PerkinElmer Life Sciences, Inc.

Transcription

1 PerkinElmer Life Sciences, Inc. Anti-dsDNA [125 I ] Radiobinding Assay Kit Catalog Number NEA103 For In-Vitro Diagnostic Use

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3 I. PROPRIETARY NAME Anti-dsDNA [ 125 I] Radiobinding Assay Kit PerkinElmer Life Sciences, Inc. Catalog Number: NEA103, 100 Tubes II. INTENDED USE This kit is designed to quantitatively determine anti-dsdna binding activity in human serum. III. EXPLANATION OF THE TEST The detection of anti-dsdna (anti-double stranded deoxyribonucleic acid) antibodies in the serum of patients is useful for the diagnosis of systemic lupus erythematosus (SLE) (1,2). Due to its close clinical resemblance to several other connective tissue diseases such as rheumatoid arthritis, scleroderma and chronic active hepatitis, SLE is difficult to diagnose (3). Anti-dsDNA antibody levels are usually significantly elevated during active SLE periods and lower during inactive periods. During treatment of SLE, anti-dsdna antibody levels have been observed to correlate with therapy and clinical status of the disease (1,2). Several laboratory tests have been used to help diagnose SLE. These tests include the LE cell test and anti-nuclear antibody (ANA) test. However, these tests have not been found to have the combination of specificity and sensitivity possessed by anti-dsdna assays (4,5). The 1982 criteria for the diagnosis of SLE established by the American Rheumatism Association listed the assay of anti-dsdna antibodies as one of the key immunological assays for diagnostic tests (6). Inclusion of this test significantly improved the diagnostic strength of the new criteria over the previous criteria. While anti-dsdna antibodies can be detected by a variety of methods including gel diffusion, agglutination and immunofluorescent techniques, only the radiobinding assay allows for both sensitive and quantitative measurement (4,7). Wold published the first radioassay for detecting anti-dsdna antibodies (8). Ammonium sulfate was utilized to separate protein bound DNA from free DNA. Other radioassays have employed filters to achieve separation (9). Since anti-ssdna (anti-single stranded deoxyribonucleic acid) and anti-protein antibodies are found in several other related connective tissue diseases, the importance of using DNA which is double stranded and free from contaminating proteins has been stressed by several investigators (10,11). The Perkin Elmer Life Sciences anti-dsdna assay utilizes DNA that has been labeled with [ 125 I] to a high specific activity. The labeling method used produces radiolabeled DNA which is double stranded as indicated by routine testing. The PerkinElmer Life Sciences antidsdna radiobinding assay is an improved modification of Wold's assay. A precipitating reagent is used for separation of free from bound labeled DNA. A set of standards is provided for preparation of a quantitative standard curve and the assay is calibrated to the First International Standard for Anti-Double-Stranded DNA, Wo-80 (12). Optimized reagents provide a reproducible assay with minimal binding to normal human serum components. 1

4 IV. PRINCIPLE OF THE METHOD The basic principle of this assay is the binding of [ 125 I] labeled dsdna to anti-dsdna antibodies (other serum proteins may participate in binding). This interaction is illustrated below. Figure 1 Labeled 125 I-dsDNA + Anti-dsDNA Antibody (in serum or standards) 125 I-dsDNA = Anti-dsDNA Antibody Complex The assay is performed by incubating a dilution of patient serum with radiolabeled dsdna. Antibody-dsDNA complexes are precipitated and collected by centrifugation. The amount of radioactivity in the pellet is determined by gamma counting. The quantity of radioactivity in the pellet is proportional to the amount of anti-dsdna activity present in the sample. A standard curve for quantitative measurement of anti-dsdna is prepared by determining the anti-dsdna binding activity of a set of standards. These standards are calibrated to the International Standard, Wo-80. Binding levels in IU/mL are obtained by interpolating unknowns from the standard curve. V. REAGENTS All necessary reagents are supplied and are intended for IN-VITRO DIAGNOSTIC USE. Kits must be stored at refrigerator temperature (2-8 C) upon receipt. The reagents are stable for the times indicated if the specific precautions given below are followed. Additional information concerning the composition of each reagent is stated on the container labels. Read each label carefully. Sodium azide has been added as an antibacterial agent where appropriate. NOTE: The National Institute for Occupational Safety and Health has issued a bulletin citing the potentially explosive hazard due to the reaction of sodium azide with copper, lead, brass, or solder in plumbing systems. Although sodium azide is added at a minimal concentration, it is still recommended that copious amounts of water be used to flush the drain pipeline after disposal of these reagents in the plumbing system. Copper-free and lead-free discharge lines should be used whenever possible. Decontamination procedures should be followed prior to maintenance on drain lines which have been used for azide-containing reagents. Recommended decontamination procedures are available from Customer Technical Support. NOTE: An excess of each reagent beyond the labeled content is added to each container in order to allow the withdrawal of the number of aliquots required to fill the stated quantity of tubes. Any reagent remaining after the stated quantity of tubes have been prepared should be discarded. Standards and controls contain human plasma. This plasma has been found to be nonreactive for HBsAg and HIV when tested with licensed reagents. However, no known test method can offer assurance that products derived from human blood will not transmit hepatitis. 2

