Human Adiponectin RIA
Adiponectin is a new member of an ever increasing family of adipocytokines and is also referred to as ACRP-30 (adipocyte compliment related protein-30), Adipo-Q, and APM-1 (adipose tissue most abundant gene transcript-1). Adiponectin is a predominant secretory protein from adipose tissue and circulates in micro-gram/ml quantities and has a structural homology with the type VIII collagen and hibernation specific protein, C1q.
In contrast to the majority of secreted proteins from adipose tissue, which are elevated in obesity, adiponectin appears to be either decreased or unaltered with degree of adiposity. More intriguingly, adiponectin seems to ameliorate the obesity related risk factors unlike other adipose tissue secretory proteins which contribute toward the health risks associated with obesity. Adiponectin also has an insulin sensitizing effect making it an excellent candidate in drug development for obesity and diabetes. Circulating adiponectin levels seem to be an excellent biochemical marker for improved insulin resistance in obese and diabetic states. This kit is for research purposes only.
In radioimmunoassay, a fixed concentration of labeled tracer antigen is incubated with a constant dilution of antiserum such that the concentration of antigen binding sites on the antibody is limited, for example, only 50% of the total tracer concentration may be bound by antibody. If unlabeled antigen is added to this system, there is competition between labeled tracer and unlabeled antigen for the limited and constant number of binding sites on the antibody. Thus, the amount of tracer bound to antibody will decrease as the concentration of unlabeled antigen increases. This can be measured after separating antibody-bound from free tracer and counting one or the other, or both fractions. A calibration or standard curve is set up with increasing concentrations of standard unlabeled antigen and from this curve the amount of antigen in unknown samples can be calculated. Thus, the four basic necessities for a radioimmunoassay system are: a specific antiserum to the antigen to be measured, the availability of a radioactive labeled form of the antigen, a method whereby antibody-bound tracer can be separated from the unbound tracer, and finally, an instrument to count radioactivity.
The LINCO Research, Inc. Adiponectin RIA assay utilizes 125I-labeled Murine Adiponectin and a Multispecies Adiponectin Rabbit antiserum to determine the level of Adiponectin in serum, plasma or tissue culture media by the double antibody/PEG technique. The Adiponectin Standard is prepared using recombinant Human Adiponectin and can be used to determine the circulating levels of adiponectin in human serum/plasma samples.
Each kit is sufficient to run 125 tubes and contains the following reagents.
A. 10X Assay Buffer
Final concentration upon dilution is 10.0 mM Phosphate Buffer, pH 7.6 containing 0.08% Sodium Azide, 0.1 % RIA Grade BSA
Quantity: 50 ml/vial
Preparation: Dilute the contents of the vial with 450 ml distilled or deionized water
B. Adiponectin Antibody
Adiponectin Antibody
Quantity: 13 ml/vial
Preparation: Ready to use
C. 125I-Adiponectin
125I-Adiponectin Label (specific activity 67.7 µCi/µg)
Lyophilized for stability. Freshly iodinated label contains < 3 µCi, (<111 kBq) calibrated to the 1st Monday of each month.
Quantity: 13.5 ml/vial upon hydration
Preparation: Contents Lyophilized. Hydrate with 13.5 ml of 1X Assay Buffer. Allow to sit at room temperature for 30 minutes, with occasional gentle mixing.
NOTE: You will observe that the adiponectin tracer displays lower B0 binding as it approaches its expiration date. This change does not alter the performance of the kit since the Quality Control Values remain within expected ranges throughout the tracer shelf life.
D. Human Adiponectin Standard
Purified Recombinant Adiponectin, 200 ng/ml
Lyophilized for stability.
Quantity: 1 ml upon hydration
Preparation: Contents Lyophilized. Hydrate with 1 ml distilled or deionized water.
E. Quality Controls 1& 2
Purified Recombinant Adiponectin
Lyophilized for stability
Quantity: 1 ml/vial upon hydration
Preparation: Contents Lyophilized. Hydrate with 1 ml distilled or deionized water.
F. Rabbit Carrier
30% Normal Rabbit Serum
Quantity: 2 ml/vial
Ready to use
G. Precipitating Reagent
Goat anti-Rabbit IgG Serum, 3% PEG and 0.05% Triton X-100 in 0.05M Phosphosaline, 0.025M EDTA,
0.08% Sodium Azide
Quantity: 130 ml/vial
Preparation: Ready to use; chill to 4°C
Prior to use, refrigerate all reagents between 2 and 8°C for short-term storage. For prolonged storage (>2 weeks), freeze at £ -20°C. Once the standards have been reconstituted, unused portions should be stored at £ -20°C. Avoid multiple freeze/thaw cycles. Refer to date on bottle for expiration when stored at £ -20°C. Do not mix reagents from different kits unless they have the same lot number.
