see above). Specimens that are hemolytic, icteric, lipemic or that contain elevated levels of IgG should not be tested by the AtheNA Multi-Lyte EBV VCA IgM Plus 8 assay. STORAGE CONDITIONS. Store the unopened kit at -8 C.. Multiplex bead suspension: Store at -8 C. Remove only the required amount of solution to analyze the specimens to be tested and return the unused portion to storage at -8 C.. Phycoerythrin conjugated goat anti-human IgM antibody: Store at -8 C.. Human controls: Store at -8 C.. SAVe Diluent: Store at -8 C.. Wash Buffer Concentrate (X). Store between - o C. Diluted Wash Buffer (X) is stable at room temperature (- o C) for up to 7 days or for days at -8 o C. SPECIMEN COLLECTION It is recommended that specimen collection be carried out in accordance with NCCLS document M9: Protection of Laboratory Workers from Infectious Disease (7). No known test method can offer complete assurance that human blood samples will not transmit infection. Therefore, all blood derivatives should be considered potentially infectious. Only freshly drawn and properly stored sera obtained by approved aseptic venipuncture procedures should be used in this assay. No anticoagulants or preservatives should be added. Do not use hemolyzed, icteric, lipemic, or bacterially contaminated sera. Store sample at room temperature for no longer than 8 hours. If testing is not performed within 8 hours, sera may be stored at -8 C for no longer than 8 hours. If delay in testing is anticipated, store test sera at - C or lower. Avoid multiple freeze/thaw cycles which may cause loss of antibody activity and give erroneous results. Specimen containers should be tightly sealed before storage. For freezing, the use of self-sealing, air tight tubes are recommended. Whenever possible, avoid the use of self-defrosting freezers because of the danger of desiccation of specimens. ASSAY PROCEDURE Materials required but not provided:. AtheNA Multi-Lyte System (Luminex R Instrument). Pipettes capable of accurately delivering to µl. Multichannel pipette capable of accurately delivering (-µl). Reagent reservoirs for multichannel pipettes. Disposable pipette tips. Laboratory timer to monitor incubation steps 7. Small bath sonicator 8. Plate shaker capable of 8 RPM (optional for mixing) REFERENCES. Rapp CE, and Heweston JF: Infectious mononucleosis and the Epstein-Barr virus. Am. J. Dis. Child. :78, Biggar RJ, Henle W, Fleisher G, Bocker J, Lennette ET, and Henle G: Primary Epstein-Barr virus infections in African infants. I: Decline of maternal antibodies and time of infection. Int. J. Cancer. :9, Fry J: Infectious mononucleosis: Some new observations from a year study. J. Fam. Prac. :87, 98.. Lennette ET: Epstein-Barr virus. In: Manual of Clinical Microbiology, th edition. Lennette ET, Balows A, Hausler WJ, Shadomy HJ, eds. Washington DC, American Society for Microbiology, p., Fleisher G, Henle W, Henle G, Lennette ET, and Biggar RJ: Primary infection with Epstein-Barr virus in infants in the United States: Clinical and Serological Observations. J. Infect. Dis. 9:, Merlin TL: Chronic mononucleosis: Pitfalls in the laboratory diagnosis. Hum. Path. 7:, Sixbey JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS: Epstein-Barr virus replication in oropharyngeal epithelial cells. New Eng. J. Med. :, Chang RS, Lewis JP, Reynolds RD, Sullivan MJ, Neuman J: Oropharyngeal excretion of Epstein-Barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts. Ann. Intern. Med. 88:, Jones JF, Ray G, Minnich LL, Hicks MJ, Kibler R, Lucus DO: Evidence of active Epstein-Barr virus infection in patients with persistent, unexplained illness. Elevated anti-early antigen antibodies. Ann. Intern. Med. :, 98.. Evans AS, Neiderman JC, Cenabre LC, West B, and Richards VA: A prospective evaluation of heterophile and Epstein-Barr virusspecific IgM antibody tests in clinical and subclinical infectious mononucleosis: Specificity and sensitivity of the tests and persistence of antibody. J. Infect. Dis. :, 97.. Henle W, Henle GE, and Horowitz CA: Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. Hum. Path. :, 97.. Lennette ET, and Henle W: Epstein-Barr virus infections: Clinical and serological features. Lab Management. p., June, Horowitz CA, Henle W, Henle G, and Schmitz H: Clinical evaluation of patients with infectious mononucleosis and development of antibodies to the R component of the Epstein-Barr virus-induced early antigen complex. Am. J. Med. 8:, 97.

