ImmunoDOT. BORRELIA DotBlot G Test BORRELIA DotBlot M Test. For In Vitro Diagnostic Use INTENDED USE SUMMARY AND EXPLANATION ASSAY PRINCIPLE REAGENTS

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1 ImmunoDOT BORRELIA DotBlot G Test BORRELIA DotBlot M Test For In Vitro Diagnostic Use INTENDED USE The Borrelia DotBlot G and DotBlot M Tests are qualitative enzyme immunoassays (EIA) for the presumptive detection of Borrelia burgdorferi antibodies (IgG or IgM, respectively) in serum Equivocal or positive results from these tests should be supplemented with a standardized western blot procedure These supplemental results can be used to support a clinical diagnosis of B burgdorferi infection (Lyme disease) SUMMARY AND EXPLANATION Symptomatic B burgdorferi disease results from infection with Borrelia burgdorferi, transmitted by the bite of an infected tick of the genus Ixoides The infection may lead to spirochetemia, with spread of the organism to multiple organs The disease resembles that caused by infection with other spirochetes, and clinical diagnosis may be difficult, particularly in the absence of a characteristic rash, erythema migrans (EM) that often follows the initial infected-tick bite Not all patients recall being bitten by ticks The disease may progress in stages, with intervals of latency between symptoms Symptoms vary according to the sites affected by the infection: joints, skin, central nervous system, heart, eye, bone, spleen, kidney, etc Late disease is most often associated with arthritis or CNS syndromes Culture of the organism is seldom attempted, and may not be readily available Antigen and nucleic acid detection methods are currently under development and evaluation, but presently are not acceptable methods for diagnosis Serological tests are the only laboratory methods readily available to support the diagnosis However, the reliability of serology for B burgdorferi has been limited by cross reactions with other Borrelia and to other less-related organisms: eg infections by many spirochetes produce antibodies cross-reactive with the B burgdorferi flagellin Commonly used serological methods are immunofluorescence (IFA), enzyme immunoassays (EIA), and western blot techniques Borrelia DotBlot G and M Tests are EIAs that use purified Borrelia burgdorferi cell lysate, and four purified borrelia antigens In 1994, the Second National Conference on Serologic Diagnosis of Lyme Disease developed a two-tier testing recommendation as one of several steps toward standardizing laboratory testing for borrelia 5 Because "screening" serologic methods were not sufficiently specific to provide support for clinical diagnosis, it was recommended that all positive or equivocal results from a sensitive EIA or IFA (first tier) should be further tested, or supplemented, by using a standardized western blot method (second tier) Guidelines from this conference state that a standardized western blot requires: The method be validated using sera from clinically (not serologically) defined patients Reagent strips that contain all relevant proteins Specified methods to prepare the gels and strips Use of specific antisera to identify band location for interpretation Results interpretation based on specified guidelines included in the 1994 report If these guidelines are followed, it is believed that the supplemental western blot testing of specimens yielding equivocal or positive results from first tier testing provides suitable assay accuracy to support clinical diagnosis Borrelia DotBlot tests, like EIA assays, use a whole borrelial antigen to screen, but the DotBlot tests also detect antibodies to specific borrelia proteins, thus improving specificity Borrelia DotBlot tests use four specific proteins: a high molecular weight protein (HMW), flagellin, a 39 kd protein (p39), and outer surface protein C (OspC) HMW is a recombinant high molecular weight protein that is reported to have a molecular weight between kd In the Borrelia DotBlot tests, the amount of HMW is adjusted to assure minimal cross-reactivity (high specificity) Antibodies to HMW are usually IgG and appear late Flagellin is a protein with many epitopes in common with other spirochetes and some other bacteria Although relatively nonspecific, the flagellin antibody is a predominant response Like HMW, the amount of flagellin is adjusted to assure minimal cross-reactivity The level of flagellin used in these tests is less than typically found in a western