A. Radioactive Materials
This radioactive material may be received, acquired, possessed and used only by research personnel or clinical laboratories for in vitro research tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the regulations of the U. S. Nuclear Regulatory Commission (NRC) or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority.
The following are suggested general rules for the safe use of radioactive material. The customer’s Radiation Safety Officer (RSO) is ultimately responsible for the safe handling and use of radioactive material.
1. Wear appropriate personal devices at all times while in areas where radioactive materials are used or stored.
2. Wear laboratory coats, disposable gloves, and other protective clothing at all times.
3. Monitor hands, shoes, and clothing and immediate area surrounding the workstation for contamination after each procedure and before leaving the area.
4. Do not eat, drink, or smoke in any area where radioactive materials are stored or used.
5. Never pipette radioactive material by mouth.
6. Dispose of radioactive waste in accordance with NRC rules and regulations.
7. Avoid contaminating objects such as telephones, light switches, doorknobs, etc.
8. Use absorbent pads for containing and easily disposing of small amounts of contamination.
9. Wipe up all spills immediately and thoroughly and dispose of the contaminated materials as radioactive waste. Inform Radiation Safety Officer.
B. Sodium Azide
Sodium Azide has been added to all reagents as a preservative at a concentration of 0.08%. Although it is at a minimum concentration, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build up.
VI. MATERIALS REQUIRED BUT NOT PROVIDED
1. Borosilicate glass tubes, 12 x 75 mm. (NOTE: Polypropylene or polystyrene tubes may be used if the investigator finds that the pellet formation is acceptably stable in their system.)
2. Borosilicate glass tubes, 12 x 100 mm, or equivalent for sample and standard dilutions
3. 10, 20, 100 and 1000 µl pipettes with disposable tips
4. 100 µl & 1.0 ml repeating dispenser
5. Refrigerated swing bucket centrifuge capable of developing 2,000 - 3,000 xg. (Use of fixed-angle buckets are not recommended.)
6. Absorbent paper
7. Vortex mixer
8. Refrigerator
9. Gamma Counter
VII. SPECIMEN COLLECTION AND STORAGE
1. Sample volumes of at least 5 µl of human serum or plasma can be used (see Sample Preparation, Section VIII. B). Sample volumes of 50 - 100 µl of Tissue Culture Media may also be used.
2. Specimens can be stored at 2-8°C if they will be tested within 24 hours of collection. For longer storage, specimens should be stored at £ -20°C. Avoid multiple freeze/thaw cycles.
3. Avoid using samples with gross hemolysis or lipemia.
For optimal results, accurate pipetting and adherence to the protocol are recommended.
A. Dilute the 10X Assay Buffer with 450 ml distilled or deionized water to prepare working concentration of 1X Assay Buffer.
B. Human Adiponectin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorff pipette, reconstitute the Human Adiponectin Standard with 1 ml distilled or deionized water into the glass vial to give a 200 ng/ml concentration of Standard. Mix well.
2. Label eight glass tubes 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 ng/ml. Add 0.5 ml Assay Buffer to each of the eight tubes. Prepare serial dilutions by adding 0.5 ml of the 200 ng/ml reconstituted standard to the 100 ng/ml tube, mix well and transfer 0.5 ml of the 100 ng/ml Standard to the 50 ng/ml tube, mix well and transfer 0.5 ml of the 50 ng/ml Standard to the 25 ng/ml tube, mix well and transfer 0.5 ml of the 25 ng/ml Standard to the 12.5 ng/ml tube, mix well and transfer 0.5 ml of the 12.5 ng/ml Standard to the 6.25 tube, mix well and transfer 0.5 ml of the 6.25 ng/ml Standard to the 3.125 ng/ml tube, mix well and transfer 0.5 ml of the 3.125 ng/ml Standard to the 1.56 ng/ml tube, mix well and transfer 0.5 ml of the 1.56 ng/ml Standard to the 0.78 ng/ml tube and mix well.