3 The summary of the precision study appears below: AtheNA Multi-Lyte EBV VCA IgM Precision Summary Panel Mean Within Run Day Within Run Day Within Run Day Between Day Between Sites Member U/mL Site SD % CV SD % CV SD % CV SD % CV SD % CV %..%. 7.8%.9 9.8%..%. 8.%.7.%. 7.9%.7.%..7% 9..9%..7%..%.8.% 9..7%.9.9% 9..% 9.78.%.8.% %. 7.%.9 7.% 7.9.%.9 7.%..9% %.97 7.%..%..8% %.7.%.8.%.79.8%..9%..%.9.7%.7 7.%..% 8..%.8.%..%.8.% % %.9.%. 9.% 8.7.%..%..7%.8.9% 7..8%. 9.%. 9.7%.8.%..8%.7 7.9%.7.%.7.8% 7.77.%.9 8.%..8%.7.% % 7..%..9%.7.% 7. 8.% 9..% 8..% 8. 9.% %.8.% 8.8.%.8 9.% 8 9..%.7 7.%.79.%. 7.% CROSS REACTIVITY AND INTERFERRING SUBSTANCES: Cross Reactivity The AtheNA Multi-Lyte EBV VCA IgM Plus 8 test system was evaluated for potential cross reactivity to other antibodies. For this study, a total of specimens were evaluated. Thirteen of the specimens were positive for IgM antibody to other infectious disease agents (Cytomegalovirus, Herpes Simplex Virus, Rubella and Toxoplasma). Of the thirteen specimens evaluated, none were reactive on the AtheNA Multi-Lyte EBV VCA IgM Plus 8 assay. Fourteen specimens were tested that possessed various autoantibodies to nuclear antigens. Of the fourteen specimens tested, none of them were reactive on the AtheNA Multi-Lyte EBV VCA IgM 8 assay. Finally, four specimens were tested that were found to be RF IgM positive. All four specimens yielded invalid results on the AtheNA Multi-Lyte EBV VCA IgM Plus 8 assay. These specimens were invalidated by Intra-Well Calibration Technology, since the non-specific bead contained in the multiplex bead suspension is designed to detect such activity and invalidate the specimens. Interfering Substances: A study was conducted to determine the potential effects of interfering substances that may be found in serum specimens. The following potentially interfering substances were spiked into serum specimens at the levels indicated: Substance Low Spike High Spike Bilirubin.9 mg/dl.8 mg/dl Human Albumin. g/dl g/dl Human IgG.8 g/dl. g/dl Cholesterol mg/dl mg/dl Triglycerides mg/dl mg/dl Hemoglobin 8 g/dl g/dl Intralipids. mg/ml 7. mg/ml It should be noted that the low and high spiked levels were in addition to the base line level of these materials that may have been present in the original sera. The levels in the original sera were not detected. For this study, three EBV IgM positive sera were evaluated in the presence of each of the substances above. Two of the sera selected were clearly positive for EBV VCA IgM and one of the samples selected was weakly reactive for EBV VCA IgM. The results of the control specimens and the low and high spiked sera are presented in the table below: 9. Vacuum aspirator and vacuum manifold for washing the microspheres Procedural Precautions:. Dilution or adulteration of these reagents may generate erroneous results.. Reagents from other sources or manufacturers should not be used.. 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 expose these reagents to strong sources of visible light unnecessarily.. 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 seconds followed by sonication of the bead suspension for seconds in a small bath sonicator.. Never pipette by mouth. Avoid contact of reagents and patient specimens with skin and mucous membranes.. 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. All reagents must be allowed to reach room temperature (- C) before starting the assay. Return unused reagents to refrigerated temperature immediately after use. 9. Avoid splashing or generation of aerosols.. Treat the waste solution with % household bleach (.% sodium hypochlorite). Avoid exposure of reagents to bleach fumes.. 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. Set-up of the Assay: Remove the individual components from storage, protect from strong light, and allow them to warm to room temperature (- C). Determine the total number of controls and samples to be tested. It is necessary to include the Negative Control and the Positive Control with each run. The Negative Control should be tested in well A and the Positive Control should be tested in well B. Each control and sample requires one microwell for processing. Note. 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 seconds and then sonicate for approximately seconds in a small bath sonicator Note. 