MBL Bion Product Availability KITS with COMPONENTS CODE NO. DSG 1 & DSG 3 ELISA KIT, 48 Tests Each RG-M7593-D
SYMBOL DEFINITIONS
= Consult Directions for Use = In Vitro Diagnostic Reagent = Store Away From Direct Light = Positive Control = Storage Temperature = Negative Control DSG 1 & DSG 3 = Expiration Date = Endpoint Titer = Number of Tests = Code Number ELISA TEST SYSTEM e = Amount = Lot Number
NOTE: Changes highlighted INTENDED USE The MBL Bion DSG 1 & DSG 3 ELISA TEST SIGNIFICANCE AND BACKGROUND SYSTEM is a semi-quantitative enzyme-linked Pemphigus includes a group of often fatal, immunosorbent assay for the detection of autoimmune, blistering diseases characterized by autoantibodies to desmoglein 1 (DSG 1) and intraepithelial lesions. Pemphigus vulgaris and its desmoglein 3 (DSG 3) in human serum. variants may present with oral or other mucosal The MBL Bion DSG 1 & DSG 3 ELISA TEST lesions alone or with mucosal plus skin lesions. SYSTEM is intended for in vitro diagnostic use as Pemphigus foliaceus and its variants present with skin an aid in the diagnosis of certain pemphigus (1, 2) lesions alone . Indirect immunofluorescent (IIF) diseases. studies reveal that both forms of pemphigus are caused by autoantibodies to cell surface antigens of PRINCIPLE OF THE PROCEDURE stratified epithelia of mucous membranes and skin (3, 4). The MBL Bion DSG 1 & DSG 3 ELISA TEST SYSTEM These antibodies bind to calcium dependent adhesion measures anti-DSG 1 and DSG 3 antibodies present molecules in cell surface desmosomes, notably in the sera by ELISA. Calibrators and patient sera are desmoglein 1 (DSG 1) in pemphigus foliaceus and added to microwell coated with DSG 1 and DSG 3 desmoglein 3 (DSG 3) in pemphigus vulgaris (5, 6). antigens, allowing antibodies to react with the Pemphigus vulgaris patients with both mucosal and immobilized antigens (Sample incubation). After skin lesions have antibodies to both DSG 3 and washing to remove any unbound serum proteins, DSG 1 (7). horseradish peroxidase conjugated IgG (Fab’) is The diagnosis of pemphigus depends on biopsy and added and incubated (Conjugate incubation). serum studies that characterize lesions and detect Following another wash step, the peroxidase the autoantibodies that cause them. Serum studies substrate is added and allowed to incubate for an afford highly sensitive diagnostic aids(4, 7-9). additional period of time (Substrate incubation). Originally they were performed by indirect Stop solution is then added to each well to terminate immunofluorescence (IIF) using monkey esophagus the enzyme reaction and to stabilize the color and other tissues sections. The identification of the development. The assay can be quantified by reactive antigens as DSG 1 and DSG 3 has made it measuring the reaction photometrically and plotting possible to develop highly specific and sensitive the results. ELISA methods (10).
MBL Bion Form 1.11.6.4.12 Rev. 10/14 DSG-1 PRINCIPLE OF THE PROCEDURE (continued) Brief Assay Procedure Add the following to microwells: Sera are reacted with antigen-coated microtiter wells.
