ANTI-dsDNA, Page 1
MICROWELL ELISA
ANTI-dsDNA TEST
Catalog No. 2553
(96 tests)
SUMMARY OF ASSAY PROCEDURE
1. Sample dilution 1:40
5 l / 200 l
2. Three incubations at room temperature
3. Stop with 50 l of acid. Read O.D. at 450 nm
NAME AND INTENDED USE
The Atlas Link Anti-dsDNA Test is intended for the quantitative determination of IgG antibodies to double stranded DNA (dsDNA, native DNA) in human sera.
SUMMARY AND EXPLANATION OF THE TEST
Anti-dsDNA is present in 50 % to 70% of patients with systemic lupus erythematosus (SLE). Circulating DNA/anti-DNA immune complexes are considered to play a part in the pathogenesis of SLE. The presence of anti-dsDNA is one of the diagnostic criteria for SLE. IgG antibodies to dsDNA are considered clinically most useful for the diagnosis and management of SLE. Antibodies to single stranded DNA (ssDNA) and IgM antibodies to DNA are found in a number of other connective diseases, liver diseases, as well as in some normal individuals. Accurate detection of Anti-dsDNA is important in the diagnosis and management of SLE.
PRINCIPLE OF THE TEST
The Atlas Link Anti-dsDNA Test is a microwell sandwich ELISA. The wells are coated with dsDNA. The sample controls, and calibrator are incubated in the wells first. After incubation, the antibodies to dsDNA, if present, will bind to coated antigens. The enzyme conjugate, goat anti-human antibody, conjugated with horseradish peroxidase, is then added to form a sandwich complex on the well. Unbound enzyme conjugate is washed off by washing buffer. Upon addition of the substrate and chromogen, the intensity of color developed is proportional to the concentration of anti-dsDNA in the samples.
MATERIALS PROVIDED
1.Microwell Strips: dsDNA coated wells (12 x 8 wells)
2.Sample Diluent: 1 bottle (22 ml)
3.Washing Concentrate: 1 bottle (100 ml, 10x)
4.Solution A: Substrate; buffer solution with H2O2. 1 vial (12 ml)
5.Solution B: Chromogen, Tetramethylbenzidine. 1 vial (12 ml)
6.Solution C: Enzyme Conjugate. 1 vial (12 ml)
7.Calibrator: Yellow Cap. (150 l/vial)
8.Negative Control: Natural Cap. (150 l/vial)
9.Positive Control: Red Cap. (150 l/vial)
10.Stop Solution: 2 N HCl (10 ml)
11.Well Holder: 1 holder for securing strips
STORAGE AND STABILITY
1.Store the kit at 2 - 8 oC.
2.Keep microwells sealed in a dry bag with desiccants.
3.The reagents are stable until expiration of the kit.
4.Do not expose test reagents to heat, sun or strong light during storage or usage.
WARNINGS AND PRECAUTIONS
1.Potential biohazardous materials:
The calibrator and controls contain human source components which have been tested and found nonreactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control / National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories." 1984
2.This test kit is designed for in vitro diagnostic use only.
3.Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
4.The components in this kit are intended for use as a integral unit. The components of different lots should not be mixed.
5.This product contains components preserved with sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.
SPECIMEN COLLECTION AND HANDLING
1.Collect blood specimens and separate the serum.
2.Specimens may be refrigerated at 2 - 8 oC for up to seven days or frozen for up to six months. Avoid repetitive freezing and thawing of serum sample.
PREPARATION FOR ASSAY
1.Bring all specimens and kit reagents to room temperature (20-25 oC) and gently mix.
2.Prepare washing buffer by adding distilled or deionized water to 10x wash concentrate to a final volume of 1 liter.
ASSAY PROCEDURE
1.Remove the desired number of wells from the sealed pouch and secure them in the holder.
2.Prepare 1:40 dilution of test samples, negative control, positive control and calibrator by adding 5 l of the sample to 200 l of sample diluent. Mix well.
3.Dispense 100 l of diluted sera, calibrator, and controls into the appropriate wells. For the reagent blank, dispense 100 l sample diluent in 1A well position. Incubate for 30 minutes at room temperature.