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 seconds at approximately 8 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 cycles. Serum Incubation. Prepare a : dilution of the Negative Control, the Positive Control and each patient sera. (Example: Combine µl of serum with µl of Sample Diluent). The sample 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 are thoroughly mixed. Mix according to Note above.. After determining the total number of wells to process, use a multichannel or a repeating pipette to dispense µl of the bead suspension into each of the wells of the filtration plate.. Transfer µl of each diluted sample (:) and control from the dilution plate to the filtration plate. For proper performance, it is important that the sample dilution and bead suspension are thoroughly mixed. Mix according to Note above.. Incubate the plate at room temperature (- C) for +/- minutes.. After the incubation, rinse 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 µl of Diluted Wash Buffer (X). c. Apply the vacuum and remove the solution. d. Repeat steps.b and.c for a total of three rinses with the Diluted Wash Buffer (X).. Following the final wash gently blot the bottom of the filter plate and allow the plate to air dry for - minutes before proceeding to the next step. Conjugate Incubation. Add µl of the Conjugate solution 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 are thoroughly mixed. Mix according to Note above. As an option, while mixing the conjugate and the beads, one may transfer the bead/conjugate mixture to empty wells of a polystyrene reaction plate.. Incubate the plate at room temperature (- C) for +/- minutes. Specimen Analysis 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.. Set the AtheNA Multi-Lyte instrument to analyze the reactions by selecting the EBV IgM template. Refer to the operator s manual for details regarding the operation of the AtheNA Multi-Lyte instrument. Results may be read from the filter plate.. The plate should be read within minutes after the completion of the conjugate incubation. If the plate is not read within minutes, the plate should be discarded and the run should be repeated. One may decide to shake the plate for approximately seconds prior to reading. This step may reduce the amount of time required to read the plate.

4 ABBREVIATED ASSAY PROTOCOL: Step Procedure Dilute specimens : in Sample Diluent. Mix well. Combine µl of bead suspension and µl of diluted specimen in an empty well. Mix well. Incubate at room temperature for +/- minutes. Rinse the microspheres X with Diluted Wash Buffer (X). Gently blot the bottom of the plate and air dry for - minutes. Add µl of conjugate to each well. Mix well. 7 Transfer to a reaction plate (optional). 8 Incubate at room temperature for +/- minutes. 9 Shake plate. Read results within minutes. CONVERSION OF FLUORESCENCE TO UNIT VALUES A. Calculations: Assay Calibration The AtheNA Multi-Lyte Plus Test System utilizes Intra-Well Calibration Technology. Intra-Well Calibration Technology includes a multipoint 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 Zeus Scientific, Inc. These values are lot specific and are encoded within the lot specific Calibration CD included in the kit box. Analyte Cut Off Values Each analyte of the AtheNA Multi-Lyte Plus Test System has an assigned cutoff value. Cut off values are determined by Zeus scientific, Inc. for each kit lot, and are encoded within the lot specific Calibration CD included in the kit box. 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. B. Quality Control Caution: The Negative and Positive controls are intended to monitor for substantial reagent failure. The Positive Control will not ensure precision at the assay cutoff.. Each time the assay is run it is necessary to include the Negative Control (in well A) and the Positive Control (in well B).. 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 Control must be positive for a predetermined group of analytes included in the multiplexed bead suspension. These ranges are lot specific and encoded within the Calibration CD. Positive Control 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 patient results. The entire run will be considered invalid and should be repeated.. 