WARNINGS AND PRECAUTIONS 1. This product is for in vitro diagnostic use only. 5. Do not use kit components beyond the stated expiration dates. 2. Calibrator sera are derived from human serum, in which HBsAg, 6. Refrigeration (2-8oC) of kit immediately upon arrival will insure HCV antibody and HIV antibody cannot be detected. No test stability until labeled expiration date. method, however, can guarantee the absence of these or any other 7. Do not expose the kit components to direct sun during storage or infectious agents. These reagents and all patient samples should be when in use during the assay. Store all kit components at 2-8oC. handled at the Biosafety Level 2 as recommended for any 8. All reagents must be brought to room temperature (20-25°C) before potentially infectious human serum or blood specimen in the starting the assay. Do not open protective envelope with antigen Center for Disease Control/National Institute of Health manual microtiter wells before equilibration to room temperature to prevent “Biosafety in Microbiology and Biomedical Laboratories”, 1993 condensation in the wells. Return unused reagents to refrigerator and FDA LABELING GUIDELINES FOR IN VITRO promptly after use. DIAGNOSTIC REAGENT MANUFACTURERS, DEC. 1985. 9. Previously frozen samples after thawing should be thoroughly 3. Matching lot numbers of microtiter antigen wells, conjugate reagent mixed prior to testing. and calibrator serum must be used together in the assay. Do not 10. Reusable glassware must be washed and thoroughly rinsed free of substitute reagents from other kits. all detergents. 4. All breakaway microtiter wells which are not immediately required 11. Assay diluent may form precipitate. Before use in test, bring should be returned to the pouch with desiccant and must be diluent to room temperature and mix well to dissolve precipitate. carefully resealed to avoid moisture absorption. Wells, which have If all precipitate does not dissolve, allow the remaining precipitate to been processed, must be discarded. settle and avoid when using diluent. It does not affect test results.
MBL Bion Rev. 10/14 DSG-2 WARNINGS AND PRECAUTIONS (continued)
12. Wash buffer concentrate may look turbid from crystal formation at 21. Blot plates free of any residual wash solution before adding conjugate 2-8°C, but this does not cause inconsistent results. Prior to or substrate solution to prevent inconsistent color development. preparation of the 1X wash buffer working solution, bring to room Do not allow wells to become dry during the assay procedure. temperature and mix thoroughly to dissolve. 22. Ensure that the bottom of the plate is clean and dry, and that no air 13. Avoid microbial contamination of all reagents and samples or bubbles are present on the surface of the liquid in the wells before incorrect results may occur. reading the plate. 14. Cross-contamination of reagents and/or samples could cause false 23. In accordance with the principles of good laboratory practice, it is results. Carefully pipette samples and reagents without foaming strongly recommended that all clinical specimens and assay or splashing to avoid cross contamination of microtiter wells. Mix materials, including the antigen microtiter wells, should be reagents by gentle inversion. Do not vortex or shake. considered potentially infectious and handled and disposed of with 15. Contamination of substrate solution with conjugate or other all necessary precautions. Disposable implements (pipettes, tips, oxidants will cause premature color change. tubes, etc.) used in the test should be soaked in 0.5% sodium 16. Do not expose any of the reagents to sodium hypochlorite (bleach) hypochlorite solution for at least one hour or autoclaved at 121oC solutions, or strong odors from these solutions. Exposure to for 30 minutes prior to discarding. bleach may cause the biological activity of these reagents to be 24. Liquid waste containing acid must be neutralized with base destroyed. (e.g. sodium bicarbonate) before decontamination with sodium 17. Prepare working solution of conjugates only in bottles provided. If hypochlorite (bleach). conjugates come into contact with residual amounts of sodium 25. Acid-containing liquid waste that has been neutralized and other azide from containers or implements, the enzymatic activity may liquid waste should be collected and decontaminated by adding a be destroyed. sufficient volume of sodium hypochlorite to obtain a final 18. Strict adherence to specified assay instructions is essential. concentration of 0.5%. A 30-minute exposure is necessary to Incubation temperatures above or below normal room temperature ensure effective decontamination. (20-25°C), incubation outside the time period range, and inaccurate 26. Do not pipette by mouth. Care should be taken to avoid splashing dilution of samples may yield erroneous results. and generation of aerosols. Avoid contact of reagents with eyes, 19. When adding reagents to antigen wells, add them at the same rate skin, mucous membranes, and clothing. Wear disposable gloves, and in the same order specimens and calibrators and were added. eye protection and lab apparel while handling specimens and Then, before incubation, cover wells with a plate cover and shake performing the assay. Wash hands thoroughly when finished. If gently on counter to ensure an even spread of samples or reagents contact occurs, immediately flush with copious amounts of water. throughout the antigen well. 27. Some reagents contain sodium azide as a preservative. Azide may 20. The wells must be rinsed with wash solution properly to avoid react with copper or lead in plumbing system to form explosive false positive or negative results. metal azides. Therefore, always flush with ample amounts of water when disposing of materials containing azide into a drain. Sodium azide is a poison and may be toxic if ingested.
