96 wells High Sensitive ELISA Kit for Human LEP
Cat.No E1559Hu
Standard Curve Range: 0.05ng/ml - 10ng/ml
Sensitivity: 0.021ng/ml
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to
the expiration date keep it at -20°C. Avoid repeated thaw cycles.
If individual reagents are opened it is recommended that the kit be
used within 1 month.
* This product is for research use only, not for use in diagnosis
procedures. It’s highly recommend to read this instruction entirely
before use.
Precision
Intra-Assay Precision (Precision within an assay) Three samples of
known concentration were tested on one plate to assess intra-assay
precision.
Inter-Assay Precision (Precision between assays) Three samples of
known concentration were tested in separate assays to assess
inter-assay precision.
CV(%) = SD/mean x 100
Intra-Assay: CV<8%
Inter-Assay: CV<10%
Intended Use
This sandwich kit is for the accurate quantitative detection of
human Leptin (also known as LEP) in serum, plasma, cell culture
supernates, cell lysates, tissue homogenates.
Reagent Provided
Components | Quantity |
Standard Solution (12.8ng/ml) | 0.5ml x1 |
Pre-coated ELISA Plate | 12 * 8 well strips x1 |
Standard Diluent | 3ml x1 |
Streptavidin-HRP | 6ml x1 |
Stop Solution | 6ml x1 |
Substrate Solution A | 6ml x1 |
Substrate Solution B | 6ml x1 |
Wash Buffer Concentrate (25x) | 20ml x1 |
Biotinylated human LEP Antibody | 1ml x1 |
User Instruction | 1 |
Plate Sealer | 2 pics |
Zipper bag | 1 pic |
Note
- Sample concentrations should be predicted before being used in the
assay. If the sample concentration is not within the range of the
standard curve, users must contact us to determine the optimal sample for their particular experiments.
- 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.
- Samples should be brought to room temperature before starting the
assay.
- Centrifuge to collect sample before use.
- Samples containing NaN3 can’t be tested as it inhibits the activity
of Horse Radish Peroxidase (HRP).
- Collect the supernatants carefully. When sediments occurred during
storage, centrifugation should be performed again.
- Hemolysis can greatly impact the validity of test results. Take
care to minimize hemolysis.
Assay Procedure
- Prepare all reagents, standard solutions and samples as instructed.
Bring all reagents to room temperature before use. The assay is
performed at room temperature.
- Determine the number of strips required for the assay. Insert the
strips in the frames for use. The unused strips should be stored at
2-8°C.
- Add 50μl standard to standard well. Note: Don’t add antibody to standard well because the standard solution
contains biotinylated antibody.
- Add 40μl sample to sample wells and then add 10μl anti-LEP antibody
to sample wells, then add 50μl streptavidin-HRP to sample wells and
standard wells (Not blank control well). Mix well. Cover the plate
with a sealer. Incubate 60 minutes at 37°C.
- Remove the sealer and wash the plate 5 times with wash buffer. Soak
wells with at least 0.35 ml wash buffer for 30 seconds to 1 minute
for each wash. For automated washing, aspirate all wells and wash 5
times with wash buffer, overfilling wells with wash buffer. Blot
the plate onto paper towels or other absorbent material.
- Add 50μl substrate solution A to each well and then add 50μl
substrate solution B to each well. Incubate plate covered with a
new sealer for 10 minutes at 37°C in the dark.
- Add 50μl Stop Solution to each well, the blue color will change
into yellow immediately.
- Determine the optical density (OD value) of each well immediately
using a microplate reader set to 450 nm within 10 minuets after
adding the stop solution.
References
Comings D.E., Gade R., Muhleman D., Peters W.R., MacMurray J.P.
Mol. Genet. Metab. 73:204-210(2001)