blot; therefore, only the stronger western blot anti-flagellin reactions are reactive in the Borrelia DotBlot tests Recombinant p39 (BmpA) constitutes a small portion of the spirochete's protein, but is a dominant immunoreactive protein Because of the limited amount of protein and its molecular weight being similar to flagellin, these antibodies are not reliably identified using the western blot method Along with the anti-flagellin immune response, anti-ospc appears early during the primary infection Patients with later disease stages rarely demonstrate a significant, lasting OspC IgG response ASSAY PRINCIPLE The Borrelia DotBlot Tests utilize an enzyme-linked immunoassay (EIA) dot technique for the detection of antibodies Various levels of antigens are present as discrete dots on an assay strip An assay strip is inserted into patient diluted serum, allowing patient antibodies reactive with the test antigen to bind to the strip s solid support membrane In the second stage, the reaction is enhanced by removal of non-specifically bound materials During the third stage, alkaline phosphatase-conjugated antihuman antibodies are allowed to react with bound patient antibodies Finally, the strip is transferred to enzyme substrate reagent, which reacts with bound alkaline phosphatase to produce an easily seen, distinct dot REAGENTS Assay Strip whole borrelial antigen (low passage B31 from ATCC), recombinant HMW 1 (isolate SON 188, California genospecies 1), flagellin, recombinant p39 (BmpA) 2,3 (isolate SH-2-82), recombinant OspC (isolate SON 188; California genospecies 1), and positive reagent control (human serum) All borrelial antigens are derived from the sensu strictu strain of B burgdorferi Diluent (#1) buffered diluent containing solubilized blocking reagent, protein stabilizers, and <01% NaN 3 Enhancer (#2) sodium chloride and <01% NaN 3 Conjugate (#3) alkaline phosphatase conjugated goat antihuman IgM or IgG (heavy chain specific) in buffered diluent and stabilizers Developer (#4) 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium chloride in buffered diluent and <01% NaN 3 Warnings and Precautions For In Vitro Diagnostic Use These reagents have been optimized for use as a system Do not interchange Borrelia DotBlot G and M assay strips Dilution or adulteration of these reagents may also affect the performance of the test Do not use kit if evidence of microbial contamination is present Do not ingest reagents Do not use any kits beyond the stated expiration date Analytic quality deionized or distilled water must be used as Clarifier Close adherence to the test procedure will assure optimal performance Do not shorten or lengthen stated incubation times since this may result in poor assay performance Some assay components contain sodium azide (NaN 3 ) that may react with lead and copper plumbing to form highly explosive metal azides On disposal, flush with a large volume of water to prevent azide buildup Warning - Potential Biohazardous Material: Human sera used in the preparation of this product were tested and found non-reactive for hepatitis B surface antigen and for antibodies to HIV-1, HIV-2, HTLV-1, and hepatitis C virus Because no test method can offer complete assurance that infectious agents are absent, handle reagents and patient samples as if capable of transmitting infectious disease 4 Storage Store reagents and assay strips at 2-8 C Reagents must be at room temperature (15-30 C) before use Avoid contamination of reagents /10

2 SPECIMEN COLLECTION Borrelia DotBlot Tests are performed on serum Hemolyzed or lipemic serum should not be used It is known that inappropriately stored samples may cause false positive OspC IgM results Presumably, this is due to microbial contamination Interpretation of IgM results using stored samples should be made cautiously Single specimens are used to assess exposure to B burgdorferi Two specimens collected at different times from the same individual are used to show seroconversion The test requires approximately 10 µl of serum Serum is collected according to standard practices and may be stored at 2-8 C for up to five days Serum may be frozen at or below -20 C for up to one year for IgM tests and up to five years for IgG tests PROCEDURE Materials Provided Assay Strips Diluent (#1) Enhancer (#2) Conjugate (#3) Developer (#4) Reaction Vessels Package Insert Materials Required But Not Provided GenBio Workstation Specimen collection apparatus Timer Analytic