SPECIMEN COLLECTION
Blood should be collected fasting or at least one hour after meals to avoid Specimens should be stored refrigerated (2-8oC) and tested within one lipemic serum. Aseptically collect 5-8 ml of blood by approved week of collection. If testing is delayed, store in aliquots at -20oC for up venipuncture procedures. Allow the blood to clot at room temperature to one month or at -70oC or lower for longer periods. Avoid repeated (20-25oC) and separate serum as soon as possible to minimize hemolysis. freezing and thawing, which may cause denaturation of protein and loss of No anticoagulants or preservatives should be added. Avoid using sera antibody activity to yield erroneous results. Do not store in a self-defrosting exhibiting a high degree of lipemia, hemolysis, icterus or microbial growth. freezer. These conditions may cause aberrant test results. Do not use heat- When testing multiple samples on the same patient to look for a significant inactivated serum as this affects test results. change in antibody level, the specimens must be tested simultaneously in the same assay. PROCEDURE
MATERIALS PROVIDED MATERIALS REQUIRED BUT NOT PROVIDED 1. Microtiter well strips in plate holder 1. Microtiter plate reader capable of reading 6. Wash bottle or plate washer instrument coated with antigens at a single wavelength of 450nm or at a 7. Distilled water-CAP Type one or equivalent 2. Conjugate (101X Concentrate) duel wavelength of 450nm and 630nm 8. One liter graduated cylinder 3. Conjugate Diluent 2. Calibrated multi-channel micropipette 9. 1.1 ml microtubes with 12 X 8 rack for sample 4. Calibrators capable of delivering 100 μl for dipsensing dilution preparation and dispensing 5. Sample Diluent conjugates, substrates and stop solution 10. Timer with alarm 6. Wash Buffer (10X Concentrate) 3. Calibrated single channel pipette capable of 11. Disposable serological pipettes, 1ml and 5 ml 7. Substrate Solution delivering 10 μl 12. Disposable basin and disinfectant (0.5% 8. Stop Solution 4. Disposable pipette tips for single and sodium hypochloride) 9. Bottle for Conjugate Working Solution multi-channel pipettors 13. Plate sealers 5. Reagent reservoirs for multi-channel 14. Paper towels pipettes 15. Felt tip marking pen
MBL Bion Rev. 10/14 DSG-3 PROCEDURE (continued) BEFORE YOU START PLATE SET-UP 1. Remove all kit test materials from refrigeration and allow them to warm to room temperature (20-25oC) prior to use. Return 1 2 3 4 5 6 7 8 9 10 11 12 unused materials to refrigerator (2-8oC) promptly after use. A DSG 1 Cal. O O O O O DSG 3 Cal. O O O O O B Neg. Cal. O O O O O Neg. Cal. O O O O O 2. Prepare Worksheet: C S1 O O O O O S1 O O O O O a. Determine number of samples and calibrators to be D S2 O O O O O S2 O O O O O tested. Each sample and calibrator requires one antigen- E S3 O O O O O S3 O O O O O coated well. F O O O O O O O O O O O O G O O O O O O O O O O O O b. On the worksheet, indicate the location the samples and H O O O O O O O O O O O O calibrators are to be placed in the microtiter wells. Note: Depending on available software, check for correct sample and calibrator configuration. TEST PROCEDURE 3. Antigen Microtiter Wells: 1. SAMPLE APPLICATION a. Remove the required number of microtiter wells and place in Using the multi-channel pipettor, transfer 100 μl of each diluted holder. sample into the appropriate microtiter wells of the antigen test Note: To prevent condensation in the wells, do not open plate. Add Calibrators directly to appropriate wells with the protective envelope with the antigen microtiter single channel pipettor. (Do not dilute Calibrators.) wells before equilibration to room temperature. b. Promptly return unused strips to their packaging with 2. SAMPLE INCUBATION o desiccant, seal and return to refrigerator (2-8 C). After the Cover wells with a plate cover and incubate at room temperature envelope has been opened, the unused strips are stable for (20-25oC) for 60 + 5 minutes. 