Lyme Igm ELISA

Lyme IgM Page 1 Atlas Link 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected]

Lyme IgM ELISA Catalog # 1424

INTENDED USE The Atlas Link (AL) Lyme (Borrelia burgdorferi) IgM Enzyme-Linked Immunosorbent Assay (ELISA) is intended for the qualitative presumptive detection of IgM antibody in human serum to Borrelia burgorferi in patients with clinical signs and symptoms of Lyme disease. This assay is intended for use as an aid in the presumptive serodiagnosis of Lyme disease. For Export Use Only.

SUMMARY Lyme disease is a spirochetal disease transmitted by ticks in the genus Ixodes. The spirochete responsible is Borrelia burgdorferi.(1) Lyme disease has worldwide distribution. In the United States the vast majority of cases occur in the northeastern and midatlantic states. Endemic transmission is also seen in the upper midwest and pacific coastal counties. (2) There are three stages of Lyme disease:

Stage I or Early Localized Skin Lesion has a characteristic skin lesion known as erythema migrans (EM) in 60-80% of patients. EM may appear up to one month after being bitten by the vector tick. Vague flu-like symptoms (fever, headache, fatigue, regional lymphadenopathy, arthralgias, myalgia) may also be present. Early treatment is very helpful in reducing the frequency of later disease manifestations.

Stage II or Early Disseminated Infection occurs when Borrelia burgdorferi spreads through the circulatory system or lymphatics. In ~50% of cases in the United States, multiple areas of EM may be indicative of hematogenous dissemination. Organs most commonly involved are: heart, joints, and central nervous system. Other organs may be involved such as: eye, bone, muscle, spleen, liver, and kidney. Neurologic, cardiac, and/or joint symptoms may last days to weeks and periods of remission and relapse may occur.

Stage III or Late Disseminated Infection is the chronic phase in which some patients may experience various symptoms including dermatologic, rheumatologic, cardiac, and neurologic while other patients may enter a latent period. Symptoms at this stage are thought to be immune mediated and treatment may be unsuccessful.

Any patient may experience one or all of the stages (3, 4). In some patients infection may not have clinical symptoms until the later stages. Diagnosis of early Lyme disease is generally based on the presence of EM. Later diagnosis is more difficult and depends on several factors: patient history, clinical symptoms, exclusion of other possible causes, and presence of antibodies to Borrelia burgdorferi. The definitive test for Lyme disease is by culture of Borrelia burgdorferi, however this test has a low yield rate and is an impractical procedure (5, 6). Initial testing should be performed by ELISA or Indirect Fluorescent Antibody IFA). Any sera that tests positive or equivocal should then be confirmed by Western-blot.

PRINCIPLE Enzyme-Linked Immunosorbent Assays (ELISA) rely on the ability of biological materials, (e.g., antigens) to adsorb to plastic surfaces such as polystyrene (solid phase). When antigens bound to the solid phase are brought into contact with a patient's serum, antigen specific antibody, if present, will bind to the antigen on the solid phase forming antigen-antibody complexes. Excess antibody is removed by washing. This is followed by the addition of goat anti-human IgM globulin conjugated with horseradish peroxidase which will bind to the antibody-antigen complexes. The excess conjugate is removed by washing, followed by the addition of Chromogen/Substrate tetramethylbenzidine (TMB). If specific antibody to the antigen is present in the patient's serum, a blue color develops. When the enzymatic reaction is stopped with 1N H2SO4, the contents of the wells turn yellow. The color, which is indicative of the concentration of antibody in the serum, can be read on a suitable spectrophotometer or ELISA microwell plate reader (7, 8, 9, 10, 11, 12, .13).

REAGENTS SUPPLIED 1. Purified Borrelia burgdorferi antigen coated microassy plate: 96 wells, configured in twelve 1x8 strips, stored in a foil pouch with desiccant/humidity indicator card. (96T: two plates). Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 2 2. B. burgdorferi Serum Diluent: Ready for use. Contains proclin (0.1%) as a preservative. (two bottle, 30 mL each) 3. Calibrator: human serum or defibrinated plasma. Sodium azide (0.1%) and pen/strep 0.01% added as preservative, with kit specific factor printed on vial label. The Calibrator is used to calibrate the assay to account for day-to-day fulctuations in temperature and other testing conditions. (96T: one vial, 0.25 mL) 4. High Positive Control: human serum or defibrinated plasma. Sodium Azide (0.1%) and pen/strep (0.01%) added as preservatives, with established range printed on vial label. The High Positive Control is utilized to indicate the upper limit of dynamic range of the assay. (96T: one vial, 0.25 mL) 5. Low Positive Control: human serum or defibrinated plasma. Sodium Azide (0.1%) and pen/strip (0.01%) added as preservatives, with established range printed on vial label. The Low Positive Control is utilized to control the assay near the cutoff of the assay. (96T: one vial, 0.25 mL)