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 will indicate the particular specimen which has been invalidated as well as a trouble shooting code. If there is too much activity detected on the non-specific control bead, the specimen results will be invalidated. One probable cause of such a result is the presence of a significant amount of rheumatoid factor IgM antibody in the original sample. 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 the AtheNA Multi-Lyte 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. Good laboratory practice recommends the use of positive and negative controls to assure functionality of reagents and proper performance of the assay procedure. Quality control requirements must be performed in conformance with local, state and/or federal regulations or accreditation requirements and your laboratory s standard Quality Control procedures. It is recommended that the user refer to CLSI EP-A and CFR 9. for guidance on appropriate QC practices 8. C. Interpretation of Results: Cutoff Determination: The cutoff for this assay was originally set against a panel of negative specimens. Each subsequent kit lot has been tested against a panel of characterized specimens, and reported values are normalized using the lot specific Calibration CD included in the kit box. Percent and s by EBV Classification; AtheNA Multi-Lyte EBV VCA IgM Assay vs. EBV VCA IgM Reference ELISA Assay EBV Classification Positive Percent % (x/n) a Prospective Specimens: 9% Exact Negative Percent % (x/n) b 9% Exact Acute 9.%(/) 79.% 99.9% N/A c N/A No infection N/A N/A %(9/9) 9.% - % Past infection N/A N/A 99.%%(/) 97.7% % Indeterminate.9%(8/) 7.7% - 9.% 9.%(/) 8.% - 99.%.7%(/7).% - 7.% 99.%%(7/8) 97.8% - 99.% a b x = the number of AtheNA Multi-Lyte EBV VCA IgM results that are confirmed positive in agreement with the reference EBV VCA IgM confirmed positive results; n = the total number of reference EBV VCA IgM results that are confirmed positive x = the number of AtheNA Multi-Lyte EBV VCA IgM results that are nonreactive in agreement with the reference EBV VCA IgM; n = the total number of reference EBV VCA IgM results that are nonreactive c resulted in / specimens. In such cases, percent agreement and 9% confidence intervals could not be calculated. Percent and s by EBV Classification; AtheNA Multi-Lyte EBV VCA IgM Assay vs. EBV VCA IgM Reference ELISA Assay EBV Classification Retrospective Samples - Expected Acute Positive Percent % (x/n) a 9% Exact Negative Percent % (x/n) b 9% Exact Acute %(8/8) 9.% - % N/A c N/A No infection N/A N/A %(/) N/A Past infection N/A N/A %(/).8% - % Indeterminate 87.%(/).7% - 98.% N/A N/A 9.9%%(/) 89.% - 99.% %(/) 9.% - % a b x = the number of AtheNA Multi-Lyte EBV VCA IgM results that are confirmed positive in agreement with the reference EBV VCA IgM confirmed positive results; n = the total number of reference EBV VCA IgM results that are confirmed positive x = the number of AtheNA Multi-Lyte EBV VCA IgM results that are nonreactive in agreement with the reference EBV VCA IgM; n = the total number of reference EBV VCA IgM results that are nonreactive c resulted in / specimens. In such cases, percent agreement and 9% confidence intervals could not be calculated. PRECISION: Assay precision was evaluated at multiple sites as follows: Six samples were identified for use in the study based upon their activity on the AtheNA Multi-Lyte Plus assay. Two sampes were selected that were clearly negative, two that were clearly positive and two samples that were near the assay cut off. This panel of six serum samples were split into three aliquots each and tested at three clinical sites. One each day of testing, each sample was diluted twice and then each dilution was run in quadruplicate, resulting in eight results per assay. This was performed on three days at each facility. EBV Analyte Interpretation: Specimen unit values for the analytes are interpreted as follows:. An AtheNA Multi-Lyte result of < AU/mL indicates no detectable IgM antibody to EBV-VCA and should be reported as non-reactive for EBV-VCA IgM antibody. 9