quality distilled or deionized water to be used as Clarifier Pipets Absorbent toweling to blot dry the assay strip Positive and Negative Control serum Set-Up 1 Turn on Workstation and adjust to appropriate temperature if necessary (Refer to Workstation Instructions) 2 Remove 4 Reaction Vessels per test from the product box and insert into appropriate slots in Workstation For the large Workstation, add water up to the fill line of the Clarifier Vessel provided For the small Workstation, use an appropriate container and sufficient water to cover all reactive windows of the assay strip 3 Place 2 ml Diluent (#1) in Reaction Vessel #1; 2 ml Enhancer (#2) in Reaction Vessel #2; 2 ml Conjugate (#3) in Reaction Vessel #3; and 2 ml Developer (#4) in Reaction Vessel #4 4 Appropriately label the Assay Strips 5 If the large Workstation is used, insert the label end of the assay strip into the Strip Holder, one per groove, taking care not to touch the assay windows Assay Procedure 1 Add 10 µl serum to Reaction Vessel #1 2 Prewet Assay Strip by immersing in Clarifier for seconds 3 Using several (5-10) quick up and down motions with the Assay Strip, mix thoroughly in Reaction Vessel #1 Let stand for minutes 4 Remove Assay Strip from Reaction Vessel and swish in the Clarifier Use a swift back and forth motion for 5-10 seconds allowing for optimal washing of the Assay Strip s membrane windows 5 Place Assay Strip into Reaction Vessel #2 Mix thoroughly with several (5-10) quick up and down motions Let stand for 5 minutes 6 Remove Assay Strip from Reaction Vessel #2 and swish in Clarifier as described (step #4) 7 Place Assay Strip into Reaction Vessel #3 Mix thoroughly with several (5-10) quick up and down motions Let stand for minutes 8 Remove Assay Strip from Reaction Vessel #3 and swish in Clarifier as described (step #4) DO NOT remove the Assay Strip from the Clarifier 9 Allow the Assay Strip to stand in the Clarifier for 5 minutes 10 Remove Assay Strip from Clarifier and place into Reaction Vessel #4 Mix thoroughly with several (5-10) quick up and down motions Let stand for 5 minutes 11 Remove Assay Strip from Reaction Vessel #4 and swish in Clarifier as described (step #4) 12 Blot and allow Assay Strip to dry It is imperative that tests of borderline specimens be interpreted after the Assay Strip has been allowed to dry Reading the Assay Strip Positive A dot with an EASILY SEEN, distinct border is visible in the center of the window The outer perimeter of the window must be white to pale gray Negative If no dot is seen or a dot is difficult to see, interpret it as negative Each window of the assay strip is read independently Please call GenBio Technical Services for further clarification Quality Control The assay is performed with reagents at C The temperature of the Workstation itself should be higher than the assay temperature in the reaction vessel The Workstation should be monitored to assure the appropriate temperature is maintained (Refer to Workstation Instructions) The Positive Control window (well 6) contains human serum and tests reagent reactivity It must be reactive but the intensity must not be used as a calibrator As a negative reagent control check, the backgrounds around dots must be white Controls are available from GenBio (IgG Positive - Product No 3905; Negative - Product No 3920; IgM Positive - Product No 3907) and should be tested in accordance with laboratory accreditation requirements and/or individual laboratory monitoring programs INTERPRETATION Reactive: To be reactive, the positive control and borrelia windows must be positive In addition, at least one of the following three dots: HMW, p39, or OspC must also be positive (Below are the patterns most commonly seen If other patterns are seen, please contact Technical Services) Please note that IgM pattern #4 frequently occurs, but in most cases represents a false positive reaction The reaction may be either caused by specimen handling or infection with Epstein-Barr virus (mononucleosis) Equivocal: To be equivocal, the positive control and whole borrelia windows must be reactive In addition, the HMW, p39, and/or OspC windows show a faint response (If needed, contact Technical Service to obtain a comparison assay strip) Note: A 1994 symposium recommended that all samples interpreted equivocal or reactive by EIA or IFA be tested by a standardized western blot procedure 5 Nonreactive: If the positive reagent control window is positive and whole