5 A. Anti-dsDNA Assay Buffer One bottle (40 ml/bottle) containing buffer are supplied. The solution contains human protein, a stabilizer, and 0.01% sodium azide in an aqueous buffer. Refer to the vial for exact expiration date of the reagent. Storage: 2-8 C. B. Anti-dsDNA Standards Six vials containing 0.2 ml per vial of anti-dsdna positive human plasma in a protein matrix containing phosphate buffered saline and 0.1% sodium azide are supplied. Refer to the vial for the exact concentration of each standard level to use for construction of the standard curve and for expiration date of the reagent. Storage: 2-8 C if used within four weeks of receipt. If the kit will be stored and used longer than four weeks, the recommended storage is -20 C. C. [ 125 I]-dsDNA Tracer One vial (2 ml) containing concentrated labeled DNA is supplied. This vial contains less than 370 KBq (10 µci) of radioactivity on the calibration date. The labeled DNA is in an aqueous buffer to which 0.01% sodium azide has been added to prevent bacterial growth. The tracer must be diluted 10 fold in Assay Buffer before use. (See Radiobinding Assay Procedure.) Storage: 2-8 C. Refer to the vial for exact expiration date of the reagent. This material is radioactive and the user should follow the precautions listed below. 3

6 INSTRUCTIONS RELATING TO THE HANDLING, USE, STORAGE, AND DISPOSAL OF THIS RADIOACTIVE MATERIAL This radioactive material may be received, acquired, possessed, and used only by physicians, veterinarians in the practice of veterinary medicine, clinical laboratories or hospitals and only for invitro clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals. Its receipt, acquisition, possession, use, and transfer are subject to the regulations and a general license of the U. S. Nuclear Regulatory Commission or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority. 1. All radioactive materials must be stored in specifically designated posted areas. Records of receipt and survey must be maintained. 2. All work with these materials must be carried out only in authorized areas. 3. Prohibit mouth pipetting of radioactive materials. 4. There must be no smoking or eating within the work area. 5. Hands must be washed after handling radioactive materials. 6. Any spilled material must be wiped up quickly and thoroughly and the contaminated substances transferred to a suitable receptacle. The surfaces involved must be washed thoroughly with an appropriate decontaminant. Monitor to ensure the area has been effectively decontaminated. 7. When use of the Tracer reagent has been completed, empty and decontaminate the vials. This radioactive material can be discarded into the sanitary sewerage system using copious amounts of water to ensure a minimal discharge concentration. 8. Prior to disposal of the empty, uncontaminated Kit and Tracer containers to unrestricted areas, remove or deface the radioactive material labels or otherwise clearly indicate that the containers no longer contain radioactive material. D. Anti-dsDNA Positive Control One vial (0.2 ml) containing anti-dsdna positive human plasma in a protein matrix containing phosphate buffered saline and 0.1% sodium azide is supplied ready-foruse. The binding activity of the control is listed on the component label. Refer to the vial for the exact expiration date on the reagent. Storage: 2-8 C if used within four weeks of receipt. If the kit will be stored and used longer than four weeks, the recommended storage is -20 C. E. Anti-dsDNA Negative Control One vial (0.2 ml) containing anti-dsdna negative serum in a protein matrix containing phosphate buffered saline and 0.1% sodium azide is supplied ready-for-use. Refer to the vial for exact expiration date of the reagent. Storage: 2-8 C if used within four 4