5 Comparison of Results in Acute, No Infection, Past Infection, and Indeterminate Populations; AtheNA Multi-Lyte EBV VCA IgM Assay versus Comparative EBV VCA IgM ELISA Assay (PROSPECTIVE SPECIMENS) Comparative Anti-EBV IgM Assay Comparative Anti-EBV IgM Assay Comparative Anti-EBV IgM Assay Negative Equivocal Positive EBV Classification AtheNA Multi-Lyte EBV VCA IgM Assay AtheNA Multi-Lyte EBV VCA IgM Assay AtheNA Multi-Lyte EBV VCA IgM Assay Reactive Nonreactive Equivocal Invalid Reactive Nonreactive Equivocal Invalid Reactive Nonreactive Equivocal Invalid Total. An AtheNA result of > AU/mL is positive for IgM antibody to EBV-VCA. A positive test result presumes a current or reactivated infection with EBV, and should be reported as reactive for EBV-VCA IgM antibody.. Specimens with AtheNA results in the equivocal range ( to AU/mL) should be retested in duplicate. Specimens that remain equivocal after repeat testing should be tested by an alternate serologic procedure, such as the Zeus Scientific, Inc. indirect fluorescent antibody (IFA) or ELISA test procedures. Additionally, specimens which remain equivocal after repeat testing should be re-evaluated by drawing another sample one to three weeks later.. If there is too much activity on the NSC (non-specific control) bead, Intra-Well Calibration Technology will invalidate that particular specimen. The most likely cause of an invalid specimen in the AtheNA Multi-Lyte EBV VCA IGM Plus 8 test system is due to the presence of RF IgM antibody in the serum sample. For the clinical study, invalid specimens were evaluated for RF IgM antibody. Thirty of the samples were positive for RF IgM. Samples that are INV NSC should be repeated. If they are repeatedly INV NSC, one may test the specimens using another method or may retest the specimens using the modified protocol as outlined below: a. Obtain Zeus product number M. b. Begin the assay as outlined above in Procedure ; however, dilute the repeatedly INV NSC samples in the M diluent at a : dilution. c. Results may be reported for those specimens that no longer generate INV NSC results. N N N N N N N N N N N N N Acute 8 No Infection 9 9 Past Infection 8 Optional procedure for testing samples that were previously found to be INV NSC Indeterminate Equivocal results following repeat testing Dilute INV NSC samples : in M Diluent Read within minutes Comparison of Results in Acute, No Infection, Past Infection, and Indeterminate Populations; AtheNA Multi-Lyte EBV VCA IgM Assay versus Comparative EBV VCA IgM ELISA Assay (RETROPROSPECTIVE SPECIMENS EXPECTED ACUTE) Comparative Anti-EBV IgM Assay Comparative Anti-EBV IgM Assay Comparative Anti-EBV IgM Assay Negative Equivocal Positive EBV Classification AtheNA Multi-Lyte EBV VCA IgM Assay AtheNA Multi-Lyte EBV VCA IgM Assay AtheNA Multi-Lyte EBV VCA IgM Assay Reactive Nonreactive Equivocal Invalid Reactive Nonreactive Equivocal Invalid Reactive Nonreactive Equivocal Invalid Total N N N N N N N N N N N N N Acute 8 No Infection Past Infection Indeterminate 7 Equivocal results following repeat testing Incubate dilutions for +/- minutes at room temperature. Centrifuge dilutions to pellet the precipate ( to 7 RPM for - minutes) Remove ul of each dilution and combine it with the EBV Bead Mix in the filter plate. Incubate for +/- minutes at room temperature. Add Conjugate to the beads Wash the beads Incubate for +/- minutes at room temperature. For purposes of percent agreement calculations, the Athena Multi-Lyte EBV VCA IgM equivocal results (n=) were assigned to the opposite clinical interpretation than that of the comparative assay result. Likewise, the comparative assay equivocal results were assigned to the opposite clinical interpretation than that of the AtheNA Multi-Lyte EBV VCA IgM result. The percent agreement between the AtheNA Multi- Lyte EBV VCA IgM assay and the comparative EBV IgM ELISA assay are summarized in the following tables by specimen EBV classification: The numeric value of the final result above the cutoff is not indicative of the amount of anti-ebv-vca IgM antibody present. Most (8%) of IM individuals have peak anti-vca IgM titers before they consult a physician(). Therefore, testing paired acute and convalescent sera for significant changes in antibody levels is not useful in most patients with IM (). The lack of detectable IgM antibodies does not exclude current EBV infection. The sample may have been collected before development of demonstrable antibody or after the antibody level is no longer detectable. Specific IgM