borrelia, HMW, p39 and OspC windows are negative, interpret the result as nonreactive Do not interpret whole borrelia reactivity in the absence of HMW, p39, or Osp C reactivity Further Details: Typically both DotBlot IgG and IgM tests must be used simultaneously to interpret the patient's seroreactivity However, IgM interpretation should only be made on samples collected from patients exposed within the previous eight weeks Only the IgG test is required if a later stage is suspected In general, stronger IgG reactions are associated with later disease stages, while IgM reactivity is associated with earlier stages of borreliosis Because samples collected within the first days after exposure usually contain little specific antibody, a second sample should be requested 1-2 weeks later if the first sample is interpreted as equivocal or non-reactive In most early cases, IgM anti-flagellin is present if a patient is interpreted as reactive However, the presence of IgM or IgG flagellin in the absence of a more specific antibody response to HMW, p39, or OspC should not be interpreted as reactive /10

3 Reporting of Results: A reactive result should be reported as presumptive evidence of exposure to B burgdorferi The user should also report that (i) it has been recommended 5 that a reactive result should be supplemented by using a standardized western blot method and (ii) a reactive result does not indicate when exposure occurred A reactive result could be reported concurrently with the western blot result An equivocal result should be reported as equivocal The user should also report that it has been recommended 5 that an equivocal result DotBlot M Patterns and Interpretations should be supplemented by using a standardized western blot method An equivocal result could be reported concurrently with the western blot result A nonreactive result should report that B burgdorferi antibodies were not detected The report should also recommend, if indicated, that another serum specimen be collected two to four weeks later and tested to detect seroconversion Pattern Interpretation NR NR R R R R R R R Whole Borrelia HMW Flagellin P39 (BmpA) Osp C Positive Control DotBlot G Patterns and Interpretations Pattern Interpretation NR NR R R R R R R R R R Whole Borrelia HMW Flagellin P39 (BmpA) Osp C Positive Control LIMITATIONS Other diseases, including others that are tick-borne, may cause syndromes similar to those of B burgdorferi infection and may result in development of detectable antibodies to B burgdorferi that are nonspecific Reactive results only for IgM antibodies to B burgdorferi are likely to be false-positives in individuals with illness of longer than one month duration In addition, specific antibodies to B burgdorferi may be present from exposure that is unrelated to the disease syndrome being evaluated All tested specimens from patients with syphilis and many specimens from patients with mononucleosis (associated with Epstein-Barr virus) contained IgM antibodies that were crossreactive with Borrelia DotBlot antigens (Table 2) Some patients with syphilis also had crossreactive antibodies of the IgG class One or more B burgdorferi antigens may also crossreact with antibodies from patients with other spirochetal diseases (yaws, pinta, leptospirosis, relapsing fever, periodontal disease), autoimmune connective-tissue diseases, rickettsial diseases, ehrlichiosis, antibody to Helicobacter pylori, and other bacterial infections Nonreactive results do not rule out the diagnosis of Bburgdorferi disease The specimen may be drawn prior to appearance of detectable antibodies (seroconversion) or early antibiotic therapy may suppress antibody response If early disease is suspected, a second sample should be collected after at least seven days If the patient has been treated with antibiotic, the second sample may be negative, IgM-reactive, and/or IgG-reactive If the patient has been treated, no antibody may form or only IgM reactivity may occur Not all patients will form IgG antibody, but reactive results for only IgM are likely to be false-positive in individuals with illness of longer than one month duration The continued presence or absence of antibodies cannot be reliably used to determine success or failure of therapy Testing should not be performed for screening of the general population The positive predictive value of a test result varies directly with the prevalence of B burgdorferi infection in the geographic region of the