60 days. 4. Wash Solution: 3. WASH Prepare a 1:10 dilution of the Wash Buffer Concentrate with distilled Remove plate cover. Aspirate or discard the well contents. To water prior to use in assay. For example: add 100 ml of 10X wash, fill the well with Wash Solution and then aspirate or concentrate to 900 ml of distilled water. Mix thoroughly to dissolve discard the contents completely. Wash 4 times. Tap the plate any crystals that may be present. The diluted wash solution on a paper towel to remove any remaining Wash Solution. is stable for 2 weeks at 2-8oC. Note: If using an automated wash instrument, set the dispensing Note: Do not dilute all of the 100 ml concentrate at one time if volume at 300-350μl. Set the wash cycle for 4 washes with no more than 2 weeks is anticipated to use up the entire kit. delay between washes. When wash cycle is complete, tap inverted plate on a paper towel to remove any remaining Wash 5. Conjugate Working Solution: Solution. a. Using the Conjugate Diluent, prepare daily a 1:101 dilution of the Conjugate Concentrate (101X) prior to use in assay. 4. CONJUGATE APPLICATION Note: Use only the bottle provided to avoid contamination. If Pour Conjugate Working Solution into new reagent reservoirs. conjugate comes into contact with residual amounts of Using the multi-channel pipette, add 100 μl of the Working sodium azide from containers or implements the Conjugate Solution to each well at the same rate and in the same enzymatic activity may be destroyed. order as the samples and calibrators. b. Dilute only the amount of conjugate needed for the number of tests being run. 5. CONJUGATE INCUBATION For example: add 100 μl (0.1 ml) of Conjugate Concentrate to 10 Cover the wells with the plate cover and incubate at room ml of the Conjugate Diluent. temperature (20-25 °C) for 60 + 5 minutes. c. Diluted conjugate must be used within a day. Discard remaining solution, rinse bottle thoroughly with distilled water, allow bottle 6. WASH to dry and recap. Remove the plate cover and wash the microtiter plate following 6. Other Reagents: Calibrators, Sample Diluent, TMB Substrate the same procedure as the first wash in step 3. Solution and Stop Solution are ready to use with no additional preparation needed. 7. SUBSTRATE APPLICATION Measure needed amount of TMB Substrate Solution directly from PREPARATION OF SAMPLES bottle with a disposable serological pipette into a new reagent reservoir. Using the multi-channel pipette, add 100 μl of the 1. If samples are refrigerated, remove and allow to reach room solution to each well at the same rate and in the same order as the temperature. If samples are frozen, after thawing, they should samples and calibrators. Discard any remaining Substrate Solution. be thoroughly mixed. Do not return any leftover solution to the reagent bottle. Note: Do not use heat-inactivated sera. Avoid using sera exhibiting a high degree of lipemia, hemolysis, icterus or 8. SUBSTRATE INCUBATION microbial growth. Cover wells with plate cover and incubate at room temperature o 2. Prepare a 1:101 dilution of each patient. To the 1.1 ml (20-25 C) for 30-35 minutes. microtubes in the 12 X 8 rack, add 10 μl of each sample to 1 ml of the Sample Diluent. Mix well. 3. Diluted samples must be used within one day.
