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Cat.No E0764Ra
Standard Curve Range: 5ng/L-1000ng/L
Sensitivity: 2.51ng/L
Size: 96 wells
Storage: Store the reagents at 2-8°C. For long term storage refer to the
expiration date keep it at -20°C. Avoid repeated thaw cycles.
*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to
read this instruction entirely before the use.
PRECICION
INTENDED USE
This sandwich kit is for the accurate quantification of Rat Tumor Necrosis Factor
Alpha(also known as TNF alpha )in serum, plasma, cell culture supernates, cell
lysates, tissue homogenates.
ASSAY PRINCIPLE
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After incubation unbound biotin-conjugated anti-Rat TNF alpha antibody is
washed away during a washing step. Streptavidin-HRP is added and binds to the
biotin-conjugated anti-Rat TNF alpha antibody. After incubation unbound
Streptavidin-HRP is washed away during a washing step. Substrate solution is
then added and color develops in proportion to the amount of Rat TNF alpha. The
reaction is terminated by addition of acidic stop solution and absorbance is
measured at 450 nm.
REAGENT PROVIDED
Components Quantity
MATERIALS REQUIRED BUT NOT SUPPLIED
Standard Solution (1280ng/L) 0.5ml
Pre-coated ELISA Plate 12 * 8 well strips 1.
Standard Diluent 3ml
3
Streptavidin-HRP 6ml
Stop Solution 6ml
Substrate Solution A 6ml
Substrate Solution B 6ml
Wash Buffer concentrate (30x) 20ml
Biotin-Conjugate Anti-Rat TNF alpha Antibody 1ml
User Instruction 1
Plate Sealer 2
Zipper bag 1
7°C±0.5°C incubator
2. Microplate reader with 450 ± 10nm wavelength filter
3. Precision pipettes and disposable pipette tips
4. Clean tubes
5. Deionized or distilled water
6. Absorbent paper
PRECAUTIONS
1. Prior to use, the kit and sample should be warmed naturally to room
temperature 30 minutes.
2. Once the desired number of strips has been removed, immediately reseal the
bag to protect the remain from deterioration. Cover all reagents when not in
use.
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3. Make sure pipetting order and rate of addition from well-to-well when
pipetting reagents.
4. This instruction must be strictly followed in the experiment.
5. Pipette tips and plate sealer in hand should be clean and disposable to avoid
cross-contamination.
6. Avoid using the reagents from different batches together.
7. Substrate solution B is sensitive to light, don’t expose substrate solution B to
light for a long time.
8. Stop solution contains acid. Please wear eye, hand and skin protection when
using this material. Avoid contact of skin or mucous membranes with kit
reagent.
Tissue: Rinse tissues in PBS(pH 7.4) to remove excess blood thoroughly and
weigh before homogenization. Mince tissues and homogenize them in PBS
(pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C.
Centrifuge at 2000-3000 RPM for approximately 20 minutes.
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Note
1. Samples to be used within 5 days should be stored at 2-8°C. Samples should
be aliquoted or must be stored at -20°C within 1 month or -80°C within 6
months. Avoid repeated freeze thaw cycles.
2. Samples should be brought to room temperature before starting the assay.
3. Samples containing NaN3 can’t be tested as it inhibits the activity of Horse
Radish Peroxidase (HRP).
4. Collect the supernatants carefully. When sediments occurred during storage,
centrifugation should be performed again.
5. Hemolysis can greatly impact the validity of test results. Take care to
minimize hemolysis.
*SAMPLE CAN'T BE DILUTED WITH THIS KIT. ONCE THE SAMPLE HAS BEEN DILUTED IT WILL RESULT
IN HIGH BACKGROUND!
REAGENTS PREPARATION
Standard S5 S4 S3 S2 S1
3. If the standard hasn't been run out, keep the remain at -20°C. Diluted
standard can’t be reused.
4. Wash Buffer: Dilute the wash buffer concentration (30x) with distilled water.
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5. Washing Method: Discard the solution in the plate without touching the
side. Blot the plate onto absorbent paper or other absorbent material. Soak
each well with at least 0.35 ml wash buffer for 1~2 minutes.
ASSAY PROCEDURE
immediately.
6. Determine the optical density (OD value) of each well immediately using a
microplate reader set to 450 nm within 30 min after adding the stop solution.
SUMMARY
Add samples, standards and ELISA solutions. Incubate for 60 minutes at 37°C
Wash the plate five times. Add substrate solution A and B. Incubate for 10
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Read the OD value within 10minutes.
Calculate
CALCULATION OF RESULTS
Construct a standard curve by plotting the average OD for each standard on the
vertical (Y) axis against the concentration on the horizontal (X) axis and draw a
best fit curve through the points on the graph. These calculations can be best
performed with computer-based curve-fitting software and the best fit line can be
concentration read from the standard curve must be multiplied by the dilution
factor.
TYPICAL DATA
This standard curve is only for demonstration purposes. A standard curve should
REFERENCES
1. Estler H.C., Grewe M., Gaussling R., Pavlovic M., Decker K.F.Biol. Chem.
Hoppe-Seyler 373:271-281(1992)
2. Furuya T., Joe B., Salstrom J.L., Hashiramoto A., Dobbins D.E., Wilder R.L.,
Remmers E.F.Genes Immun. 2:229-232(2001)
www.bt-laboratory.com | 1713 Junjiang International Bldg. 218Ningguo Rd. Yangpu Dist. SH.China | Tel: 86 21
31007137| Fax: 86 21 65109711 816 | Email:save@bt-laboratory.com
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3. Warle M.C., van der Laan L.J., Kusters J.G., Pot R.G., Hop W.C., Segeren K.C.,
Ijzermans J.N., Metselaar H.J., Tilanus H.W.Transpl. Immunol. 14:77-82(2005)
TROUBLESHOOTING
DATA TEMPLATE
Possible Case Solution
1
High Background 2 3 4 5 6 7 8 9 10 11 12
A
Improper washing Increasing duration of soaking steps
B
Substrate was contaminated Replace.Substrate should be clean and avoid crossed
CNon-specific binding of antibody contamination by using the sealer
Plate are not be sealing incompletely Replace another purified antibody or blocking buffer
D
Incorrect incubation temperature Make sure to follow the instruction strictly
ESubstrate exposed to light prior to use Incubate at room temperature
Contaminated wash buffer Keep substrate in a dark place
F Use a clean buffers and sterile filter
G
H
Weak Signal
No Signal
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31007137| Fax: 86 21 65109711 816 | Email:save@bt-laboratory.com
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If you have any technical issue or need any assistance please contact us via e-mail:
support@bt-laboratory.com.
www.bt-laboratory.com | 1713 Junjiang International Bldg. 218Ningguo Rd. Yangpu Dist. SH.China | Tel: 86 21
31007137| Fax: 86 21 65109711 816 | Email:save@bt-laboratory.com