4.Remove liquid from all wells and repeat washing five times with washing buffer.
5.Dispense 100 l of enzyme conjugate to each well and incubate for 30 minutes at room temperature.
6.Remove enzyme conjugate from all wells. Repeat washing five times with washing buffer.
7.Dispense 100 l of solution A and 100 l of solution B and incubate for 30 minutes at room temperature.
8.Add 50 l of 2 N HCl to stop reaction.
Make sure there are no air bubbles in each well before reading
9.Read O.D. at 450 nm with a microwell reader.
CALCULATION OF RESULTS
1.Calculate the mean of duplicate calibrator value xc.
2.Calculate the mean of duplicate positive control and patient samples.
3.Calculate the dsDNA IU/ml of each determination by dividing the mean values of each sample by calibrator mean value, xc, and multiplying by the calibrator IU/ml.
Example of typical results:
Calibrator dsDNA IU/ml = 100
Calibrator O.D. = 0.895, 0.910xc= 0.903
Positive control O.D. = 0.738, 0.809xp=0.774
dsDNA IU/ml = (0.774/0.903) x 100 = 86 IU/ml
Patient sample O.D. = 1.245, 1.295xs=1.270
dsDNA IU/ml = (1.270/0.903) x 100 = 140 IU/ml
QUALITY CONTROL
The test run may be considered valid provided the following criteria are met:
1.The O.D. value of the reagent blank against air from a microwell reader should be less than 0.250.
2.If the O.D. of the Calibrator is lower than 0.250, the test is not valid and must be repeated.
3.The dsDNA IU/ml for Positive Control should be in the range stated on the labels.
INTERPRETATION
The following is intended as a guide to interpretation of Atlas Link Anti-dsDNA Test results; each laboratory is encouraged to establish its own criteria for test 03interpretation based on sample populations encountered.
dsDNA IU/mlInterpretation
< 40.0dsDNA negative; no further dsDNA testing is required
40.0 - 50.0Borderline dsDNA positive
50.0 - 60.0Low Positive
> 60.0dsDNA positive
PERFORMANCE CHARACTERISTICS
154 patient samples were tested by independent investigators on both the IFA dsDNA test system and the ELISA Anti-dsDNA test system. The results for the ELISA Anti-dsDNA compared to IFA dsDNA are as follows:
true positives100%Therefore:
false positives4.9%Specificity = 95.1%
true negatives95.1%Sensitivity = 100 %
false negatives0%
Clinical diagnoses of 52 true positives were as follows:
11 SLE
9 Mixed Connective Tissue Disease
6 Rheumatoid Arthritis
4 Scleroderma
3 Sjogren's Syndrome
3 Other
16 pooled positive specimens
LIMITATIONS OF THE PROCEDURE
For diagnostic purpose, the dsDNA values should be used as an adjunct to other data available to the physician. Certain drugs such as p-aminosalicylic acid, phenytoin, isoniazio, hydralazine, procainamide, etc. may induce auto-antibody formation. Positive results may also result in apparently healthy people due to a host of other factors. A positive test suggests certain diseases, but is not diagnostic and should be confirmed by clinical findings.
REFERENCES
1.Reichlin, M. Current perspectives on serological reactions in SLE patients, Clin. Exp. Immunol. 44:1-10, 1981.
2.Venables, P.J.W., Erhardt, C.C. and R.N. Maini. Antibodies to extractable nuclear antigens in rheumatoid arthritis: relationship to vasculitis and circulating immune complexes. Clin. Exp. Immunol. 39:16, 1980.
3.Harmon, C.E. Anti-nuclear antibodies in autoimmune diseases; significance and pathogenecity. Medical Clinics of North America 69:547, 1985.
4.Rothschild, B.M., Jones, J.V., Chesney, C., Pifer, D., Thompson, L.D., James, K.K. and H. Badger. Relationship of clinical findings in Systemic Lupus Erythematosus to sero-reactivity. Arthritis and Rheumatism 26: 45-51, 1983.
Atlas Link
12720 Dogwood Hills Lane
Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664
12/21/97
Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA
Phone: (703) 266-5667, FAX: (703) 266-5664