Biohit Oyj. Anti-Telomere IgG. ELISA kit for the detection of antitelomere IgG antibodies based on the double-stranded telomeric DNA recognition

Transcription

1 Biohit Oyj Anti-Telomere IgG ELISA kit for the detection of antitelomere IgG antibodies based on the double-stranded telomeric DNA recognition For in vitro Diagnostic Use Store at 2-8 o C Upon Receipt INSTRUCTIONS FOR USE REF Biohit Oyj Laippatie 1 FIN Helsinki, Finland Tel: Fax: info@biohit.fi IVD 1

2 Anti-Telomere IgG ELISA Cat. No CONTENTS 1. INTENDED USE SUMMARY AND EXPLANATION OF THE TEST PRINCIPLE OF THE TEST WARNINGS AND PRECAUTIONS SPECIMEN COLLECTION AND HANDLING KIT CONTENTS, REAGENT PREPARATION AND STABILITY FOR MATERIALS PROVIDED Microplate Washing Buffer Concentrate (x 10) Diluent Buffer Standard Negative Control Conjugate Solution Substrate Solution Stop Solution Incubation Covers Instructions for Use MATERIALS REQUIRED BUT NOT PROVIDED STORAGE AND STABILITY TEST PROCEDURE RESULTS Quality Control Values Calculation of the Results Interpretation of the Results LIMITATIONS OF THE PROCEDURE PERFORMANCE CHARACTERISTICS REFERENCES DATE OF ISSUE WARRANTY ORDERING INFORMATION EXPLANATION OF THE SYMBOLS USED IN LABELS SHORT OUTLINE OF THE PROCEDURE

3 1. INTENDED USE Anti-Telomere IgG enzyme immunoassay test kit is intended for the detection of human IgG-class antibodies to telomeric dsdna in serum. FOR IN VITRO DIAGNOSTIC USE. 2. SUMMARY AND EXPLANATION OF THE TEST Systemic lupus erythematosus (SLE) is a prototype of autoimmune diseases that affects practically every organ in the body. The presence (70%) of multiple antibodies, especially IgG-class antibodies directed against native or double-stranded DNA, are typical of SLE and correlate well with disease activity. Anti-dsDNA antibodies are shown to be highly enriched in glomerural eluates and the possibility exists that DNA-anti-DNA complexes contribute to the pathogenesis of renal injury in SLE (1). Measurement of high titers of anti-telomere dsdna antibodies in an ELI- SA (enzyme-linked immunosorbent assay) is a sensitive screening test for SLE (1, 2). Criteria to distinguish SLE from other connective-tissue diseases, such as rheumatoid arthritis, established by the American Rheumatism Association and last revised in 1982, also include the detection of autoantibodies to dsdna (3). Unlike in the conventional enzyme immunoassay, which uses purified calf thymus dsdna as capture antigens for autoantibodies, the newly developed Biohit anti-telomere antibody assay is based on the use of double-stranded telomeric DNA as capture antigen for the binding of SLE antibodies. Human antibodies against telomeric DNA are more specific and sensitive test for SLE. The telomeres, which represent approx. 0.15% of the human genome, are found at the ends of all human chromosomes. The highly conserved human telomeric sequence (TTAGGG)n is shared by all vertebrates. The telomeres protect the cell from the loss of genomic information on the chromosomes. 3. PRINCIPLE OF THE TEST This anti-telomere-elisa is based on an enzyme immunoassay technique with double-stranded telomeric DNA adsorbed on a microplate and detection antibody labeled with horseradish peroxidase (HRP). The assay proceeds according to following reactions: 3

4 1. Specific double-stranded telomeric DNA attached to polystyrene surface binds anti-telomere antibodies present in human serum. 2. Microwells are washed to remove residual sample. 3. HRP-conjugated monoclonal anti-human IgG is added to microwells and binds to the anti-telomere antibodies. 4. The wells are washed to remove the residual conjugate and TMB-substrate is added. The substrate is oxygenized by the enzyme and a colored end product is produced. 5. The enzyme reaction is terminated with stop solution. The intensity of color developed is directly related to the anti-dsdna concentration of the sample. 4. WARNINGS AND PRECAUTIONS For in vitro diagnostic use. CAUTION: Handle serum samples as potential biohazardous material. All samples should be regarded as potentially contaminated and treated as if they were infectious. Please refer to the U. S. department of Health and Human Services (Bethesda, MD., USA) publication Biosafety in Microbiological and Biomedical Laboratories, 1999, 4th ed. (CDC/NIH) and No. (CDC) on reports of laboratory safety procedures on different diseases or any other local or national regulation. This kit contains reagents manufactured from human blood components. The source materials provided in this kit have been tested for the presence of antibodies to hepatitis B and C as well as antibodies to HIV, and found to be negative. However, as no test method can offer absolute assurance that these pathogens are absent, all recommended precautions for the handling of blood derivative should be observed. Always use protective gloves when handling patient samples. Use a safety pipetting device for all pipetting. Never pipette by mouth. Read all instructions prior to performing this assay. All reagents provided in the kit can be disposed by pouring into a sink and flushing with an excess of tap water. The components of the kit are provided at precise concentrations. Further dilution or other alterations of the reagents could cause incorrect results. Return the reagents to 2-8 C for storage immediately after use. 4