7 weeks of receipt. If the kit will be stored and used longer than four weeks, the recommended storage is -20 C. F. Anti-dsDNA Precipitator One bottle (100 ml) containing an aqueous salt solution is supplied ready-for-use. Storage: 2-8 C. Refer to the bottle for exact expiration date of the reagent. VI. SAMPLE COLLECTION Serum is recommended and the usual precautions for venipuncture suffice. Separate the serum from the clot and store the serum at 4 C if it will be assayed the same day as collected. Sera should be stored at -20 C to -70 C for later assay. Equilibrate samples to room temperature and vortex gently before assay. VII. PROCEDURE A. Reagents Supplied - sufficient for 100 tubes 1. 1 bottle Anti-dsDNA Assay Buffer, (40 ml/bottle) 2. 1 vial [ 125 I]-dsDNA Tracer, concentrate, (2 ml/vial) 3. 6 vials Anti-dsDNA Standards (0.2 ml/vial) - Refer to the vial for the exact concentration of each standard level to use for construction of the standard curve vial Anti-dsDNA Positive Control, (0.2 ml/vial) 5. 1 vial Anti-dsDNA Negative Control, (0.2 ml/vial) 6. 1 bottle Anti-dsDNA Precipitator, (100 ml/bottle) B. Equipment and Reagents Required In addition to the reagents supplied with the kit, the following materials are required: 1. Pipettors/or pipets that can accurately and precisely deliver the required volumes. 2. Gamma scintillation counter 3. Laboratory vortex mixer 4. Test tube rack 5. Centrifuge (swinging bucket head) 6. Test tubes - 12 x 75 mm polystyrene 5

8 7. Absorbent paper for blotting 8. Radioactive waste container C water bath C. Anti-dsDNA Radiobinding Protocol NOTE: Equilibrate all reagents to room temperature and mix before use. The reagents should always be added at room temperature in the order specified. All Standards, Controls, and clinical samples should be run in duplicate. 1. In a test tube rack set up and label 18 tubes for the Standards, Controls, and Total Counts and 2 additional tubes for each clinical sample to be tested. (See Table 1.) 2. Prepare the working tracer by adding one volume of tracer to nine volumes of Assay Buffer. Calculate how much working tracer will be needed for an assay and only prepare the theoretical amount needed. 3. Pipet 10 µl of standard into the bottom of each of the standard tubes. 4. Pipet 10 µl of Negative and Positive Control into the bottom of each control tube. 5. Pipet 10 µl of sample into the bottom of each sample tube. 6. Pipet 200 µl of working tracer into each tube (Standards, Controls, Samples and Total Counts Tubes). Set the Total Counts Tubes aside to be counted at the end of the assay. 7. Vortex the remaining test tubes for 2-5 seconds. 8. Place the tubes into a 37 C water bath and incubate for 2 hours. 9. Remove the tubes from the water bath and add 1 ml of Anti-dsDNA Precipitator into each tube. 10. Vortex each tube gently for 2-5 seconds. 11. Centrifuge the tubes at room temperature or 4 C for 20 minutes at a minimum of 1,200 x g. 12. Remove the supernatant by decanting or aspirating. Decant by gently inverting all tubes once in a smooth motion, preferably at the same time, discarding the supernatants into a radioactive waste container. Keeping the tubes inverted, place them on absorbent paper for blotting. To facilitate removal of remaining droplets, press the rims of the tubes into the paper. Allow tubes to drain about 1-2 minutes, If preferred, the supernatants may be aspirated, taking care not to disturb the antibody complex at the bottom of the tubes. 6

9 13. Wipe the outside of each tube and count in a gamma counter. At the usual efficiencies of 50-70%, a counting time of 1 minute per tube is generally adequate. TABLE 1 - RADIOBINDING ASSAY PROTOCOL (All volumes in microliters) Tube Standards Samples Tracer Precipitator Total Count 1, Mix. - Standards Incubate 1000 Controls for 2 hr Sample 19, at 37 C Mix. Centrifuge at RT. or 4 C for 20 minutes at a minimum of 1200 x g. Decant or aspirate and blot tubes. Count pellets D. Precautions - Limitations 1. Incubation conditions should be standardized for proper day-to-day internal quality control. 2. As with all radiobinding assay procedures, pipetting is crucial. It is essential that pipetting be accurate and reproducible. A positive displacement pipet is recommended for the 10 µl standard and sample additions. 3. Samples with concentrations above the range of the standard curve should be diluted with the Assay Buffer and reassayed. The values obtained are then multiplied by the appropriate dilution factor. 4. The use of grossly hemolyzed or lipemic samples should be avoided. 5. The presence of exogenous radioactivity in clinical samples may lead to erroneous results. 6. Avoid using a centrifuge which may overheat due to prolonged use. 7. The reagents in this kit should be used as a unit. Do not mix different lots of any component within a given assay. 8. This product has not been tested for use with any method other than that stated in this Instruction Manual. 9. It is possible that a serum specimen will not have the same avidity as the material used for the preparation of the Standards (7). Differences in avidity between the Standards and the sample will result in non-linear dose-response curves. Thus a two-fold dilution of the sample will not produce one-half of the initial value. However, the binding activity of such a specimen is still a useful and reproducible value. 7

