Protocol for ELISA Kit
KBB-120
Human IFNγ ELISA Kit
Preparation Before Experiment
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Methods for Plate Washing
By handwork: Absorb or throw the liquid that in the plate. Don’t touch wall. Mat several layers of sop papers on
experiment desk, patting the plate downwards for several times. Infuse PBS or TBS buffer (at least 0.3ml) into wells,
sopping 1-2 min. Repeat this process several times if necessary.
By automation: If you have the automated plate washer, you should use it proficiently before in formal experiments.
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Sample Preparation and Storage
If not analyzed shortly, samples should be aliquoted and stored frozen at –20°C. And avoid multiple freeze-thaw
cycles.
Cell culture supernates, tissue lysates, body fluids – Remove particulates by centrifugation and analyze
immediately or aliquot and store frozen at -20℃.
Serum – Use a serum separator tube (SST) and allow samples to clot for 30 min at room temperature, then,
centrifuge at approximately 1000 X g for 15 min of collection. Remove serum and analyze immediately or
aliquot and store frozen at -20℃.
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Principles for Sample Dilution
Users should estimate contents of the sample purpose proteins, determining proper dilution multiples, in order that
the concentration of the diluted sample purpose proteins could arrive at the optimal detection range for ELISA kit.
According to differences of contents in purpose proteins, taking the diluted methods respectively.
High – The concentration of purpose proteins is 10-100ng/ml, and the working dilution is 1:100. Add 3μl sample to
297μl sample diluent buffer.
Middle – The concentration of purpose proteins is 1-10ng/ml, and the working dilution is 1:10. Add 25μl sample to
225μl sample diluent buffer.
Low – The concentration of purpose proteins is 15.6-1000pg/ml, and the working dilution is 1:2. Add 100μl sample
to 100μl sample diluent buffer.
Especially Low – The concentration of purpose proteins is ≤15.6pg/ml, and the sample dilution is unnecessary, or
the working dilution is 1:2. The above data is only for your reference, and the working dilution
should be noted detailedly.
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Protocol for ELISA Kit
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Reagent Preparation and Storage
A. Dilution and usage of human IFNγ standard: IFNγ standard solution should be prepared within 2 hours prior to
usage. The kit provides two tubes of IFNγ standard (10ng per tube). One tube was used for each experiment.
1.
Preparation of 10,000pg/ml of human IFNγ standard: Add 1 ml sample diluent buffer into one tube
containing 10ng of IFNγ standard and stand at room temperature for 10 min and mix evenly. This will
produce IFNγ standard solution at a concentration of 10ng/ml.
2.
Preparation of 1000pg/ml of human IFNγ standard: Take 0.1 ml of above IFNγ standard solution into
0.9 ml sample diluent buffer and mix, that will result in IFNγ solution at a concentration of 1ng/ml.
3.
Preparation of 500pg/ml→ 15.6pg/ml of human IFNγ standard: Labeled 6 Eppendorf tubes in 500pg/ml,
250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml respectively. Aliquot 0.3 ml of sample diluent
buffer into each tube. Add 0.3 ml of 1000pg/ml of IFNγ standard solution into first tube and mix. And
then, take 0.3 ml of IFNγ solution from first tube to second tube and mix. Use the same way to make
serial dilutions of IFNγ and end at last tube.
Notice:
The diluted standards (10,000pg/ml) should be used within 12 hours at 4℃. And it can also be used
within 2 days in the condition of storing frozen at -20℃. And avoid multiple freeze-thaw cycles.
B． Preparation of biotinylated anti-human IFNγ antibody working solution: The solution should be prepared within
2 hours prior to usage.
1.
Based on the volume of 0.1ml per well, the total volume was calculated (allowing 0.1-0.2 ml more than
calculated volume).
2.
Biotinylated anti-human IFNγ antibody was diluted in 1:99 with antibody diluent buffer and mix evenly.
C．Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared 1
hour prior to usage.
1.