antibodies are usually detected in patients with recent primary infection, but may be found in patients with reactivated or secondary infections, and they are sometimes found in patients with no other detectable evidence of recent infection. LIMITATIONS. Test results should be interpreted in conjunction with the clinical evaluation and the results of other diagnostic procedures.. Specimens with elevated IgG concentrations may interfere with the outcome of this assay. Use of these types of specimens should be avoided.. Performance characteristics of this device have not been established with EBV-associated disease other than infectious mononucleosis.. Testing should not be performed as a screening procedure for the general population. The predictive value of a positive or negative result depends on the prevalence of analyte in a given patient population. Testing should only be done when clinical evidence suggests the diagnosis of EBV-associated infectious mononucleosis. 8

6 . Performance characteristics of this device have not been established for matrices other than serum. Distribution of Patient Age EXPECTED RESULTS The clinical study for the product included a total of 9 prospectively collected specimens. Aside from the samples tested at Zeus Scientific, Inc., specimens were tested at three other facilities; a university medical center located in Eastern U.S. and two hospitals located in Northeastern U.S. Of the 9 specimens tested, included the age and sex of the patient. These included specimens tested at Zeus Scientific and the university medical center. The two hospitals did not include age/sex data with their test results. The Athena Multi-Lyte EBV VCA IgM Plus 8 results for these specimens by age group and gender are summarized in the following table: Age Gender (years) -9 Female -9 Female -9 Female -9 Female -9 Female -9 Female -9 Female 7+ Female Total Female Reactive Non-Reactive Equivocal Invalid 7 7 Total The table below shows a breakdown of the specimen age/sex information. The following frequency distribution shows the age distribution of all specimens that included the age of the patient. Age Information: Summary of Female Specimens Summary of Specimens n=female n=male 9 mean.8 mean.8 median. median. min min max 8 max 8 Frequency As with all in vitro diagnostic assays, each laboratory should determine its own reference range(s) for the diagnostic evaluation of patient results 9. PERFORMANCE CHARACTERISTICS Results by Specimen Classification There were a total of 7 specimens tested. Of the 7 specimens tested, 9 were prospective specimens and 7 were retrospective specimens. The retrospective specimens were purchased as expected acute specimens based on clinical signs and symptoms of acute infectious mononucleosis. The actual EBV infection classification of both the prospective and retrospective specimens was determined as described below,. The EBV infection classification for each patient in the acute infection, no infection, past infection and indeterminate populations (7 patients total) was determined by a heterophile antibody latex agglutination assay, plus serological assessment using EBV marker profiles obtained from results of commercially available, FDA- approved ELISA reference assays. The serological assessment included the following EBV markers: IgG antibody to Epstein Barr virus viral capsid antigen (EBV VCA IgG), IgG antibody to Epstein Barr virus nuclear antigen (EBNA- IgG), and IgM antibody to Epstein Barr virus viral capsid antigen (EBV VCA IgM). The individual Athena Multi-Lyte EBV VCA IgM assay result was compared to the reference EBV VCA IgM assay result and to the patient classification. Each patient's EBV infection was classified based on the reactive (+) or nonreactive(-) patterns of the EBV reference serological markers and the heterophile antibody assay. These patterns are presented in the following table: EBV infection classification by reference assays: Prospective Specimen Group Retrospective Specimen Group Heterophile VCA IgG VCA IgM EBNA- IgG EBV Classification Acute infection No infection 9 n/a Past infection n/a + + Indeterminate n/a + Total: = Reactive = Nonreactive n/a = not available Note: When a reference assay result was equivocal, it was assumed to be nonreactive (-) for classification purposes. Age