产品介绍
Introduction
Adiponectin, also known as Adipocyte Complement-Related Protein of 30kDa
(ACRP30), is a secreted serum protein expressed exclusively in differentiated
adipocytes. Studies indicated that decreased plasma adiponectin concentration is
associated with obesity, insulin resistance (1), essential hypertension (2),
inflammation and atherosclerosis (3), and acute myocardial infarction (4). On
the other hand, increased adiponectin level leads to nephrotic syndrome (5,
6).
Principal of the Assay
The AssayMax Rat Adiponectin ELISA Kit is designed for detection of
adiponectin in rat urine, plasma, serum and cell culture supernatants. This
assay employs a quantitative sandwich enzyme immunoassay technique that measures
adiponectin in less than 4 hours. A polyclonal antibody specific for adiponectin
has been pre-coated onto a microplate. Adiponectin in standards and samples is
sandwiched by the immobilized antibody and biotinylated polyclonal antibody
specific for adiponectin, which is recognized by a streptavidin-peroxidase
conjugate. All unbound material is then washed away and a peroxidase enzyme
substrate is added. The color development is stopped and the intensity of the
color is measured.
Caution and Warning
? This kit is for research use only.
? The kit should not be used beyond the expiration date.
? The Stop Solution is an acid solution.
Reagents
? Adiponectin Microplate:A 96-well polystyrene microplate (12 strips
of 8 wells) coated with a polyclonal antibody against rat adiponectin.
? Sealing Tapes:Each kit contains 3 pre-cut, pressure-sensitive
sealing tapes, which can be cut to fit the format of the individual assay.
? Adiponectin Standard:Rat adiponectin in a buffered protein base
(1.6 μg, lyophilized).
? Biotinylated Adiponectin Antibody (100x):A 100-fold concentrated
biotinylated polyclonal antibody against adiponectin (80 μl).
? MIX Diluent Concentrate (10x): A 10-fold concentrated buffered
protein base (30 ml).
? Wash Buffer Concentrate (20x): A 20-fold concentrated buffered
surfactant (30 ml, 2 botlles).
? Streptavidin-Peroxidase Conjugate (SP Conjugate):A 100-fold
concentrate (80 μl).
? Chromogen Substrate: A ready-to-use stabilized
peroxidase chromogen substrate tetramethylbenzidine (8 ml).
? Stop Solution: A 0.5 N hydrochloric acid to stop the
chromogen substrate reaction (12 ml).
Storage Condition
? Store kit at 2-80C or -200C upon arrival up to the expiration
date.
? Opened MIX Diluent may be stored for up to 1 month at 2 - 80C.
Store reconstituted reagents at -200C or below.
? Opened unused strip wells may return to the foil pouch with
the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a
vacuum desiccator.
Other Supplies Required
? Microplate reader capable of measuring absorbance at 450
nm.
? Pipettes (1-20 μl, 20-200 μl, 200-1000μl and multiple
channel).
? Deionized or distilled reagent grade water.
Sample Collection and Storage
? Plasma:Collect plasma using one-tenth volume of 0.1 M
sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10
minutes and assay. Dilute samples 1:400 into MIX Diluent. The undiluted samples
can be stored at -200C or below for up to 3 months. Avoid repeated freeze-thaw
cycles. (EDTA or Heparin can also be used as anticoagulant.)
? Serum:Samples should be collected into a serum
separator tube. After clot formation, centrifuge samples at 2000 x g for 10
minutes. Remove serum and assay. Dilute samples 1:400 into MIX Diluent. The
undiluted samples can be stored at -200C or below for up to 3 months. Avoid
repeated freeze-thaw cycles.
? Cell Culture Supernatants:Centrifuge cell culture media
at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay.
Dilute samples 1:10 into MIX Diluent. Store samples at -200C or below. Avoid
repeated freeze-thaw cycles.
? Urine:Collect urine using sample pot. Centrifuge
samples at 600 x g for 10 minutes and assay. Store samples at -200C or below for
up to 3 months. Avoid repeated freeze-thaw cycles.
Reagent Preparation
? Freshly dilute all reagents and bring all reagents to room
temperature before use. If crystals have formed in the concentrate, mix gently
until the crystals have completely dissolved.
? MIX Diluent Concentrate (10x): Dilute the MIX Diluent
Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2 -
80C.
? Adiponectin Standard:Reconstitute the 1.6 μg of rat
adiponectin Standard with 4 ml of MIX Diluent to generate a standard stock of
400 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior
to making dilutions. Prepare duplicate or triplicate standard points by diluting
stock (400 ng/ml) 1: 4 to produce a standard solution of 100 ng/ml. Prepare
duplicate or triplicate standard points by serially diluting the Standard
solution (100 ng/ml) 1:2 with equal volume of MIX Diluent to produce 50, 25,
12.5, 6.25, 3.125, 1.56 and 0.78 ng/ml. MIX Diluent serves as the zero standard
(0 ng/ml). Any remaining solution should be frozen at -200C.