6. Negative Control: human serum or defibrinated plasma. Sodium Azide (0.1%) and pen/strep (0.01%) added as preservatives, with established range printed on vial label. The Negative Control is utilized to control the negative range of the assay. The Negative Control is also used as an indication of the performance of the Absorbent. The sera is a RF positive specimen spiked with specific anti-B. burgdorderi IgG. If the IgG is not removed the specimen will be positive due to RF. (96T: one vial, 0.25 mL) 7. Horseraddish peroxidase (HRP) Conjugate: Ready to use. Goat anti-human IgM, containing proclin (0.1%) as a perservative. (96T: two bottle, 16 mL each) 8. Chromogen/Substrate Solution: Tetramethylbenzidine (TMB) ready to use. (96T: two bottle, 15 mL each) 9. Absorbent Solution: Ready to use. Contains goat/sheep anti-human IgG with protien stabilizers and proclin (0.1%) as preservative. When stored at 2° -8° C, the solutions is stable until the labeled expiration date. (96T: two bottles, 12 mL each) 10. Wash Buffer (20X concentrate): dilute 1 part concentrate and + 19 parts deionized or distilled water. Contains TBS, Tween and proclin (0.1%) as preservative, pH 7.2 + 0.2. (96T: one bottle, 60 mL)

The following components are not kit lot # dependent and may be used interchangeably within the DA ELISA IgM assays: Chromogen/Substrate Solution, Wash Buffer. The Serum Diluent is specific for the B. burgdorferi kit. Do not interchange the Serum Diluent with other kits.

PRECAUTIONS 1. For Export Use Only. 2. The human serum components used int eh preparation of the Controls and Calibrators in this kit have been tested by an FDA approved method for the presence of antibodies to human immuodeficiency virus 1 & 2 (HIV 1 & 2), hepatitis C (HVC) as we;; as hepatitis B surface antigen and found negative. Because no test methods can offer complete assurance that HIV, HCV, hepatitis B virus, or other infecipos agents are absent, specimens and human-based reagents should be handled as if capable of transmitting infectious agents. 3. The Centers for Disease Control and the National Institutes of Health recommend that postentially infectious agents be handled at the Biosafety Level 2 (23). 4. The components in this kit have been quality cotrol tested as a Master Lot unit. Do not mix components from different lot numbers except Chromogen/Substrate Solution and Wash Buffer. Serum Diluent supplied with B. burgdorferi kits can be used only with other B. burgdorferi kits. Do not mix with components from other manufacturers. 5. Do not use reagents beyond the stated expiration date marked on the package label. 6. All reagents must be a room temperature (21o-25o C) before running assay. Remove only the volume of reagents that is needed. Do not pour reagents back into vials as reagent contamination may occur. 7. Before opening Controls ane Cutoff Calibrator vials, tap firmly on the benchtop to ensure that all liquid is at the bottom of the vial. 8. Use only distilled or deionized water and clean glassware. 9. Do not let wells dry during assay; add reagents immediately after completing wash steps. 10. Avoid cross-contamination of reagents. Wash hands before and after handling reagents. Cross-contamination of reagents and/or samples could cause false results. 11. If washing steps are performed manually, wells are to be washed three times. Up to five wash cycles may be necessary if a washing manifold or automated equipment is used. 12. Sodium azide inhibits Conjugate activiry. Clean pipette tips must be used for the Conjugate addition so that sodium azide is not carried over from other reagents. 13. It has been reported that sodium azide may react with lead and copper in plumbing to form explosive compounds. When disposing, flush drains with water to minimize build-up of metal azide compounds. 14. Never pipette by mouth or allow reagents or patient sample to come into contact with skin. Reagents containg proclin, sodium azide, and TMB may be irritating. Avoid contact with skin and eyes. In case of contact, flush with plenty of water.