10 10. Always express results in IU/mL in order to assure reproducibility. As the AntidsDNA Radiobinding Assay Kit ages, there may be some movement of the standard curve as the result of reduced %B values for each standard. However, values of control and patient samples will not change when results are expressed in IU/mL. 11. CAUTION: HUMAN SOURCE MATERIAL. HANDLE AS POTENTIALLY INFECTIOUS. Each donor unit of human sera or plasma used in the preparation of this product was tested by FDA approved methods for the presence of the antibody to HIV as well as for hepatitis B surface antigen and found to be negative (not repeatedly reactive). Because no test method can offer complete assurance that human immunodeficiency virus (HIV), hepatitis B virus or other infectious agents are absent, this material should be handled using good laboratory practice to avoid skin contact and ingestion. VIII. PROCEDURE FOR CALCULATING UNKNOWNS After counting has been completed, the anti-dsdna activity of the serum can be determined by interpolation from a standard curve. The following method is given. A. If all tubes have been counted for the same period of time, use the total accumulated counts; otherwise, correct all counts to a common count rate. B. Average the counts for each set of duplicates. C. Subtract the averaged 0 standard counts from all averaged total, standard, control, and sample counts to obtain the average net counts. D. Calculate the % Bound for each Standard, Control, and Sample by dividing the average net counts by the average net total counts and multiply by 100. % B = Average Net Counts of Standard, Control or Sample x 100 Average Net Total Counts E. Using semi-log graph paper plot % Bound for each Standard against the corresponding anti-dsdna activity in IU/mL. F. Determine the anti-dsdna activity in the control and patient samples by interpolation from the standard curve. Since identical volumes are used for standards and samples and the standard curve is expressed as IU/mL of anti-dsdna activity, samples can be read directly as IU/mL. See Table 2 and Figure 2 for typical standard curve and calculations. NOTE: Alternative methods can be used to plot data: 8

11 1. Using semi-log paper, plot the average Net Counts for each standard, instead of %B, against the corresponding anti-dsdna activity. This method reduces the number of calculations required. 2. Using linear paper, plot %B or Net Counts vs. binding activity. 3. Using log-logit paper plot %B vs. binding activity. TABLE 2 - SAMPLE CALCULATIONS Average Counts Bound Net Counts Bound Tube Number Counts Bound % Bound Total Counts 1 35,583 35,891 32, ,198 Standard A (0 IU/mL) 3 2,822 2, ,068 Standard B (1.4 IU/ml) 5 3,550 3, ,548 Standard C (3.8 IU/mL) 7 5,421 5,384 2, ,347 Standard D (13.8 IU/mL) 9 11,588 11,719 8, ,850 Standard E (45 IU/mL) 11 24,999 24,854 21, ,708 Standard F (98 IU/mL) 13 31,416 31,276 28, ,135 IU/mL: From Curve Negative Control Serum 15 2,955 2, ,987 Positive Control Serum 17 21,583 21,534 18, ,484 blue = counts in fluid phase Exact concentrations of standards B - F may change with component lot. Refer to the vial for the exact concentration of each standard level to use for construction of the standard curve. 9

12 Figure Two Typical Standard Curve (DO NOT USE TO CALCULATE SAMPLES) %B Anti-dsDNA, IU/mL IX. PERFORMANCE CHARACTERISTICS Precision Study Precision was determined by multiple duplicate analyses of various serum pools and controls. Typical results are given in IU/mL (Table 3). TABLE 3 Within Assay Variation Between Assay Variation Coeff. of Coeff. of Sample n Mean + 1 S.D. Var. (%) Sample n Mean + 1 S.D. Var. (%) A A B B C C D D E E F F G X. EXPECTED VALUES The PerkinElmer Anti-dsDNA [ 125 I] Radiobinding Assay Kit was used to determine the antidsdna binding activity for a total of 210 serum samples from clinically normal individuals, 10