patient: a low-prevalence population will nearly always yield a lower positive predictive value (higher proportion of false-positive results) than a high-prevalence population Therefore, serology should be used only when a clinical diagnosis of B burgdorferi infection has been established; positive results should be interpreted with caution when obtained for a patient from a low-prevalence population EXPECTED RESULTS Regional differences in the prevalence of human infections with B burgdorferi are related to the ecology of the tick vector species and their rate of B burgdorferi infections Human infections are most likely to occur where the vector population is highly infected Early Stage Reactivity: During the first few days after infection (tick bite), antibody is not present, yet disease symptoms (EM) may occur A second sample taken at least one week later may be used to identify B burgdorferi seroconversion Flagellin IgM antibody is sometimes present alone This may indicate an early true response, but may as frequently /10

4 indicate a false positive reaction In some cases, presumably when therapy prevents systemic infection, no antibody (IgG or IgM) response occurs The most frequently seen DotBlot IgM antibody combinations are whole borrelia with: 1) OspC and flagellin, 2) OspC, or 3) OspC, flagellin and p39 Evaluate these combinations according to the Interpretation section Late Stage Reactivity: During late stages (sequelae) a strong, long lasting IgG response to p39, HMW protein, and flagellin is typically seen with DotBlot The IgG reaction against OspC is of short (weeks) duration In many cases, IgM reactivity persists The relationships between various patterns of specific antibody reactivity and diagnostic interpretations have not yet been established Normals: One of 100 normal subjects evaluated from an endemic region was IgM reactive and one subject was IgM equivocal Sera from two of ninety-seven normal people in a non endemic region showed an IgG equivocal result, but none were classified as either IgG or IgM reactive Overall IgG or IgM assay specificity was 99% in the endemic region and was 100% overall in the non-endemic region PERFORMANCE CHARACTERISTICS In general, it is difficult to assess the performance characteristics of EIAs and IFAs in the recommended two-tier testing protocol 5 because such performance depends on the characteristics of the standardized western blot method that is chosen by the user ImmunoDOT DotBlot Test has been tested at three outside laboratories and within the GenBio laboratory Both prospective and retrospective studies were conducted to determine the test s performance ImmunoDOT DotBlot sensitivity is summarized in Table 1 and the specificity is summarized in Table 2 The first three groups in Table 1 are collected from subjects presenting with EM All samples from the Culture Confirmed cases were from primary cases with recent exposure (tick bites) Samples received from CDC are classified as unknown since the time of sample collection is typically not correlated to the time the patient first presented with disease All samples identified as positive by serology in the prospective studies are also classified as unknown Table 1: DotBlot Sensitivity Disease Stage Total # IgM IgG IgM or IgG Culture Confirmed (EM) 59 54% 8% 56% First Visit (EM) 73 30% 23% 45% Any Visit (EM) % 31% 56% Early Neuroborreliosis 43 74% 42% 84% Cardiomyopathy 17 94% 59% 100% Arthritis 76 64% 71% 89% Late Neuroborreliosis 33 70% 94% 97% ACA* 18 39% 61% 72% Unknown 47 51% 53% 85% * Acrodermatitis Chronica Atrophicans Table 2: DotBlot Specificity Disease Total # IgM IgG IgM or IgG Endemic Normals % 100% 99% Non-endemic Normals % 100% 100% Autoimmune 49 96% 100% 96% Mononucleosis 44 93% 100% 93% H pylori reactive 15 93% 100% 93% Syphilis 20 0% 90% 0% Unknown % 97% 92% Comparison to Other Methods The following information is from a serum panel obtained from the US Centers for Disease Control and Prevention (CDC) and tested by GenBio The results are presented as a means to convey further information on the performance of this assay with a masked, characterized serum panel This does not imply an endorsement of the assay by the CDC The panel consists of 46% positive and 54% negative samples (classified by two-tier methods) 5 The relative performance of the DotBlot results (IgG and IgM) and the results of a currently marketed product (predicate device) are shown in Table 3 Table 3: Comparison to