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 3 15. If a sodium hypochlorite (bleach) solution is being used as a disinfectant, do not expose to work area during actual test procedure because of potential interference with enzyme activity. 16. Avoid contact of sulfuric acid with skin or eyes. If contact occurs, immediately flush area with water. 17. Caution: Liquid waste at acid pH must be neutralized prior to adding sodium hypochlorite solutions (bleach) to avoid formation of poison gas. It is recommended to dispose of reacted, stopped plates in biohazard bags. See Precaution 3. 18. The concentrations of anti-B. burgdorferi IgM in a given specimen determined with assays from different manufacturers can vary due to differences in assay methods and reagents specificity.

MATERIALS REQUIRED BUT NOT SUPPLIED 1. Graduated cylinder (100 mL). 2. Flask (1L). 3. Timer - 0 to 60 minutes. 4. Micropipettes capable of accurately delivering 10-200 mL volumes (less than 3% CV). 5. Deionized or distilled water. 6. Paper towels. 7. Wash bottle, semi-automated or automated wash equipment. 8. Single or dual wavelength microplate reader with 450 nm filter. If dual wavelength is used, set the reference filter to 600-650 nm. Read the Operator's Manual or contact the instrument manufacturer to establish linearity performance specifications of the reader. 9. Test tubes for serum dilution. 10. Disposal basin and disinfectant (e.g., 0.5% sodium hypochlorite). Note: Use only clean, dry glassware.

STORAGE AND SHELF LIFE OF REAGENTS

1. Store unopened kit between 2° and 8° C. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Unopened microassay plates must be stored between 2° and 8° C. Unused strips must be immediately resealed in a sealable bag with desiccant/humidity indicator and returned to storage at 2° and 8° C. 3. Store HRP Conjugate between 2° and 8° C. 4. Store the Calibrator, High Positive Control, Low Positive Control and Negative Control between 2° and 8° C. 5. Store Serum Diluent and 20X Wash Buffer between 2° and 8° C. 6. Store the Chromogen/Substrate Solution between 2° and 8° C . 7. Store 1X (diluted) Wash Buffer at room temperature (21° to 25° C) for up to 5 days, or 1 week between 2° - 8° C. Note: If constant storage temperature is maintained, reagents and substrate will be stable for the dating period of the kit. Refer to package label for expiration date. Precautions were taken in the manufacture of this product to protect the reagents from contamination and bacteriostatic agents have been added to the liquid reagents. Care should be exercised to protect the reagents in this kit from contamination. Do not use if evidence of microbial contamination or precipitation is present.

SPECIMEN COLLECTION 1. Handle all blood and serum as if capable of transmitting infectious agents. 2. Optimal performance of the kit depends upon the use of fresh serum samples (clear, non-hemolyzed, non-lipemic, non-icteric). A minimum volume of 50 mL is recommended, in case repeat testing is required. Specimens should be collected aseptically by venipuncture (16). Early separation from the clot prevents hemolysis of serum. 3. Store serum between 2° and 8° C if testing will take place within two days. If specimens are to be kept for longer periods, store at -20° C or colder. Do not use a frost-free freezer because it may allow the specimens to go through freeze-thaw cycles and degrade antibody. Samples that are improperly stored or are subjected to multiple freeze-thaw cycles may yield spurious results. 4. The NCCLS provides recommendations for storing blood specimens, (Approved Standard - Procedures for the Handling and Processing of Blood Specimens, H18A. 1990).

PREPARATION OF REAGENTS 1. Allow pouched plate, reagents, Calibrator, Controls, buffers and patient sera to equilibrate to room temperature (21°-25° C) to avoid condensation. 2. Wash Buffer should be diluted 1:20 to 1.2 liters with distilled and/or deionized water. 3. Calibrator, Controls and patient sera should be diluted 1:41 (i.e., 10 mL + 400 mL) with B. burgdorferi Serum Diluent.

SERUM TREATMENT

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 4 Solid phase immunoassays for the detection of virus-specific IgM are known to be sensitive to interfering factors. This kit overcomes interferences by treating samples prior to running the assay. The Absorbent Solution diminishes competing virus-specific IgG, which would be responsible for false negative reactions. False positives are similarly minimized by removing the IgG, thus neutralizing the bound rheumatoid factor in the samples.