13 patients with active and inactive systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis and scleroderma. Table 4 summarizes these results. Results from additional clinical studies can be obtained by contacting Customer Technical Support. In a group of normal individuals 97% had anti-dsdna binding activities less than 3.6 IU/mL. Results higher than 3.6 IU/mL were observed in a small fraction of normal individuals and in several patients with osteoarthritis and scleroderma. Elevated levels of antibodies to dsdna were found in all serum samples of SLE patients in this study, both clinically active and inactive. It is important to note, however, that in a small number of SLE patients anti-dsdna antibodies may not be detected. In this study it was found that anti-dsdna binding activites greater than 3.6 IU/mL are highly indicative of SLE. This cutoff level is provided as a guide and is derived from the mean of results from the normal samples plus two standard deviations. We strongly recommend that each laboratory determine its own level of diagnostic significance by performing an analysis of clinical specimens from its own population. Results from a single serum specimen should not be used as the sole criterion of diagnosis. More importantly, trends or changes in anti-dsdna levels are predictive of SLE disease activity. TABLE 4 Expected Values (IU/mL) Patient Group n Number of Values < 3.6 Range Normal Adults ND Active SLE Inactive SLE > Rheumatoid Arthritis Osteoarthritis Scleroderma ND ND ND 6.3 ND - Not Detectible XI. REFERENCES 1. Pincus, T., Schur, P.H., Rose, J.A., Decker, J.L., and Talal, N., New Eng. J. Med. 281 (13), (1969). 2. Hughes, G.R.V., Cohen, S.A., and Christian, C.L., Amm. Rheum. Dis. 30, (1971). 3. Reichlin M., in "The Clinical Management of Systemic Lupus Erythematosus" Grune & Stratton, Inc., New York, Maehara, K.T., Am. J. Med. Tech. 49 (7), (1983). 11

14 5. Webb, J., Edmonds, J.P., Hughes, G.R.V., Tan, E.M., Wells, J.V., Whittingham, S., and Zilko, P., Eval. Report Aus. Rheum. Assoc. J. Path. 13, (1981). 6. Steinman, C.R., Tan, E.M., Cohen, A.S., Fries, J.F., Masi, A.T., McShane, D.J., Rothfield, N.F., Schaler, J.G., Talal, N., and Winchester, R.J., Arth. & Rheum. 25 (11), (1982). 7. Holian, J., Griffiths, I.D., Glass, D.N., Maini, R., and Scott, J.T., Ann. Rheum. Dis. 34, (1975). 8. Wold, R.T., Young, F.E., Tan, E.M., and Farr, R.S., Science, 161, (1968). 9. Ginsberg, B. and Keiser, H., Arth. & Rheum. 16 (2), (1973). 10. Locker, J.D., Medof, M.E., Bennett, R.M., and Sukhupunyaraksa, S., J. Immunol. 118 (2), (1977). 11. Nakamura, R.M. and Tucker, E.S., Clin. Immuno. Newsletter 2 (16), Feltkamp, T.E.W., Kirkwood, T.B.L., Maini, R.N., and Aarden, L.A., Ann. Rheum. Dis., 47, (1988). 13. Sharp, G.C., in Advances in Systemic Lupus Erythematosus, Hayslett, J.P. and Hardin, J.A., Eds., Grune & Stratton, New York, XII. MANUFACTURER PerkinElmer Life Sciences, Inc. 549 Albany Street Boston, MA, USA Toll free: International: XIII. DATE OF ISSUANCE OF THIS LABELING November, 2001 XIV. ORDERING INFORMATION In-vitro laboratory testing with the PerkinElmer Anti-dsDNA [ 125 I] Radiobinding Assay Kit requires either a general license or a specific license from the U. S. Nuclear Regulatory Commission or from a State with which the NRC has entered into an agreement for the exercise of regulatory authority. A general license is issued pursuant to Title 10 CFR Part 31, Section 31.11, USNRC regulations to any physician, veterinarian in the practice of veterinary medicine, clinical laboratory or hospital which has filed and receives from the Commission a validated copy of Form NRC-483 with a registration number assigned, or has a license that authorizes the 12

15 medical use of byproduct material that was issued under Title 10 CFR Part 35 of the USNRC or equivalent Agreement State regulations. In accordance with the regulatory requirements, the general licensee shall not possess at any one time, at any one location of storage or use, a total amount of Iodine-125, Iodine-131, Selenium-75 and/or Iron-59 in excess of 200 microcuries. NEA103 Anti-dsDNA [ 125 I] Radiobinding Assay Kit, 100 Tubes - less than 370 KBq (10 µci) per kit. Test Materials NEA103T 1 vial [ 125 I]-DNA, 2 ml

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