Product Description

MICROWELL ELISA, Anti-ds DNA ELISA Test
Catalog No. (*6 Tests)

NAME AND INTENDED USE
The anti-dsDNA test is a quantitative enzyme immunoassay
(EIA) intended to screen for the presence of dsDNA
antibodies in human serum as an aid in the diagnosis of
systemic lupus erythematosus (SLE).
WARNINGS AND PRECAUTIONS
PRINCIPLE OF THE TEST
Purified dsDNA is bound to microwells. The DNA retains its
antigenicity and remains double stranded. Antibodies to
dsDNA, if present in diluted serum, bind in the microwells.
Washing of the microwells removes unbound serum
antibodies. Horseradish peroxidase (HRP) conjugated antihuman
IgG immunologically binds to the bound patient
antibodies forming a "conjugate - anti-dsDNA - dsDNA"
sandwich. Washing of the microwells removes unbound
conjugate. An enzyme substrate in the presence of bound
conjugate hydrolyzes to form a blue color. The addition of an
acid stops the reaction forming a yellow end product. The
intensity of the color is measured photometrically at **0nm.
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 dsDNA are found
in a number of other connective diseases, liver diseases, as
well as in some normal individuals. Accurate detection of antidsDNA
is important in the diagnosis and management of SLE.
EIA tests for anti-dsDNA have demonstrated greater sensitivity
than standard IFA and RIA tests allowing for improved detection
of low titer antibodies to dsDNA.
1. For in vitro diagnostic use only.
2. Do not pipette by mouth.
3. Avoid contact with open skin.
4. Components containing human serum have tested negative
for HBsAg and HIV antigens. This does not assure the
absence of these antigens; sera should be
considered potentially hazardous. These materials should be
handled as recommended in the CDC/NIH Health manual,
Biosafety in Microbiological and Biomedical Laboratories,
***4.
5. Sodium azide is a toxic substance and is used in some
reagents. In case of contact with eyes and skin, flush
immediately with copious amounts of water. Sodium azide
may react with lead and copper plumbing to form explosive
metal azides. On disposal of reagents flush with a large
volume of water to help prevent azide buildup.
6. The Stop solution contains a dilute acid solution. Use with
care to avoid contact
with skin and eyes. Avoid exposure to bases, metals, or other
compounds which
may react with acids. Spills should be cleaned up immediately.
STEPS
1
2
3
4
5
6
Sample Dilution
Diluted Sample and
prediluted Controls
Washing
Enzyme conjugate
Washing
TMB Color Reagent
7 Stop solution
8 Reading OD **0 nm
**0 μl
**0 μlx3minutes
**0 μl
**0 μl
**0 μl
*0minutes
*0minutes
*5minutes
SUMMARY OF ASSAY PROCEDURE
VOLUME INCUBATION TIME
1: **0(*0 μl/ 1ml)
**0 μlx3minutes
****5 oC RT
SUMMARY AND EXPLANATION OF THE TEST
Antinuclear antibodies (ANAs) directed against a variety of
macromolecules occur in extraordinarily high frequency in
systemic rheumatic diseases. Many rheumatic diseases
are characterized by the presence of one or more of these
Antinuclear antibodies.Therefore, the identification of the
specific antibody is useful in the detection and diagnosis of the
disease.
Anti-dsDNA is present in *0% to *0% of patients with 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
SPECIMEN COLLECTION
Collect blood aseptically in untreated tubes. Allow blood to clot,
separate serum immediately. Avoid use of lipemic, hemolyzed
or contaminated sera. Store sera at **8°C. Freeze
MATERIALS REQUIRED
1. EIA Reader (set to **0 nm)
2. Micropipettors (*0μl & **0μl)
3. *-channel Repeating Pipettor (for washing)
4. Deionized (DI) Water
5. Pipettes (1ml & *0ml)
6. 1 Liter Container (for Wash Solution)
7. Test Tubes (4ml & *5ml)
8. Countdown timer
RECOMMENDED MATERIALS
1. Automatic Washer
2. **0 ul *-channel Micropipettor (for reagent delivery)
3. 1ml Mini-Tubes (for sample dilutions)
MATERIALS SUPPLIED
1. dsDNA Microtiter plate: *6 antigen-coated wells, sealed in a
resealable foil pouch with desiccant. Ready to use.
2. Wash Concentrate (*0X): *0 ml. Add to **0 ml DI water.
Save for future use.
3. Sample Diluent: **0 ml. Ready to use. Use to dilute patient
samples 1:**0.Use for blanking control. Avoid unnecessary
contamination. Contains sodiumazide as a preservative.
4. Conjugate Reagent: *2 ml. Ready to Use.
5. Anti-dsDNA Positive Control: 1.0 ml. Ready to Use.
6. Anti-dsDNA Calibrator: 1.0 ml. Ready to Use.
7. Anti-dsDNA Negative Control: 1.0 ml. Ready to Use.
8. TMB Substrate: *2 ml. Ready to use.
9. Stop Solution: *2 ml. Contains sulfuric and hydrochloric
acid.
STABILITY AND STORAGE OF REAGENTS
The kit is stabilized for ambient shipment. All kit components
should be stored at **8ºC and can be used until the expiration
date printed on the labels. Once the antigen-coated well foil
pouch has been opened, the wells are stable for *0 days.
sera at **0°C if not tested within *4 hours; avoid repeated
freezing.
CAUTION: Serum samples should NOT be heat-inactivated as
this may cause false positive results.
PROCEDURE NOTES
1. All materials must be at room temperature (*0~*5oC).
2. Do not use calibrators from different kit lots. Do not use
expired reagents.
3. Avoid contamination of reagents, dispensing pipettes, and