PROCEDURE FOR SERUM ABSORPTION

1. In a set of test tubes, dilute Calibrator, Controls and patient samples 1:41 in B. burgdorferi Serum Diluent (i.e., 400 mL Diluent + 10 mL of Calibrator, Control or serum sample). 2. For each assay, run the Cutoff Calibrator in triplicate, in both the antigen and control antigen wells. Add 350 mL of the 1:41 dilution (step 1) to 350 mL Absorbent Solution. Mix well. This will give enough for 3 antigen wells and 3 control antigen wells. (Final dilution 1:81). 3. For the Controls and test samples, add 150 mL of the 1:41 dilution (step 1) to 150 mL Absorbent Solution. Mix well. This will give enough for 1 antigen well and 1 control antigen well. (Final dilution 1:81). 4. Incubate all absorbent dilutions at room temperature (21°-25° C) for 20 minutes + 5 minutes.

GENERAL PROCEDURE

1. Bring all reagents and microwells to room temperature (21°-25° C). 2. Remove desired number of strips from the sealed foil pouch. Replace unused strips back in the pouch with desiccant/humidity indicator card and seal tightly. Store unused strips and reagents at 2°-8° C. 3. The Calibrator, High Positive, Low Positive and Negative Controls, and each patient serum must be run in both an antigen (Ag) and control antigen (CAg) coated well. A reagent blank (RB) should be run on each assay. Check software and reader requirements for the correct Calibrator/Control configurations.

Example Configuration:

1A Reagent Blank (RB) to zero instrument 2A blank well 1B Negative Control Ag well 2B Negative Control CAg well 1C Calibrator Ag well 2C Calibrator CAg well 1D Calibrator Ag well 2D Calibrator CAg well 1E Calibrator Ag well 2E Calibrator CAg well 1F High Positive Control Ag well 2F High Positive Control CAg well 1G Low Positive Control Ag well 2G Low Positive Control CAg well 1H Patient sera Ag well 2H Patient sera CAg well

4. Record all steps of assay and label test tubes with appropriate identification for kit Controls, Calibrator and patient samples to be tested. 5. Pipette 100 mL of diluted and absorbed patient, Calibrator and Control sera into corresponding wells of the antigen strip and control antigen strip. Add 100 mL of B. burgdorferi Serum Diluent to the reagent blank well. 6. Incubate each well at room temperature (21°-25° C) for 20 minutes + 2 minutes. 7. Aspirate or shake out liquid from all wells. If using semi-automated or automated washing equipment, add 250- 300 mL of diluted Wash Buffer to each well. Aspirate or shake out and turn plate upside down and blot on paper toweling to remove all liquid. Repeat the wash procedure two times (for a total of three (3) washes) for manual or semi-automated equipment or four (4) times (for a total of five (5) washes) for automated equipment. After the final wash, blot the plate on paper toweling to remove all liquid from the wells.

**IMPORTANT NOTE: Regarding steps 7 and 10 - Insufficient or excessive washing will result in assay variation and will affect validity of results. Therefore, for best results the use of semi-automated or automated equipment set to deliver a volume to completely fill each well (250-300 mL) is recommended. A total of up to five (5) washes may be necessary with automated equipment. Please contact Wampole Laboratories with any question regarding appropriate wash equipment. Complete removal of the Wash Buffer after the last wash is critical for the accurate performance of the test. Also, visually ensure that no bubbles are remaining in the wells.

8. Add 100 mL Conjugate to each well, including reagent blank well. Avoid bubbles upon addition as they may yield spurious results. 9. Incubate each well at room temperature (21°-25° C) for 20 minutes + 2 minutes. 10. Repeat wash as described in step 7. 11. Add 100 mL of Chromogen/Substrate Solution into each well, including reagent blank well, maintaining a constant rate of addition across the plate. 12. Incubate each well at room temperature (21°-25° C) for 10 minutes + 2 minutes.

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 5

13. Stop the reaction the addition of 100 mL of 1N H2SO4 Stop Solution following the same order of Chromogen/Substrate Solution addition, including reagent blank well. Tap the plate gently along the outsides, to mix the contents of the wells. Wait a minimum of 5 minutes and read. The plate may be held up to 1 hour after addition of the Stop Solution before reading. 14. The developed color should be read on an ELISA plate reader equipped with a 450 nm filter. If dual wavelength is used, set the reference filter to 600-650 nm. The instrument should be blanked on air. The reagent blank must be less than 0.150 Absorbance at 450 nm. If the reagent blank is >0.150 the run must be repeated. Blank the reader on the reagent blank well and then continue to read the entire plate. Dispose of used plates after readings have been obtained.

