K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Human Adiponectin ELISA
For the quantitative determination of Adiponectinin human biological samples

Cat. No. KT-1865
For research use only.

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KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATION

Human Adiponectin ELISA
Cat. No. KT-1865
INTENDED USE
The Human Adiponectin ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative
determination of Adiponectin (ADP) in human biological samples. For research use only.

PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the ADP present in thesample reacts with the anti-ADP antibodies which have been adsorbed to the surface of polystyrene microtiter wells. Afterthe removal of unbound proteins by washing, the Detection Antibody, biotin conjugated anti-ADP, is added and complexes
are formed. Following a wash step, the horseradish peroxidase (HRP) conjugated Streptavidin is added and complexes
are formed. After another washing step, the complexes are assayed by the addition of a chromogenic substrate, 3,3’,5,5’tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of ADP in the sample
tested; thus, the absorbance, at 450 nm, is a measure of the concentration of ADP in the test sample. The quantity ofADP in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected forsample dilution.

Figure 1.

Anti-ADP Antibodies Bound To Solid Phase│
Calibrators and Samples Added│ADP * Anti-ADP Complexes Formed│
Unbound Sample Proteins Removed│
Detection Antibody Added│
Complexes Formed│
Unbound Protein Removed│
HRP Streptavidin Added│
Complexes Formed│
Unbound HRP Removed│
Chromogenic Substrate Added│
Determine Bound Enzyme Activity

COMPONENTS
1. Diluent Concentrate
One bottle containing 50 mL of a 5X concentrated diluent running buffer.
2. Wash Solution Concentrate
One bottle containing 50 mL of a 20X concentrated wash solution.
3. Detection Antibody 100X
One vial containing 150 µL of affinity purified anti-Human ADP antibody conjugated with biotin in a stabilizing buffer.
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4. HRP-Streptavidin 100X
One vial containing 150 µL of HRP conjugated streptavidin in a stabilizing buffer.
5. TMB Substrate Solution
One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
6. Stop Solution
One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
7. Microtiter Plate
Twelve removable eight-well micro strips in well holder frame. Wells are coated with affinity-purified anti-human ADP.
8. Human ADP Calibrator
One vial containing a lyophilized Human ADP Calibrator.

MATERIALS REQUIRED BUT NOT PROVIDED•••••••

Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Test tubes
Microplate washer/aspirator
Distilled or de-ionized H2O
Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer

PRECAUTIONS
1.
2.
3.
4.
5.
6.

Read the instructions carefully before beginning the assay.
This kit is for research use only.
Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain
cases, unusual results may be obtained due to high levels of interfering factors.
No additives or preservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.
Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction.
Other precautions: Do not interchange kit components from different lots.
 Do not use kit components beyond the expiration date.
 Protect reagents from direct sunlight.
 Do not pipette by mouth.
 Do not eat, drink, smoke or apply cosmetics where reagents are used.
 Avoid all contact with the reagents by using gloves.
 Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water aftercontact.

REAGENT PREPARATION
1. Diluent Solution
The Diluent Solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
2. Wash Solution Concentrate
The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal
formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to30-35°C before dilution can dissolve crystals.
3. Detection Antibody 100X
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL
detection antibody to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoidfoaming.
4. HRP-Streptavidin 100X
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL HRPStreptavidin to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoid foaming.

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5. TMB Substrate Solution
Ready to use as supplied.
6. Stop Solution
Ready to use as supplied.
7. Microtiter Plate
Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will
not be used in the assay and place back in pouch and re-seal along with desiccant.
8. Human ADP Calibrator
Add 1.0 mL of distilled or de-ionized water to the lyophilized Human ADP Calibrator and mix gently until dissolved. Thecalibrator is now at a concentration of 425 ng/mL (the reconstituted calibrator should be aliquoted and frozen if
future use is intended). Human ADP Calibrators need to be prepared immediately before use (see chart below). Mix
well between each step. Avoid foaming.

7Concentration(ng/mL)
1065432
105
2.5
1.25
0.63
0.31
0.16
0Calibrator

Calibrator Volume added
to 1X Diluent
20 µL Human ADPCalibrator
0.3 mL Calibrator 70.3 mL Calibrator 60.3 mL Calibrator 50.3 mL Calibrator 40.3 mL Calibrator 30.3 mL Calibrator 2