Standard Point | Dilution | [Adiponectin] (ng/ml) |
P1 | 1 part Stock (200 ng/ml) + 3 parts MIX Diluent | 100.00 |
P2 | 1 part P1 + 1 part MIX Diluent | 50.00 |
P3 | 1 part P2 + 1 part MIX Diluent | 25.00 |
P4 | 1 part P3 + 1 part MIX Diluent | 12.50 |
P5 | 1 part P4 + 1 part MIX Diluent | 6.25 |
P6 | 1 part P5 + 1 part MIX Diluent | 3.13 |
P7 | 1 part P6 + 1 part MIX Diluent | 1.56 |
P8 | MIX Diluent | 0.00 |
? Biotinylated Adiponectin Antibody (100x):Spin down the
biotinylated antibody briefly and dilute the desired amount of the antibody
1:100 with MIX Diluent. Any remaining solution should be frozen at -200C.
? Wash Buffer Concentrate (20x):Dilute the Wash Buffer
Concentrate 1:20 with reagent grade water.
? SP Conjugate (100x):Spin down the SP Conjugate briefly
and dilute the desired amount of the conjugate 1:100 with MIX Diluent. Any
remaining solution should be frozen at -200C.
Assay Procedure
? Prepare all reagents, working standards and samples as
instructed. Bring all reagents to room temperature before use. The assay is
performed at room temperature (20-300C).
? Remove excess microplate strips from the plate frame and
return them immediately to the foil pouch with desiccant inside. Reseal the
pouch securely to minimize exposure to water vapor and store in a vacuum
desiccator.
? Add 50 μl of Standard or sample per well. Cover wells with a
sealing tape and incubate for one hour. Start the timer after the last sample
addition.
? Wash five times with 200 μl of Wash Buffer manually. Invert
the plate each time and decant the contents; hit it 4-5 times on absorbent paper
towel to completely remove the liquid.
If using a machine wash six times with 300 μl of Wash Buffer and
then invert the plate, decant the contents; hit it 4-5 times on absorbent paper
towel to completely remove the liquid.
? Add 50 μl of Biotinylated Adiponectin Antibody to each well
and incubate for one hour.
? Wash a microplate as described above.
? Add 50 μl of Streptavidin-Peroxidase Conjugate per well and
incubate for 30 minutes. Turn on the microplate reader and set up the program in
advance.
? Wash a microplate as described above.
? Add 50 μl of Chromogen Substrate per well and incubate for
about 10 minutes or till the optimal blue color density develops. Gently tap
plate to ensure thorough mixing and break the bubbles in the well with pipette
tip.
? Add 50 μl of Stop Solution to each well. The color will change
from blue to yellow.
? Read the absorbance on a microplate reader at a wavelength of
450 nm immediately. If wavelength correction is available, subtract
readings at 570 nm from those at 450 nm to correct optical imperfections.
Otherwise, read the plate at 450 nm only. Please note that some unstable black
particles may be generated at high concentration points after stopping the
reaction for about 10 minutes, which will reduce the readings.
Data Analysis
? Calculate the mean value of the duplicate or triplicate readings for each
standard and sample.
? To generate a Standard Curve, plot the graph using the standard
concentrations on the x-axis and the corresponding mean 450 nm absorbance on the
y-axis. The best-fit line can be determined by regression analysis using log-log
or four-parameter logistic curve-fit.
? Determine the unknown sample concentration from the Standard Curve and
multiply the value by the dilution factor.
Performance Characteristics
? This assay recognizes both natural and recombinant rat adiponectin. It
can detect both globular domain and full-length adiponectin.
? The minimum detectable dose of adiponectin is typically 1.5 ng/ml.
? Intra-assay and inter-assay coefficients of variation were 4.3 % and 7.5
% respectively.
Linearity Average Percentage of Expected Value |
Sample Dilution | Plasma | Serum |
1:200 | 101% | 99% |
1:400 | 99% | 101% |
1:800 | 104% | 99% |
Average Percentage of Expected Value | |
Sample Dilution | Cell Culture Media | |
1:10 | 100% | |
1:20 | 99% | |
1:40 | 103% | |
1:80 | 105% | |
Average Percentage of Expected Value | |
Sample Dilution | Urine | |
1:5 | 106% | |
1:10 | 98% | |
1:20 | 93% | |
Recovery Standard Added Value | 2 ? 25 ng/ml | |
Recovery % | 89-113 % | |
Average Recovery % | 102 % | |
| | | | |
Reference Value
? The normal blood levels of Adiponectin (ACRP30) range from 8.3-13.9
ug/ml.
References
(1) Tsao, T.S. et al.(2002) EJP440:213-221
(2) Adamczak, M. et al. AJH16:72-75
(3) Matsubara, M. et al.(2003) Eur J Endocrinol.148(6):
657-62
(4) Kojima, S. et al.(2003) Heart89(6): 667
(5) Zoccali, C. et al.(2003) Kidney Int Suppl.84:
S98-102
(6) Pannacciulli, N. et al.(2003) J Clin Endocrinol Metab.
88(4): 174
Version 2.3
Related products
? EMA2500-1 AssayMax Mouse Adiponectin ELISA Kit (Plasma, Serum, Urine and
Cell Culture Supernatant samples)
? EA2500-1 AssayMax Human Adiponectin ELISA Kit (Plasma, Serum, Urine and
Cell Culture Supernatant samples)
P3
1 part P2 + 1 part MIX Diluent
25.00
1:400
99%
101%
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