QUALITY CONTROL For the assay to be considered valid the following conditions must be met: 1. Reagent blank (when read against air blank) must be <0.150 Absorbance (A) at 450 nm. 2. Negative Control must be <0.250 A at 450 nm (when read against reagent blank). 3. Each Calibrator must be >0.250 A at 450 nm (when read against reagent blank). 4. High Positive Control must be >0.500 A at 450 nm (when read against reagent blank). 5. The ISR (Immune Status Ratio) Values for the High, Low, and Negative Controls should be in their respective ranges printed on the vials. If the Control values are not within their respective ranges, the test should be considered invalid and the test should be repeated. 6. Additional controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. 7. Refer to NCCLS C24-A for guidance on appropriate Quality Control practices. 8. If above criteria are not met on repeat, contact DA Technical Service.

INTERPRETATION OF RESULTS

1. The patient’s ISR (Immune Status Ratio) are interpreted as follows:

ISR Results Interpretation <0.90 Negative No detectable IgM antibody by the ELISA test.

0.90-1.10 Equivocal Currently the CDC recommends that all equivocal samples be tested by immunoblotting.

>1.10 Positive Indicates presence of detectable IgM antibody. Recommend supplemental testing by immunoblot.

False positive results can occur with syphilis patients. RPR, VDRL or TPHA tests can rule out patients with syphilis. Also, false positive results may occur with sera from patients with relapsing fever, other spirochetal diseases, Rocky Mountain Spotted Fever, autoimmune diseases, EBV and CMV infections. Epidemiology of case, symptoms and other laboratory tests can help in differentiating these conditions from B. burgdorferi disease (16).

The following are recommendations for reporting results: a. Negative for anti-B. burgdorferi IgG/IgM antibodies, repeat testing in 2-4 weeks if suspected clinical diagnosis of early Lyme disease. b. Equivocal for anti-B. burgdorferi IgG/IgM antibodies, supplemental testing by immunoblot pending, if negative repeat testing in 2-4 weeks if suspected clinical diagnosis of early Lyme disease. c. Presumptively positive for anti-B. burgdorferi IgG/IgM antibodies, supplemental testing by immunoblot pending. d. The magnitude of the measured result (ISR), above the cutoff is not indicative of the total amount of antibody present and should not be reported. e. A negative antibody result does not rule out Lyme Disease. False negative results may occur when samples are drawn too early after onset. Production of detectable antibody levels is often delayed. Also, antibiotic therapy

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 6 early after EM may diminish or abrogate antibody response. Some patients may never generate detectable antibody levels. Patients with symptoms suggestive of Lyme disease with negative results should be retested in 4-6 weeks (19). f. False positive results can occur with syphilis patients. RPR, VDRL, or TPHA tests can rule out patients with syphilis. Also, false positive results may occur with sera from patients with relapsing fever, other spirochetal diseases, RockyMountain Spotted Fever, autoimmune diseases, EBV, and CMV infections. Epidemiology of case, symptoms, and other laboratory tests can help in differentiating these conditions from Lyme disease (16).

3. Samples that remain equivocal after repeat testing should be retested on an alternate method, e.g., immunofluorescence assay (IFA).

EXPECTED VALUES

1. The incidence of Lyme disease has been reported to be 1.4/100,000 specimens tested. (20) 92% of the cases reported were from New York, New Jersey, Pennsylvania, Connecticut, Massachusetts, Rhode Island, Wisconsin, and Minnesota. 2. Other non-specific laboratory results in Lyme disease may include: an increase in total IgM, slightly elevated leukocytes and liver transaminases, and the presence of circulating immune complexes and cryoglobulins. (19)