Predicate Device CDC Positive CDC Negative DotBlot Positive 16 1 DotBlot Negative 5 24 Comparative Test Positive 19 4 Comparative Test Negative 2 21 Prospective Studies Two US sites conducted prospective studies using their "two tier" test system Site 1 uses a commercial microtiter ELISA screening kit, and supplements with an IgG western blot interpreted using criteria based on sera from a panel of culture defined subjects Site 2 uses an in-house microtiter ELISA and tests all positives with an IgM and IgG western blot method that has been validated and interpreted according to the CDC recommended guideline Table 4 presents DotBlot vs ELISA screening results Table 4: DotBlot vs Microtiter ELISA Assay Type Sensitivity Specificity Site 1 IgM 15% (6-31%) 99% (92-100%) IgG 23% (11-39%) 97% (90-100%) IgG or IgM 31% (17-48%) 96% (87-99%) Site 2 IgM 39% (17-64%) 93% (85-97%) IgG 39% (17-64%) 100% (96-100%) IgG or IgM 61% (36-83%) 93% (85-97%) The low sensitivity of DotBlot relative to microtiter ELISA is due to the low specificity of the microtiter ELISA Site 1 reported 39 ELISA positives of which only 10 were found positive by western blot At Site 2, only 7 of the 18 ELISA positives were also western blot positive Table 5 presents DotBlot vs western blot results for ELISA positives Table 5: DotBlot vs Western Blot Assay Type Sensitivity Specificity Site 1 IgM 50% (19-81%) 97% (82-100%) IgG 70% (39-93%) 93% (77-99%) IgG or IgM 90% (56-100%) 90% (73-98%) Site 2 IgM 71% (29-96%) 81% (48-98%) IgG 71% (29-96%) 81% (48-98%) IgG or IgM 100% (59-100%) 63% (31-89%) Agreement between DotBlot and the "two tier" results was 93% at Site 1 and 90% at Site 2 Reproducibility Twelve blinded samples were supplied to two outside laboratories that conducted the prospective studies Low positive samples were prepared by dilution into specimen diluent Each site tested the samples in duplicate daily for three days The IgG results are shown in Table 6 and the IgM results in Table 7 These data show that samples with low levels of reactivity may be marginally positive in one run and negative in a second run Based on final interpretation, IgM reproducibility was 97% and IgG reproducibility was 99% /10

5 Table 6: IgG Reproducibility ID Reactivity Whole HMW P41 P39 OspC 1 Positive 6/6 6/6 3/6 6/6 0/6 2 Negative 0/6 0/6 0/6 0/6 0/6 3 Weak Positive 6/6 0/6 6/6 5/6 0/6 4 Weak Positive 6/6 2/6 3/6 6/6 0/6 5 Negative 0/6 0/6 0/6 0/6 0/6 6 Weak Positive 4/6 0/6 0/6 2/6 0/6 Table 7: IgM Reproducibility ID Reactivity Whole HMW P41 P39 OspC 1 Negative 0/6 0/6 0/6 0/6 0/6 2 Weak Positive 1/6 0/6 0/6 0/6 1/6 3 Weak Positive 4/6 0/6 6/6 3/6 0/6 4 Positive 6/6 0/6 0/6 0/6 6/6 5 Negative 0/6 0/6 0/6 0/6 0/6 6 Negative 0/6 0/6 0/6 0/6 0/6 Bibliography 1 LaFebre R US Patent 5,324,630 2 Simpson WJ, ME Schrumpf, TG Schwann Reactivity of human Lyme borreliosis sera with a 39-kilodalton antigen specific to Borrelia burgdorferi J Clin Microbiol 28:1329 (1990) 3 Simpson WJ, W Burgdorfer, ME Schrumpf, RH Karstens, TG Schwann Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals J Clin Microbiol 29:236 (1991) 4 Centers for Disease Control/National Institutes of Health Manual Biosafety in Microbiological and Biomedical Laboratories (1983) 5 Proceedings of the Second National Conference on Serologic Diagnosis of Lyme Disease, October 27-29, 1994, Dearborn, Michigan QUICK REFERENCE PROCEDURE Borrelia DotBlot G Borrelia DotBlot M Set-Up Make sure Workstation is at temperature Place reaction Vessels into slots in Workstation and add water to the Clarifier Vessel Place 2 ml Diluent (1) in Vessel #1; 2 ml Enhancer (2) in Vessel #2; 2 ml Conjugate (3) in Vessel #3; and 2 ml Developer (4) in Vessel #4 Procedure Add 10 µl serum to Vessel #1 Prewet assay strip in Clarifier for seconds Place strip in Vessel #1, mix, let stand min Remove strip, place in Clarifier, swish 5-10 sec Place strip in Vessel #2, mix, let stand 5 min Remove strip, place in Clarifier, swish 5-10 sec Place strip in Vessel #3, mix, let stand min Remove strip, place in Clarifier, let stand 5 min Place strip in Vessel #4, mix, let stand 5 min Remove strip, place in Clarifier, swish, blot, dry, and read Whole Borrelia HMW Flagellin P39 Osp C Positive Control To place an order for ImmunoDOT products, contact your local distributor or call GenBio directly for the distributor nearest you and for additional product information For assistance, please call toll-free GenBio A Avenue of Science San Diego, CA /10