Volume of 1X Diluent
830 µL
0.3 mL
0.3 mL
0.3 mL
0.3 mL
0.3 mL
0.3 mL
0.6 mLSTORAGE AND STABILITY
1. Complete Kit
The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: Seelong term storage recommendations below for the Human ADP Calibrator.
2. Diluent Solution
The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one weekfrom the date of preparation. Both solutions should be stored at 4°C.
3. Wash Solution
The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least
one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Detection Antibody 100X
Undiluted Biotin conjugated anti-ADP should be stored at 4°C and diluted immediately prior to use. The working
conjugate solution is stable for up to 1 hour when stored in the dark.
5. HRP-Streptavidin 100X
Undiluted horseradish peroxidase conjugated streptavidin should be stored at 4°C and diluted immediately prior touse. The working conjugate solution is stable for up to 1 hour when stored in the dark.
6. TMB Substrate Solution
The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
7. Stop Solution
The Stop Solution should be stored at 4°C and is stable until the expiration date.
8. Microtiter Plate
Anti-human ADP coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouchwith a desiccant pack.
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9. Human ADP Calibrator
The lyophilized Human ADP calibrator should be stored at 4°C or frozen until reconstituted. The reconstitutedcalibrator should be aliquoted out and stored frozen (Avoid multiple freeze-thaw cycles). The working calibrator
solutions should be prepared immediately prior to use.

INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expectedvalues.

SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture and the serum separated from the cells, after clot formation, by centrifugation.
For plasma samples, blood should be collected into a container with an anticoagulant and then centrifuged. Care should
be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay immediately or aliquot and store
samples at -20°C. Avoid repeated freezing/thawing.
For any sample that might contain pathogens, care must be taken to prevent contact with open wounds. No additives orpreservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.

ASSAY PROTOCOL
Dilution of Samples
The assay for quantification of ADP in samples requires that each test sample be diluted before use. For a single step
determination a dilution of 1:20 is appropriate for most serum/plasma samples. For absolute quantification of samples thatyield results outside the range of the calibration curve, a lesser or greater dilution might be required. If unsure of sample
level, a serial dilution with one or two representative samples before running the entire plate is highly recommended.
To prepare a 1:20 dilution of serum/plasma sample, transfer 25 µL of sample to 475 µL of 1X Diluent. This gives you a
1:20 dilution. Mix thoroughly. Dilute samples immediately prior to use.

Procedure
1. Bring all reagents to RT before use.
2. Pipette 100 µL of
Calibrator 0 (0.0 ng/mL) in duplicate
Calibrator 1 (0.16 ng/mL) in duplicate
Calibrator 2 (0.31 ng/mL) in duplicate
Calibrator 3 (0.63 ng/mL) in duplicate
Calibrator 4 (1.25 ng/mL) in duplicate
Calibrator 5 (2.5 ng/mL) in duplicate
Calibrator 6 (5 ng/mL) in duplicate
Calibrator 7 (10 ng/mL) in duplicate
3. Pipette 100 µL of diluted sample (in duplicate) into pre-designated wells.
4. Incubate the Microtiter Plate at 22°C (RT) for sixty (60 ± 2) minutes. Keep plate covered and level during incubation.
5. Following incubation, aspirate the contents of the wells.
6. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of fourwashes. If washing manually; completely fill wells with wash buffer, invert the plate and pour/shake out the contents ina waste container. Follow this by sharply striking the wells on absorbent paper to remove residual buffer. Repeat 3times for a total of 4 washes.
7. Pipette 100 µL of appropriately diluted detection antibody to each well. Incubate at 22°C (RT) for thirty (30 ± 2)
minutes. Keep plate covered in the dark and level during incubation.
8. Wash and blot the wells as described in Steps 5 and 6.

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9. Pipette 100 µL of appropriately diluted HRP-streptavidin to each well. Incubate at 22°C (RT) for thirty (30 ± 2) minutes.
Keep plate covered in the dark and level during incubation.
10. Wash and blot the wells as described in Steps 5 and 6.
11. Pipette 100 µL of TMB Substrate Solution into each well.
12. Incubate in the dark at RT for precisely ten (10) minutes.
13. After ten (10) minutes, add 100 µL of Stop Solution to each well.
14. Determine the absorbance at 450 nm of the contents of each well. Zero the plate reader to air.
The absorbance of the final reaction mixture can be measured up to 2 hours after the addition of the Stop Solution.
However, good laboratory practice dictates that the measurement be made as soon as possible.

RESULTS
1. Subtract the average background value from the test values for each sample.
2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a fourparameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at ADP concentrationin original sample.

QUALITY CONTROLIn accord with good laboratory practice, the assays for specific ADP require meticulous quality control. Each laboratory
should use routine quality control procedures to establish inter- and intra-assay precision and performance characteristics.

LIMITATION OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the information contained in the package insert instructions and with adherence to good laboratory
practice.
2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware,
quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation
time or temperature.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068
Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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