Based on the volume of 0.1ml per well, the total volume was calculated (allowing 0.1-0.2 ml more than
calculated volume).
2. Avidin- Biotin-Peroxidase Complex (ABC) was diluted in 1:99 with ABC dilution buffer and mix evenly.
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Protocol for ELISA Kit
Assay Procedure
The diluted ABC working solution and TMB color developing agent should be warmed up at 37℃ for 30 min before
usage. When diluting samples and reagents, they must be mixed completely and evenly. Standard IFNγ detection
curve should be prepared for each experiment. Applicant will decide sample dilution fold by crude estimation of
IFNγ amount in samples.
1. Aliquot 0.1ml per well of 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml of
prepared IFNγ standard into precoated 96-well. Add 100μl of sample diluent buffer into control well (Zero
well). The testing samples from human sera, plasma, body fluids, tissue lysates or cell culture
supernates were prepared in sample diluent buffer with different dilutions based on their IFNγ contents.
100μl of diluted samples were added directly into each well. It is recommended that each dilution of IFNγ
standards and samples should be added into duplicated wells.
2.
Seal plate with cover and incubate at 37℃ for 90 min.
3. Remove cover, discard plate contents into receptacle, and strike plate on fresh towels to remove residual
solution in each well, but don’t let wells completely dry at any time.
4. Add 0.1ml of biotinylated anti- human IFNγ antibody working solution into each well and incubate the
plate at 37℃ for 60 min.
5.
Wash plate three times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay for 1 min.
6.
Add 0.1ml of prepared ABC working solution into each well and incubate the plate at 37℃ for 30 min.
7.
Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay for 1-2 min.
8.
Add 90 μl of prepared TMB color developing agent into each well, and incubate plate at 37℃ for 10-15
min (blue color gradient can be visually observed in the first 1 to 4 low dilution wells of IFNγ standard
sample dilutions, the rest wells show no obvious color difference).
9.
Add 0.1ml of prepared TMB stop solution into each well. The color changed into yellow immediately.
10. Read O.D. absorbance at 450nm microplate reader within 30 min after adding stop solution.
For calculation, the absolute O.D.450= O. D.450 of IFNγ standard or sample – O.D.450 of Zero well. The IFNγ standard
graphics can be draw using IFNγ concentration (X) vs absolute O.D. 450 value (Y). The IFNγ concentration of samples
can be obtained from the IFNγ standard curve. Notice: the real IFNγ concentration of sample = IFNγ concentration of
sample obtained from standard curve × sample dilution fold (N).
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Protocol for ELISA Kit
Conclusion
1.
Add samples and standards and incubate the plate at 37℃ for 90 min. Do not wash.
2.
Add biotinylated antibodies and incubate the plate at 37℃ for 60 min. Wash plate 3 times with 0.01M
TBS.
3.
Add ABC working solution and incubate the plate at 37℃ for 30 min. Wash plate 5 times with 0.01M
TBS.
4.
Add TMB color developing agent and incubate the plate at 37℃ for 10-15 min.
5.
Add TMB stop solution and read.
Typical Data obtained using IFNγ
(TMB reaction incubate at 37℃ for 10 min)
Concentration
O.D
0.0pg/ml
15.6pg/ml
31.3pg/ml
62.5pg/ml
125pg/ml
250pg/ml
500pg/ml
1000pg/ml
0.036
0.070
0.099
0.145
0.274
0.496
0.962
1.936
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Protocol for ELISA Kit
Human IFNγ ELISA Kit - 1 x 96 Well Plate Images
O.D.
Human IFNγ ELISA Kit
2.1
3
1.7
7
1.4
2
1.0
6
0.7
1
0.3
6
0.0
0
0.0
183.3
366.7
550.0
733.3
916.7
1100.0
Concentration (pg/ml)
FOR RESEARCH USE ONLY.
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NOT FOR DIAGNOSTIC AND CLINICAL USE.
+46 (0)8 5444 33 40
Denmark 800 10595
Norway 800 10301
Finland 0800 111333
[email protected]