MBL Bion Rev. 10/14 DSG-4 PROCEDURE (continued)
9. STOP REACTION 11. CALCULATION OF RESULTS Remove the plate cover. Using a disposable serological pipette, measure out the Stop Solution into a new reagent reservoir For results calculated by reader program or manually, use the (different from the conjugate or substrate reservoirs). Using the following formula: multi-channel pipette, add 100 μl of the solution to each well at Unit value (U/ml) = ( A
In order for the test results to be considered valid, the criteria If the results for calibrators do not meet their respective listed below must be met. If either of these is not met, the results specifications, the assay should be considered invalid and should are invalid and the test should be repeated. be repeated. If, upon repeat testing, the calibrator results are still incorrect, do not report patient results. Negative Calibrator must be: < 0.10 (OD at 450 nm) Additionally, MBL Bion recommends that the user run in-house DSG Calibrators must be: > 0.70 (OD at 450 nm) controls with each run. These should include at least two levels of each antibody with one of each close to the positive decision point, each with established reproducible values. INTERPRETATION OF RESULTS DSG 1
The following is intended only as a guide for interpretation. Each Less than 18 Negative for anti-DSG-1 Ab laboratory is recommended to establish its own criteria for test interpretation based on sample populations typically encountered. 18 - 36 Indeterminate Greater than or equal to 36 Positive for anti-DSG-1 Ab MBL Bion recommends repeat testing of indeterminate samples, either with a fresh sample drawn at a later time or the original sample DSG 3 tested by another method. Less than 19 Negative for anti-DSG-3 Ab
19 - 37 Indeterminate
Greater than or equal to 37 Positive for anti-DSG-3 Ab TROUBLESHOOTING To ensure consistent performance, possible problems, their causes Conjugate may have become contaminated which could also and the solutions to correct/ prevent them are mentioned below: cause deterioration. Review proper storage instructions and the handling and preparation of reagents. 1. Calibrator values incorrect - Before repeating assay, review the procedural items such as reagent temperatures, 3. Substrate color too light - Use of kit beyond its stated incubation temperatures, incubation time periods, and expiration date can cause poor color development in the washing times or set-up of autowasher. Review handling wells. Also, it can be caused by preparing an incorrect techniques such as drying of antigen wells after wash steps dilution of conjugate. and splashing of reagents to cause cross contamination. 4. Substrate color too dark - The appearance of darker colors Re-QC pipetting device if volume accuracy is in question. throughout the antigen wells is usually an indication that 2. No color development in substrate - Make sure samples the wells were not properly washed particularly after the and calibrators have been added during dilution incubation with the enzyme conjugate. Review preparation. Make sure conjugate has been added instructions. properly; or, perhaps kit was stored at elevated temperatures for an extended period of time causing deterioration of conjugate.
MBL Bion Rev. 10/14 DSG-5 LIMITATIONS OF THE PROCEDURE
1. As with other diagnostic test procedures, the results 3. The assay performance characteristics have not been obtained with the MBL Bion DSG 1 & DSG 3 ELISA Test established for matrices other than serum. System serve only as an aid to diagnosis and should not 4. A positive result indicates the presence of antibodies to be interpreted as diagnostic by themselves. The results recombinant DSG 1 or DSG 3 and does not specifically should be interpreted in conjunction with clinical evaluation identify a certain type of pemphigus. of the patient along with other diagnostic procedures by medical authorities. 5. A negative result does not rule out the presence of pemphigus disease. 2. Performance of these assays in the pediatric population has not been established.
EXPECTED VALUES
Sera from 179 healthy male and female adult healthcare workers were tested with the MBL Bion DSG 1 & DSG 3 ELISA Test System. The results are summarized below. A negative result is the expected value for a normal patient; however, each laboratory should establish and maintain its own expected values based on samples from the populations they typically encounter.
Mean SD Mean + 3SD Max.
DSG 1 2.15 2.55 9.80 21.58
DSG 3 2.35 2.48 9.79 16.65
Further, in another study, the incidence of DSG 1, DSG 3 and IIF Positives were examined in four populations, with the following results.
POPULATION (number) DSG 1 POSITIVE DSG 3 POSITIVE IIF POSITIVE Normal blood donors (n = 36) 1 (3%) 0 (0%) 4 (11%) Bullous pemphigoid (n = 40) 2 (5%) 0 (0%) 7 (17%) Pemphigus vulgaris (n = 28) 21 (75%) 28 (100%) 28 (100%) Pemphigus foliaceus (n = 23) 23 (100%) 6 (26%) 21 (92%)
CLINICAL STUDY SUMMARY
IIF-MONKEY DSG 1 DSG 3 DSG 1 &/or DSG 3 ESOPHAGUS True Negatives 73 76 73 65
False Negatives 7 15 0 2
True Positives 44 36 51 49
False Positives 3 0 3 11
Specificity 73/76 = 96.1% 76/76 = 100% 73/76 = 96.1% 65/76 = 85.5%
Sensitivity 44/51 = 86.3% 36/51 = 70.6% 51/51 = 100% 49/51 = 96.1%
MBL Bion Rev. 10/14 DSG-6 EXPECTED VALUES (continued)
Amagai et al. reported similar incidences of DSG 1 and DSG 3 in a different study.(9) SPECIFIC PERFORMANCE CHARACTERISTICS I. COMPARATIVE STUDIES To investigate overall agreement of the MBL Bion DSG 1 and DSG 3 ELISA TEST SYSTEM, 127 serum samples were compared with an immunofluorescent test system. The comparison included the following groups: normal subjects (n=36), bullous pemphigoid (n=40), pemphigus vulgaris (n=28) and pemphigus foliaceus (n=23). The result of this comparison is summarized below.