Note: Do not use a Repeater pipette. Change tip for every dilution. Wet tip with Standard before dispensing. Unused portions of standard should be stored at £ -20°C. Avoid multiple freeze/thaw cycles.
| Standard Concentration ng/ml |
Volume of Deionized Water to Add |
Volume of Standard to Add |
| 200 | 1 ml | 0 |
| Standard Concentration ng/ml |
Volume of Assay Buffer to Add |
Volume of Standard to Add |
| 100 | 0.5 ml | 0.5 ml of 200 ng/ml |
| 50 | 0.5 ml | 0.5 ml of 100 ng/ml |
| 25 | 0.5 ml | 0.5 ml of 50 ng/ml |
| 12.5 | 0.5 ml | 0.5 ml of 25 ng/ml |
| 6.25 | 0.5 ml | 0.5 ml of 12.5 ng/ml |
| 3.125 | 0.5 ml | 0.5 ml of 6.25 ng/ml |
| 1.56 | 0.5 ml | 0.5 ml of 3.125 ng/ml |
| 0.78 | 0.5 ml | 0.5 ml of 1.56 ng/ml |
C. Human Adiponectin Quality Control 1 and 2 Preparation
1. Use care in opening the lyophilized Quality Control vials. Using an Eppendorff pipette, reconstitute each of the Human Adiponectin Quality Control 1 and Quality Control 2 with 1 ml distilled or deionized water into the glass vials. Mix well.
D. Sample Preparation
1. Human serum/plasma samples
Circulating concentrations of Adiponectin in human sera are in mg/ml levels, whereas the assay range is 1 – 200 ng/ml. It is advisable to dilute the serum or plasma samples 1:500 prior to use in this Adiponectin. If the values do not fall within the standard curve range, then the dilution should be adjusted appropriately.
RIA. Example to dilute human serum/plasma samples: Pipet 10 µl serum/plasma into 4990 µl Assay Buffer to prepare a 1:500 dilution.
Note: When analyzing diluted samples, it is important to adjust the final result by the appropriate dilution factor to compensate for the dilution of sample.
2. Tissue Culture Medium
50 – 100 µl tissue culture medium per tube is required for analysis. Since Adiponectin levels in tissue culture medium depend on incubation conditions, it is advisable to pilot test for appropriate volume and/or dilution before assaying all the samples.
Use unconditioned tissue culture medium to determine the background or basal level.
E. Assay Set-Up, Day One
1. Pipet 300 µl of Assay Buffer to the Non-Specific Binding (NSB) tubes (3-4), 200 µl to the Reference (Bo) tubes (5-6), and 100 µl to tubes 7 through the end of the assay.
2. Pipet 100 µl of Standards and Quality Controls in duplicate (see flow chart). For preparation, see Section VIII, Part B and C.
3. Pipet 100 µl of each diluted Sample in duplicate. Refer to Section IX for calculation modification of diluted samples.
4. Pipet 100 µl of 125I-Adiponectin to all tubes. Important: For preparation, see Section III, Part C.
5. Pipet 100 µl of Adiponectin antibody to all tubes except Total Count tubes (1-2) and NSB tubes (3-4).
6. Vortex, cover, and incubate overnight (20-24 hours) at room temperature (20-25° C).
F. Day Two
7 Add 10 µl of Rabbit Carrier to all tubes except Total Count tubes (1-2).
8. Add 1.0 ml of cold (4°C) Precipitating Reagent to all tubes except Total Count tubes (1-2).
9. Vortex and incubate 20 minutes at 4°C.
10. Centrifuge, 4°C, all tubes [except Total Count tubes (1-2)] for 20 minutes at 2,000-3,000 xg. NOTE: If less than 2,000 xg is used or if slipped pellets have been observed in previous runs, the time of centrifugation must be increased to obtain a firm pellet (e.g., 40 minutes). Multiple centrifuge runs within an assay must be consistent.
Conversion of rpm to xg:
xg = (1.12 x 10-5) (r) (rpm)2
r = radial distance in cm (from axis of rotation to the bottom of the tube)
rpm = rotational velocity of the rotor
11. Immediately decant the supernate of all tubes except Total Count tubes (1-2), drain tubes for at least 15-60 seconds (be consistent between racks), and blot excess liquid from lip of tubes. NOTE: Invert tubes only one time. Pellets are fragile and slipping may occur.
12. Count all tubes in a gamma counter for 1 minute. Calculate the ng/ml of Adiponectin in unknown samples using automated data reduction procedures (see Section IX).