Human Adiponectin（ADP）TSZ原裝品牌上海恒遠供應

ELISA Kit Instructions                                                              

Use:

This ELISA kit is for quantitative detecting Human Adiponectin（ADP） content in specimens of cells culture supernates, body fluids, tissues homogenate, serum, and plasma. The kit is for scientific research use only. Not for diagnostic or therapeutic procedures.

Principle:

This ADP enzyme linked immunosorbent assay employs the quantitative sandwich enzyme immunoassay technique. The microplate provided in this kit has been pre-coated with a monoclonal antibody specific for ADP. Standards or samples are then pipetted into the microplate wells, and ADP present in the samples or standards binds to antibodies adsorbed to the microplate wells. In order to quantitatively determine the amount of ADP present in the samples, the horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for ADP are added to each well. The microplate is incubated for 1h, and then the wells are thoroughly washed to remove any unbound components. The substHumane solution A and B is respectively added to each well. After the enzyme (HRP) and substHumane reacting over a short period, this reaction is stopped by addition of a sulphuric acid solution and the color change is measured at a wavelength of 450 nm. The proportion to amount of ADP bound in the initial step develops in the color change.

Reagents in kit:

Experiment materials and reagent prepaHumanion:

1.          Instruments and materials: microplate reader capable of measuring absorbance at 450 nm (preheat 30 minutes before use), micropipette, pipette tips, distilled water or deionized water, filter paper.

2.          Washing solution: prepare washing solution in the proportion of 1:30. Mix gently to avoid foaming.

Sample collection and storage:

1.          Cell culture supernatants: After centrifugation at 1000xg for 15 minutes, collect supernatants with a sterile tube.

2.          Cells: After washing cells 3 times with PBS, adjust the cell concentHumanion to 10⁴-10⁶/ml; cells are broken down either through repeated freeze-thaw cycles or by using ultrasound treatment, and then cell ingredients are released. Collect the supernatants after centrifugation.

3.          Serum: allow samples to clot before centrifugation for 15 minutes at 1000 x g, collect the supernatants.

4.          Plasma: Collect plasma using EDTA or citric acid sodium as anticoagulation; after mixing, stand for 10 to 20 minutes, and then centrifuge at 1000 x g for 15 minutes, collect the supernatants.

5.          Body Fluid: include fluid from thoracic or abdominal cavity, cerebrospinal fluid, secretion and so on. Collect supernatants with tubes (without pyrogen and endotoxin) after centrifugation at 1000 x g for 15 to 20 minutes.

6.          Tissue samples: After weighing 0.5mg specimens and add into 500ul PBS, specimen homogenate can be prepared by manual grind, a homogenate device, or ultrasound. After centrifugation for 20 minutes (at 2000-3000 RPM), collect the supernatants.

7.          Samples should not contain NaN3 because NaN3 inhibit horseradish peroxidase (HRP) activity.

8.          Storage: Assay immediay or aliquot and store samples at -70℃, avoid repeated freeze-thaw cycles. Precipitation appears during storage, must centrifuge again. As far as possible, do not use blood samples with hemolysis or hyperlipid. Samples containing a visible precipitate must be clarified prior to use in the assay. Don't thaw specimens at 37 ℃ or higher. Prior to assay, the frozen sample should be brought to room tempeHumanure slowly and mixed gently.

Assay procedure:

1.          The kit and samples are brought to room tempeHumanure (20-25℃) for 30 minutes before use.

2.          Group: take out 96 microplate and determine microplate strips according to quantity of the specimens and the standard samples, and then remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack and reseal. Set up the standard group (six concentHumanions), blank control group, and the sample group respectively. To minimize the error and guarantee the accuracy, it is suggested that eADP sample is arranged with three or more wells.