LIMITATIONS

1. The user of this kit is advised to carefully read and understand the package insert. Strict adherence to the protocol is necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of the incubation steps are essential for accurate results. 2. Specific IgG may compete with the IgM for sites and may result in a false negative. Conversely, rheumatoid factor in the presence of specific IgG may result in a false positive reaction. The Absorbent Solution diminishes competing B. burgdorferi-specific IgG and minimizes rheumatoid factor interference in samples. Studies indicate that the maximum amount of IgG which can be removed by the kit Absorbent Solution is in excess of the expected high end of the normal range for IgG > 1340 mg/dL. 3. The state of present-day technology does not provide a recommended reference standard. Because of the current inconsistencies in various test methodologies, physicians and laboratories must rely on a combination of test methods and results, and clinical symptoms when making a diagnosis of Lyme disease. 4. Kit procedures or practices outside those in this package insert may yield questionable results. 5. Icteric, lipemic, hemolyzed or heat inactivated sera may cause erroneous results and should be avoided. 6. Patients with syphilis may have antibodies which will cross-react with the Borrelia burgdorferi antigen in the Lyme IgM ELISA kit. Serological testing is only a diagnostic tool and must be interpreted in combination with other serologic and clinical observations. Rapid plasma reagin (RPR), microhemaglutination assay for antibodies to Treponema pallidum (MHA-TP), and the venereal disease Research Laboratory (VDRL) are positive for patients with treponemal disease and negative for those with Lyme Disease (17). False positive Fluorescent Treponemal Antibody - absorption (FTA - Abs) tests may occur in patients with Lyme Disease. Negative results for RPR, MHA- TP, or VDRL along with clinical manifestations can be used as an aid to exclude possible syphilic infections (18). 7. False positive results may occur with sera from patients with relapsing fever, other spirochetal diseases, Rocky Mountain Spotted Fever, autoimmune diseases, EBV and CMV infections. Epidemiology of case, symptoms and other laboratory tests can help in differentiating these conditions from Lyme disease. (16). 8. A negative antibody result does not rule out Lyme Disease. False negative results may occur when samples are drawn too early after onset. Production of detectable antibody levels is often delayed. Also, antibiotic therapy early after EM may diminish or abrogate antibody response. Some patients may never generate detectable antibody levels. Patients with symptoms suggestive of Lyme disease with negative test results should be retested in 4 - 6 weeks. (19) 9. A positive result only indicates prior exposure to B. burgdorferi. The level of antibody response or class of antibody response may not be used to determine active infection or disease stage. 10. Screening of the general population should not be performed. The positive predictive value depends on the likelihood of Lyme disease being present. Testing should only be performed when clinical symptoms are present or exposure is suspected. 11. The results of ELISA immunoassays performed on serum from immunosupressed patients must be interpreted with caution. 12. The assay performance characteristics have not been established for matrices other than sera

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 7 13. This assay detects immunoglobulin M specific to B. burgdorferi, whereas other available tests may detect total immunoglobulins (IgG/M/A). The analytical sensitivity for each immunoglobulin class has not been defined for this product. 14. The affinity and/or avidity of anti-B. burgdorferi IgM for the B. burgdorferi antigen used have not been determined for this assay.

PERFORMANCE CHARACTERISTICS Relative sensitivity and specificity

The data in Table 1 illustrates good sensitivity and specificity of the Lyme IgM ELISA relative to an alternate ELISA.

Table 1 Lyme IgG/IgM ELISA Sensitivity & Specificity Relative to Alternate Lyme ELISA

Diagnostic Automation Lyme IgG/IgM ELISA

+ E - Total

+ 38 5 3 46 Alternate ELISA 0 3 5 8

- 0 0 201 201

Total 38 8 209 255

Sensitivity = 38/41 = 92.7% 95% Confidence Interval = 84.5-100.0%* Specificity = 201/201 = 100.0% 95% Confidence Interval = 98.5-100% Agreement = 239/242 = 98.82% 95% Confidence Interval = 97.3-100.0% Equivocals are not included in the above calculations. The 95% Confidence Intervals were calculated using the normal method.

*The 95% Confidence Interval for sensitivity was calculated assuming one false negative.

PRECISION Seven sera were assayed ten times each on three different assays. With appropriate technique the user should obtain precision of <15% CV.

