MULTI-SPECIES LEPTIN RIA
Linco's Multi-Species Leptin Radioimmunoassay (RIA) Kit has been developed to quantitate leptin in plasma or serum from many animals. The antibody used in this kit was raised against Human Leptin but displays broad crossreactivity to leptin molecules of many, but not all, species. It is recommended that investigators determine the suitability of this assay for the analysis of leptin in the species of interest. 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 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 Multi-Species Leptin assay utilizes 125I-labeled Human Leptin and a Multi-Species Leptin antiserum to determine the level of Leptin in serum, plasma or tissue culture media by the double antibody/PEG technique.
Each kit is sufficient to run 250 tubes and contains the following reagents.
A. Assay Buffer
0.05M Phosphosaline, pH 7.4, containing 0.025M EDTA, 0.08% Sodium Azide, 0.05% Triton X-100, and 1% RIA Grade BSA
Quantity: 40 ml/vial
Preparation: Ready to use
B. Antiserum
Guinea Pig anti-Multi-Species Leptin Antibody in Assay Buffer
Quantity: 26 ml/vial
Preparation: Ready to use
C. 125I-Human Leptin
125I-Human Leptin Label, HPLC purified (specific activity 135 mCi/mg).
Lyophilized for stability. Freshly iodinated label contains <3 µCi, (<111 kBq) calibrated to the 1st Monday of each month.
Quantity: 27 ml/vial upon hydration
Preparation: Contents Lyophilized. Hydrate with entire contents of Label Hydrating Buffer. Allow to set at room temperature for 30 minutes, with occasional gentle mixing.
D. Label Hydrating Buffer
Assay Buffer containing Normal Guinea Pig IgG as a carrier. Used to hydrate 125I-Human Leptin
Quantity: 27 ml/vial
Preparation: Ready to use
E. Standards
Purified Recombinant Human Leptin in Assay Buffer at the following concentrations:
1, 2, 5, 10, 20, 50 ng/ml.
Since the Multi Species Leptin antibody was raised against Human Leptin, Human Leptin standards are used in this assay. The crossreactivity of this antibody to leptin molecules of many other species is unknown. Therefore, it is recommended that investigators use ng/ml Human Equivalent (ng/ml HE) as the unit of measure.
Quantity: 1ml/vial
Preparation: Ready to use
F. Quality Controls 1 & 2
Purified Recombinant Human Leptin in Assay Buffer
Quantity: 1 ml each
Preparation: Ready to use
G. Precipitating Reagent
Goat anti-Guinea Pig IgG Serum, 3% PEG and 0.05% Triton X-100 in 0.05M Phosphosaline, 0.025M EDTA,
0.08% Sodium Azide
Quantity: 260 ml/vial
Preparation: Ready to use; chill to 4°C.
Refrigerate all reagents between 2 and 8°C for short term storage. For prolonged storage (>2 weeks), freeze at £ -20°C. Avoid multiple (>5) 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 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 work station 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. 100 µl pipet with disposable tips
3. 100 µl & 1.0 ml repeating dispenser
4. Refrigerated swing bucket centrifuge capable of developing 2,000 - 3,000 xg. (Use of fixed-angle buckets are not recommended.)
5. Absorbent paper
6. Vortex mixer
7. Refrigerator
8. Gamma Counter
VII. SPECIMEN COLLECTION AND STORAGE
1. A maximum of 100 µl per assay tube of serum or plasma can be used, although, 50 µl per assay tube is adequate for most applications. Tissue culture and other media may also be used.
2. Care must be taken when using heparin as an anticoagulant, since an excess will provide falsely high values.4 Use no more than 10 IU heparin per ml of blood collected.
3. Specimens can be stored at 4°C if they will be tested within 24 hours of collection. For longer storage, specimens should be stored at £ -20°C. Avoid multiple (>5) freeze/thaw cycles.
4. Avoid using samples with gross hemolysis or lipemia.
For optimal results, accurate pipetting and adherence to the protocol are recommended.
A. 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 Assay Procedure Flow Chart).
3. Pipet 100 µl of each sample in duplicate. (NOTE: Smaller volumes of sample may be used when leptin concentrations are anticipated to be elevated or when sample size is limited. Additional Assay Buffer must be added to compensate for the difference so the volume is equivalent to 100 µl, e.g., when using 50 µl sample, add 50 µl Assay Buffer.)
4. Pipet 100 µl of Multi Species Leptin antibody to all tubes except Total Count tubes (1-2) and NSB tubes (3-4).
5. Vortex, cover, and incubate overnight (20-24 hours) at 4°C.
B. Day Two
6. Pipet 100 µl of 125I-Human Leptin to all tubes.
7. Vortex, cover, and incubate overnight (20-24 hours) at 4°C.
C. Day Three
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 firmer, more stable pellet (e.g., 40 minutes). A swing-bucket rotor is recommended. Conversion of rpm to xg:
xg = (1.11 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 from all tubes except Total Count tubes (1-2), drain tubes for 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 HE of leptin in unknown samples using automated data reduction procedures (see the following "Calculations" section).