Dilute standard: Prepare sixs test tube ,make number successively,add Standard diluent100ul to ervery test tube,add Original density Standard 100ul to the first test tube, Gentlymix;then take out 100ul from the first test tube and add to the second test tube, Gentlymix; then take out 100ul from the second test tube and add to the third test tube, Gentlymix; then take out 100ul from the third test tube and add to the forth test tube, Gently mix;then take out 100ul from the forth test tube and add to the fifth test tube, Gently mix; takeout 100ul from the fifth test tube and Discard,make the sixth test tube as standard 0. The density Standard of every test tube is : 1000pg/ml,500 pg/ml,250 pg/ml,125 pg/ml,62.5pg/ml,0 pg/ml.

3.          Set Standard wells on the Microelisa Stripplate , add different concentHumanions of standard50 ul successively .

4.          Add specimens: in accordance with the order of the standard, add 50ul standard solution in eADP well of standard group; 50ul distilled water in eADP well of blank control group; add Sample 40μl to testing sample well, then add Biotinylated anti –ADP -antibody 10μl , don’t touch the well wall as far as possible, and Gently mix.

5.          Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.

6.          washing：Uncover Closure plate membrane, discard Liquid, dry by swing, still for 30s then drain, repeat 5 times, dry by pat.

7.          Add Enzyme conjugate: add 50ul of Enzyme conjugate to the standard group and specimen group respectively (with the exception of blank control group).

8.          Incubation: Plates are sealed tightly with plate sealing membrane, incubated in wet box at 37 ℃constantly for1 hour.

9.          Wash plate: filling eADP well full with diluted washing solution, rest for 15-30 seconds, repeat for 5 times, thoroughly pat dry with absorbent paper.

10.       Color development: to eADP well add SubstHumane A 50ul, followed by SubstHumane B 50ul.

11.       Color termination: after reaction at 25-37 ℃ in dark for 10-15 minutes, add Stop solution 50uL to eADP well.

12.       Read plate: Determine the optical density (OD) of eADP well within 30 minutes, using a microplate reader set to 450 nm. Please wipe away the residual liquid and fingers trace in advance

Data calculation

1.          This standard curve is used to determine the amount of ADP in an unknown sample. The standard curve is geneHumaned by plotting the mean O.D. (450 nm) obtained for eADP of the six standard concentHumanions on the vertical (Y) axis versus the corresponding concentHumanion on the horizontal (X) axis.

2.          First, calculate the mean O.D. for eADP standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.

3.          To determine the amount in eADP sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentHumanion.

4.          Any variation in opeHumanor, pipetting and washing technique, incubation time or tempeHumanure, and kit age can cause variation in result. EADP user should obtain their  own standard curve.

5.          The sensitivity by this assay is 1.0 pg/ml.

6.          Standard curve

Note:

1.          To avoid cross contamination, disposable tips should be used for one-time only.

2.          Add samples: to minimize the interval between the first well and the last one, the speed of adding sample should be controlled. Otherwise, it will lead to difference of the incubation time, thus affecting the accuracy of the experiment.

3.          Incubation: in strict accordance with the demands about the instruction of incubation time and tempeHumanure.

4.          Control of the reaction time: monitor the color change in the well in fixed time after substHumane is added, if the color is darker, please add Stop solution ahead of schedule.

5.          It is suggested to get preliminary data before a formal test if the concentHumanion of the specimen is too high. Dilute the specimen and times the results accordingly when calculation.

6.          It is suggested to get a preliminary test done on the kit (namely get standard curve made first, trial on a few specimens), if there is any doubt about this kit, or the failure is resulted from transportation, please contact customer service of company or agent for replacement; but it shall not be subject to any loss related to the product itself.

Safety:

1.          Avoid direct contact the human body with color-display reagent A, B and the terminating reaction solution. Once it happened, please wash out with water as soon as possible.

2.          Don't eat or drink, smoke or make up during experiment.

3.          Don't suck up any reagents in the kit with mouth.

Preservation conditions and validity:

1.          Storage: 2-8 ℃

2.          Period of Validity: six months

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