5 5. SPECIMEN COLLECTION AND HANDLING No fasting is required prior to blood sampling. The blood sample is collected by venipuncture into e.g. a plastic serum tube without additives. Blood tubes are mixed immediately by turning them upside down 5-6 times and tubes for serum allowed to clot (for minimum 30 minutes) at room temperature (20-25 C). Serum after clotting is separated by centrifugation (e.g. plastic tube, acceleration up to 2000 G, minutes). Serum can be stored refrigerated (2-8 C). For longer storage the samples should be stored frozen (preferably at -70 C, alternatively at -20 C). Mix the samples thoroughly after thawing. Avoid repeated freezing and thawing of the samples. Grossly hemolysed, lipemic or turbid specimen should be avoided. 6. KIT CONTENTS AND REAGENT PREPARATION The reagents are sufficient for 96 wells, and three separate runs. Reagents of different kit lots should not be mixed Microplate Contents: 12 x 8 strips in frame coated with double-stranded telomeric DNA. Preparation: Ready for use. Stability: Stable until expiry date Washing Buffer Concentrate (x 10) Contents: 120 ml of 10 x PBS concentrate containing Tween 20, and 0.1% Proclin 300 as preservative. Preparation: Dilute 1:10 (e.g. 100 ml ml) with distilled water. Stability: Prepare only the amount of buffer needed for the analysis. The diluted solution is stable for two weeks refrigerated (2-8 C) Diluent Buffer Contents: 100 ml of phosphate buffer containing bovine serum albumin, inert red dye extract, Tween 20, and 0.1% Proclin 300 as preservatives. Preparation: Ready for use. Stability: Stable until expiry date. 5

6 6.4. Standard Contents: One vial containing 1.5 ml of anti-telomere standard plasma. The concentration (IU/ml) of the standard is printed on the label of the vial. Contains 0.1% Proclin 300 as preservative. Preparation: Ready for use. Stability: Stable until expiry date Negative Control Contents: One vial containing 1.5 ml of anti-telomere negative control plasma with 0.1% Proclin 300 as preservative. The exact concentration (IU/ml) of the negative control is labeled on the vial. Preparation: Ready for use. Stability: Stable until expiry date Conjugate Solution Contents: 0.2 ml of HRP-conjugated monoclonal anti-human IgG in stabilizing buffer. Preparation: Dilute 1:100 (e.g. for 4 strips 40 µl µl) with the diluent buffer. Stability: Prepare the dilution before use and prepare only the amount needed Substrate Solution Contents: 15 ml of tetramethylbenzidine (TMB) in aqueous solution. Preparation: Ready for use. Stability: Stable until expiry date. Avoid direct exposure to light Stop Solution Contents: 15 ml of 0.1 mol/l sulphuric acid. Preparation: Ready for use. Stability: Stable until expiry date Incubation Covers Four plastic sheets to cover the microplate during incubation Instructions for Use 6

7 7. MATERIALS REQUIRED, BUT NOT PROVIDED Distilled or deionized water Micropipettes and disposable tips, to accurately deliver µl Pipettes to accurately deliver 1-10 ml 8-channel pipette delivering 100 µl Graduated cylinder, 1000 ml Vortex mixer for sample dilutions Test tubes for specimen dilutions Microplate washer Paper towels or absorbent paper Timer Microplate reader, 450 nm e.g. plastic blood collection tube for serum Container for ice 8. STORAGE AND STABILITY Store the Anti-Telomere IgG kit refrigerated (2-8 C). When stored at these temperatures the kit is stable until the expiration date printed on the box label and the label of each individual kit component. Do not freeze or expose the kit to high temperatures or store at above 8 C when not in use. The substrate solution is sensitive to light. The microplate or individual strips should not be removed from the foil pouch until equilibrated to room temperature (20-25 C). Unused strips must be returned to the foil pouch, sealed and stored at 2-8 C. Do not use reagents after the expiration date printed on the label. Do not use reagents from kits with different lot numbers or substitute reagents from kits of other manufacturers. Use only distilled or deionized water. The components of the kit are provided at precise concentrations. Further dilution or other alterations of the reagents may cause incorrect results. Indication of Kit Deterioration Liquid components should not be visibly cloudy or contain precipitated material. At 2-8 C, the washing buffer concentrate may, however, partially crystallize, but the crystals will dissolve by mixing at room temperature (20-25 C). The substrate solution should be colorless or pale blue. Any other color indicates deterioration of the substrate solution. 7