Table 2 Lyme IgG/IgM ELISA Precision

Assay 1 (n=10) Assay 2 (n=10) Assay 3 (n=10) Inter-Assay(n=30) X SD CV X SD CV X SD CV X SD CV 1 3.44 0.301 8.75% 3.40 0.266 7.82% 3.50 0.342 9.77% 3.45 0.295 8.55% 2 2.14 0.170 7.94% 2.09 0.096 4.59% 2.24 0.192 8.66% 2.16 0.165 7.64% 3 2.84 0.229 8.06% 3.07 0.287 9.67% 3.30 0.418 12.66% 3.13 0.380 12.14% 4 1.91 0.150 7.85% 1.88 0.105 5.59% 2.06 0.230 11.16% 1.95 0.183 9.39% 5 0.11 0.036 32.73% 0.10 0.032 32.0% 0.14 0.054 38.57% 0.12 0.044 36.67% 6 0.37 0.056 15.14% 0.36 0.048 13.33% 0.46 0.071 15.43% 0.40 0.074 18.50%

REFERENCES

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected] Lyme IgM Page 8

1. Miller, G.L., R.B. Craven, R.E. Bailey, and T.F. TSAI. 1987-1988. The Epidemiology of Lyme Disease in the United states Lab Med 1990; 21:285-289. 2. CDC: Lyme Disease - United States, 1987 and 1988. MMWR 1989; 38: 668-672. 3. Schmid, G.P. 1989. Epidemiology and Clinical Similaritites of Human Spirochetal Diseases. Rev Inf Dis. 11S6: 1460-1468. 4. Dattwyler, R.J. 1990. Lyme borreliosis: An Overview of the Clinical Manifestations. Lab Med. 21: 290-291. 5. Golightly, M.G., J.A.Thomas, A.L. Viciana. 1990. The Laboratory Diagnosis of Lyme borreliosis. Lab Med. 21: 299-304. 6. Tilton, R.C., R.W. Ryan. 1991. Laboratory Detection of Borrelia burgdorferi Infection. Clin Microbiol Newsltr. 13: 68-71. 7. Engvall, E., K. Jonsson, and P. Perlman. 1971. Enzyme-Linked Immunosorbent Assay, (ELISA) Quantitative Assay of Immunoglobulin G. In: Immunochemistry. 8:871-874. 8. Engvall, E. and P. Perlman. 1971. Enzyme-Linked Immunosorbent Assay, ELISA. In: Protides of the Biological Fluids, H. Peeters, Ed., Proceedings of the Nineteenth Colloquium, Brugge Oxford. Pergamon Press. p. 553-556. 9. Engvall, E., K. Jonsson, and P. Perlman. 1971. Enzyme- Linked Immunosorbent Assay. II. Quantitative Assay of Protein Antigen, Immunoglobulin-G, By Means of Enzyme-Labeled Antigen and Antibody-Coated Tubes. In: Biochem. Biophys. Acta., 251:427-434. 10. Van Weeman, B. K. and A.H.W.M. Schuurs. 1971. Immunoassay Using Antigen-Enzyme Conjugates. FEBS Letter. 15:232-235. 11. Bakerman, S. 1980. Enzyme Immunoassays. Lab. Mgmt. August, p. 21-29. 12. Voller, A., and D. E. Bidwell. 1975. In: Brit. J. Exp. Pathology. 56:338-339. 13. Engvall, E. and P. Perlman. 1972. Enzyme-Linked Immunosorbent Assay, ELISA. III. Quantitation of Anti-Immunoglobulins in Antigen- Coated Tubes. J. Immunol. 109: 129-135. 14. National Committee for Clinical Laboratory Standards. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture Approved Standard. NCCLS Publication H18-A. 1990. 15. CDC/NIH Guidelines: 1993. Biosafety in Microbiological and Biomedical Laboratories. 3rd Edition. 16. Magnarelli, L., et al. 1987. Cross reactivity in serologic tests for Lyme disease and other spirochetal infections. J. Infec. Dis. 156:183-188. 17. Dammini, G.J. 1986. Lyme Disease: Its Transmission and Diagnostic Features. Lab Mgmt. 33-38. 18. Hunter, E.F., H. Russell, C. Farshy, J. Sampson, S. Larsen. 1986. Evaluation of Sera from Patients with Lyme Disease in the Fluorescent Treponemal Antibody-Absorption tests for Syphilis. Sexually Transmitted Diseases. 13:232-236. 19. Barbour, A. 1988. Laboratory aspects of Lyme borreliosis. Clin. Micro. Rev.1:399-414. 20. Tsai, T., R. E. Bailey, P. S. Moore. 1989. National Surveillance of Lyme Disease, 1987-1988. Connecticut Medicine. 53: 324-326.

Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA Phone: (703) 266-5667, FAX: (703) 266-5664 http://www.atlaslink-inc.com, [email protected]