IIF IIF POS NEG POS NEG DSG 1 POS 47 2 DSG 3 POS 34 0 NEG 11 67 NEG 26 67 Overall Agreement = 89.8% Overall Agreement = 79.5%
II. REPRODUCIBILITY 3 SAMPLES TESTED IN SIX WELLS THREE TIMES ON THREE DIFFERENT LOTS INTRA-ASSAY DSG 1 DSG 3
Lot A 1st test 2nd test 3rd test 1st test 2nd test 3rd test
S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 mean 31.2 47.6 99.1 35.1 57.2 117.2 35.3 57.4 127.3 21.7 72.7 97.9 21.8 72.5 98.9 24.4 75.0 99.0 SD 1.3 2.0 3.2 1.2 2.1 3.8 1.1 2.0 3.5 0.4 1.6 1.8 0.4 0.6 0.6 1.1 1.2 1.1 CV (%) 4.2 4.2 3.2 3.4 3.6 3.3 3.0 3.4 2.8 1.7 2.3 1.8 1.7 0.9 0.7 4.4 1.6 1.1
Lot B 1st test 2nd test 3rd test 1st test 2nd test 3rd test
S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 mean 24.9 45.4 92.9 31.1 58.5 119.7 31.2 59.8 135.2 27.1 78.5 101.3 25.8 76.8 100.7 28.6 79.5 107.1 SD 0.5 2.0 2.2 1.2 2.4 2.0 1.8 1.6 2.1 0.6 2.3 0.9 0.3 0.6 0.9 0.7 1.8 1.8 CV (%) 2.2 4.3 2.4 3.8 4.2 1.7 5.6 2.6 1.5 2.2 3.0 0.9 1.1 0.8 0.9 2.5 2.2 1.7
Lot C 1st test 2nd test 3rd test 1st test 2nd test 3rd test
S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 S1 S2 S3 mean 29.3 51.7 104.7 32.1 58.7 117.7 31.8 57.5 125.2 25.8 78.7 111.5 24.1 77.3 117.0 25.1 76.3 108.4 SD 0.9 2.1 2.7 1.0 2.1 1.6 0.6 1.8 1.9 0.7 1.0 1.9 0.2 0.7 1.9 0.6 1.8 1.2 CV (%) 3.2 4.0 2.6 3.2 3.5 1.4 2.0 3.1 1.5 2.6 1.2 1.7 0.6 1.0 1.6 2.5 2.4 1.1
MBL Bion Rev. 10/14 DSG-7 SPECIFIC PERFORMANCE CHARACTERISTICS (continued)
3 SAMPLES TESTED ON 6 DIFFERENT DAYS USING THREE DIFFERENT LOTS INTER-ASSAY
Lot A Lot B Lot C DSG 1 1st test 1st test 1st test S1 S2 S3 S1 S2 S3 S1 S2 S3 mean 19.5 107.8 189.1 17.9 107.2 204.3 16.9 103.6 199.1 SD 1.8 7.4 12.3 1.0 7.0 22.4 0.9 4.4 16.2 CV (%) 9.3 6.9 6.5 5.3 6.5 11.0 5.2 4.2 8.1
DSG 3 1st test 1st test 1st test S1 S2 S3 S1 S2 S3 S1 S2 S3 mean 26.8 70.6 142.0 31.5 75.6 138.0 33.2 74.3 140.7 SD 1.4 3.8 5.8 1.7 2.1 1.8 1.2 1.6 2.1 CV (%) 5.3 5.3 4.1 5.4 2.8 1.3 3.7 2.1 1.5
3 SAMPLES TESTED SIX TIMES ON THREE DIFFERENT LOTS LOT COMPARISON DSG 1 DSG 3
S1 S2 S3 S1 S2 S3
LotABCABCABCLotABCABCABC
mean 35.1 31.1 32.1 57.2 58.5 58.7 117.2 119.7 117.7 mean 22.0 27.1 25.8 72.7 78.5 78.7 100.1 101.8 111.5
Mean 32.8 58.1 118.2 Mean 25.0 76.6 104.5
SD 2.1 2.3 2.9 SD 2.2 2.8 5.0
CV (%) 6.3 3.9 2.4 CV (%) 8.7 3.6 4.8
III. CROSS-REACTIVITY STUDIES