8 9. TEST PROCEDURE PRELIMINARY PREPARATIONS Allow all the reagents and the microplate to reach room temperature (20-25 C). Dilute the washing buffer concentrate 1 to 10 with distilled water (e.g. 100 ml ml). Read the complete assay procedure before starting. It is recommended that the control sera and samples are applied on the plate as duplicates. It is necessary to use control sera in each test run. Mix all the reagents well before use. STEP 1: SPECIMEN DILUTION Dilute the mixed serum samples 1 to 100 (10 µl µl) with the diluent buffer, mix well. STEP 2: SAMPLE Mix and pipette 100 µl of diluent buffer (Blank), negative control (NC), standard (STD) and diluted samples (S1, S2, S3 etc.) into the wells as duplicates (see Figure 1). Cover the plate with the incubation cover. Incubate for 30 minutes at room temperature (20-25 C). Note: It is recommended that the samples are dispensed into the wells within 10 minutes to avoid assay drift within the plate A Blank Blank B STD STD C NC NC D S1 S1 E S2 S2 F S3 S3 G etc. etc. H etc. etc. Figure 1. Pipetting Order. STEP 3: WASHING Wash the strips with 3 x 350 µl of the diluted (1 to 10) washing buffer and gently tap the inverted plate a few times on a clean paper towel.

9 STEP 4: CONJUGATE Pipette 100 µl of the mixed dilution (1:100) of conjugate solution into the emptyed wells with an 8-channel pipette. Cover the plate with the incubation cover. Incubate for 30 minutes at room temperature (20-25 C). STEP 5: WASHING Wash the strips with 3 x 350 µl of the diluted (1 to 10) washing buffer and gently tap the inverted plate a few times on a clean paper towel. STEP 6: SUBSTRATE Pipette 100 µl of the mixed substrate solution into the wells with an 8- channel pipette. Start the incubation time after pipetting the substrate solution into the first well and continue the incubation for 30 minutes at room temperature (20-25 C). Avoid exposure to light during incubation. STEP 7: REACTION STOP Pipette 100 µl of the mixed stop solution with an 8-channel pipette into the wells. STEP 8: MEASURING OF RESULTS Measure the absorbance at 450 nm within 30 minutes. 10. RESULTS Quality Control Values The standard and the negative control included in the kit should be run each time the kit is used. The negative control value should fall within the appropriate confidence interval established in each laboratory Calculation of the Results The standard in the test kit has been calibrated against the WHO International Standard (Wo/80). The International Units (IU/ml) has been printed on the label of controls. Calculate the mean absorbance of each pair of patient samples. Subtract the mean absorbance of the blank from the mean absorbance of the patient samples, if your reader does not allow zero adjustment with the blank wells. 9

10 Calculate the results as follows: Patient Sample (IU/ml) = Standard (IU/ml) Interpretation of the Results Negative 64 IU/ml Positive > 64 IU/ml A value at or less than 64 IU indicates a negative result, or that no telomeric dsdna IgG antibodies have been detected, or that the antibodies are below detectable level. A value of over 64 EIU indicates that telomeric dsdna IgG antibodies are detected, and the result is thus positive. For quality purposes, the negative control provided with the kit must be tested in each assay. Expected control result must be obtained in order to accept test results. The magnitude of the measured result above may reflect the severity of the disease, e.g. presence of nephritis (1, 2). The cut-off values have been determined using the Biohit Anti-Telomere IgG ELISA kit. Each laboratory should establish its own range of expected values for the clinical situation where telomeric dsdna IgG antibody values are used for diagnosis. Also the telomeric dsdna IgG results determined for a given specimen or with assays from different manufacturers may vary due to differences in assay methods and reagent specificity. Results obtained by other assay methods from other manufacturers should not be used interchangeably. 11. LIMITATIONS OF THE PROCEDURE As with any diagnostic procedure the Biohit Anti-Telomere IgG ELISA test results must be interpreted together with the patient s clinical presentation and any other information available to the physician. 12. PERFORMANCE CHARACTERISTICS Percent Agreements The Biohit Anti-Telomere IgG ELISA kit was evaluated in a study with 379 patient samples (1). In this study, the prevalence and significance of 10

11 IgG antibodies against telomeric dsdna in SLE and in other autoimmune rheumatic diseases was investigated. Patients were classified as having SLE according to the ACR criteria (3). The results are shown in Tables 1 A and 1 B. Table 1 A. Anti-telomeric dsdna IgG antibodies in SLE and in other autoimmune rheumatic diseases. Biohit Anti-Telomere IgG ELISA Positive, N Negative, N SLE (N=275) 164 (60%) 111 (40%) Rheumatoid arthritis (N=76) 4 (5%) 72 (95%) Primary Sjögren s syndrome (N=13) 4 (30%) 9 (70%) Mixed connective tissue disease (N=8) 1 (13%) 7 (87%) Scleroderma (N=4) 0 (0%) 4 (100%) Others (N=3) 0 (0%) 3 (100%) One case of polymyositis, one of primary biliary chirrosis, and one of fibrosine alveolitis, the two last both with Sjögren s syndrome. Table 1 B. Performance of the Biohit anti-telomere IgG ELISA assay in diagnosis of SLE (N=275). Clinical Sensitivity 60% Clinical Specificity 91% Positive Predictive Value 95% Negative Predictive Value 46% 13. REFERENCES 1. Salonen E-M, Ruuskanen L, Friman C. Anti-telomere antibodies in SLE. Arthr & Rheum, 1996; 39: Wallace DJ, Salonen E-M, Avaniss-Aghajani E, Morris R, Metzger AL, Pashinian N. Anti-telomere antibodies in systemic lupus erythematosus: a new ELISA test for anti-dna with potential pathogenic implications. Lupus, 2000; 9: Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum, 1982; 25:

12 14. DATE OF ISSUE Anti-Telomere IgG kit insert. Version 05, WARRANTY Biohit shall remedy those defects discovered in any Product (the Defective Product ) that result from unsuitable materials or negligent workmanship and which prevent the mechanical functioning or intended use of the Products including, but not limited to, the functions specified in the Biohit s specifications for the Products. ANY WARRANTY WILL, HOWEVER, BE DEEMED AS VOID IF FAULT IS FOUND TO HAVE BEEN CAUSED BY MALTREATMENT, MISUSE, ACCIDENTIAL DAMAGE, INCORRECT STORAGE OR USE OF THE PRODUCTS FOR OPERATIONS OUT- SIDE THEIR SPECIFIED LIMITATIONS OR OUTSIDE THEIR SPECI- FICATIONS, CONTRARY TO THE INSTRUCTIONS GIVEN IN THE IN- STRUCTION MANUAL. The period of this warranty for the Distributor is defined in the instruction manual of the Products and will commence from the date the relevant Product is shipped by Biohit. In case of interpretation disputes the English text applies. All Biohit diagnostic kits have been manufactured according to ISO 9001 / ISO quality management protocols and have passed all relevant Quality Assurance procedures related to these products. 16. ORDERING INFORMATION Anti-Telomere IgG ELISA. Cat. No Headquarters BIOHIT OYJ Laippatie Helsinki, Finland Tel: Fax: info@biohit.fi 12

13 17. EXPLANATION OF THE SYMBOLS USED IN LABELS For in vitro diagnostic use Catalogue number Batch code Use by Consult instructions for use C Temperature limitation. Store at +2-8 C 96 determinations Do not re-use 13

14 14 Notes:

15 Notes: 15

16 18. SHORT OUTLINE OF THE PROCEDURE Allow all the reagents to reach room temperature (20-25 C) Remember to mix all the reagents and samples well just before pipetting After mixing pipette 100 µl of blank solution, the negative control, the standard and diluted (1 to 100) patient samples into the wells Incubate for 30 min at room temperature (20-25 C) Wash 3 times with 350 µl of the diluted washing buffer Pipette 100 µl of the diluted (1 to 100 dilution) and mixed conjugate solution into the wells Incubate for 30 min at room temperature (20-25 C) Wash 3 times with 350 µl of the diluted washing buffer Pipette 100 µl of the mixed substrate solution into the wells Incubate for 30 min at room temperature (20-25 C) 16 Pipette 100 µl of the mixed stop solution into the wells Read at 450 nm within 30 minutes