To investigate the potential for positive reactions due to cross-reactive antibodies, thirty-six (36) samples were tested using the MBL Bion DSG 1 & DSG 3 ELISA TEST SYSTEM. Twenty-five (25) of these samples were positive for infectious disease antibodies against antigens such as EBV-VCA, HSV, CMV, Measles, Mumps, VZV, Toxo, Borrelia (Lyme’s Disease); and, eleven (11) samples were positive for antibodies to various other autoimmune diseases such as ANCA, AMA, ASMA, and APCA. All samples tested were negative on the MBL Bion DSG 1 & DSG 3 ELISA TEST SYSTEM. Interfering substances Hemoglobin (up to 490mg/dL), Bilirubin F (up to 18.2mg/dL), Bilirubin C (up to 22mg/dL), chyle (up to 2800 unit as Formazine) and/or Rheumatoid factor (up to 965 IU/mL) did not affect the assay result, but avoid using highly hemolized samples or highly lipemic samples. BIBLIOGRAPHY 1. Lever WF. Pemphigus. Medicine 1953; 32:1-123. 7. Amagai M, Tsunoda K, Zillikens D, Nagai T, Nishikawa T. The clinical phenotype of 2. Lever WF. Pemphigus and Pemphigoid. Charles Thomas. Springfield, IL pemphigus is defined by the anti-desmoglein autoantibody profile. J Am Acad Dermatol 1999, 1965. 40:167-70. 3. Beutner EH, Jordon RE, Chorzelski TP. The immunopathology of 8. Sabolinski ML, Beutner EH, Krasny S, Kumar V, Huang J, Chorzelski TP, et al. Substrate pemphigus and bullous pemphigoid. J Invest Dermatol 1968; 51:63-80. specificity of antiepithelial antibodies of pemphigus vulgaris and pemphigus foliaceus sera in 4. Beutner EH, Chorzelski TP, Jablonska S. Clinical significance of immunofluorescence tests on monkey and guinea pig esophagus sections. J Invest Dermatol immunofluorescent tests of sera and skin in bullous diseases: A 1987, 88:545-49. cooperative study. In “Immunopathology of the skin” (EH Beutner, TP 9. Amagai M, Komai A, Hashimoto T, Shirakata Y, Hashimoto K, Yamada T, et al. Usefulness of Chorzelski and V Kumar ed.) 3rd Ed. 1987, 177-205. enzyme linked immunosorbent assay using recombinant desmogleins 1 and 3 for sero-diagnosis 5. Stanley JR, Koulu L, Thivolet C. Distinction between epidermal antigens of pemphigus. Brit J Dermatol 1999, 140: 351-357. binding pemphigus vulgaris autoantibodies and pemphigus foliaceus 10. Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, et al. Characterization of autoantibodies. J Clin Invest 1984, 74:313-320. autoantibodies in pemphigus using antigen specific Elisa’s with baculovirus expressed 6. Stanley JR. Cell adhesion molecules as targets of autoantibodies in recombinant desmogleins. J Immunol 1997, 159: 2010-2017. pemphigus and pemphigoid, bullous disease due to defective epidermal 11. Harman KE, Gratin MJ, Bhogal SJ, Challecomebe SJ, Black M. The clinical significance of adhesion. Advances in Immunol 1992, 53:291-325. autoantibodies to desmoglein 1 in 78 cases of pemphigus vulgaris. J Invest Derm 1999, 112:568(Abstr). 12. Data on file at MBL Bion, Inc.
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MBL Bion Rev. 10/14 DSG-8