Intended Use
This Sensitive Human Leptin Radioimmunoassay (RIA) Kit utilizes an antibody made specifically against Human Leptin. Sensitivity of 0.05 ng/ml can be achieved when using a 100 µl sample of serum, plasma, or tissue culture media, in a two-day, disequilibrium assay (400 µl Total Volume). This kit is for research purposes only.

Principles Of Procedure
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 Sensitive Human Leptin assay utilizes 125I-labeled Human Leptin and a Sensitive Human Leptin antiserum to determine the level of Leptin in serum, plasma or tissue culture media by the double antibody/PEG technique.

Reagents Supplied

Each kit is sufficient to run 250 tubes and contains the following reagents.

  1. Assay buffer
    0.05M Phosphosaline pH 7.4 containing 0.025M EDTA, 0.08% Sodium Azide, 1% RIA grade BSA and 0.05% Triton X-100
    Quantity: 40 ml/vial
    Preparation: Ready to use
  2. Antiserum
    Guinea Pig anti-Sensitive Human Leptin Serum in Assay Buffer
    Quantity: 26 ml/vial
    Preparation: Ready to use
  3. 125I-Human Leptin
    125 I-Human Leptin Label, HPLC purified (specific activity 135 µCi/µg)
    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 sit at room temperature for 30 minutes, with occasional gentle mixing
  4. 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
  5. Standards
    Purified Recombinant Human Leptin in Assay Buffer at the following concentrations:
    0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 ng/ml
    Quantity: 1 ml/vial
    Preparation: Ready to use
  6. Quality Controls 1 and 2
    Purified Recombinant Human Leptin in Assay Buffer
    Quantity: 1 ml/vial
    Preparation: Ready to use
  7. 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.

Storage And Stability

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.

Reagent Precautions

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 there from, to human beings or animals. Its receipt, acquisition, possession, use and transfer are subject to the regulations of the U. S. Nuclear Regulatory Commission 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 customers Radiation Safety Officer (RSO) is ultimately responsible for the safe handling and use of radioactive material.

      1. Wear appropriate personnel 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 RSO.

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.

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

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. 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 hrs. 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.

Assay Procedure

For optimal results, accurate pipetting and adherence to the protocol are recommended.

Day One

  1. Pipet 300 µl of Assay Buffer to the Non-Specific Binding (NSB) tubes (3-4), 200 µl to 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).
  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 should be added to compensate for the difference so that the volume is equivalent to 100 µl, e.g., when using 50 µl of sample, add 50 µl of buffer).
  4. Pipet 100 µl of Sensitive Human 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 room temperature.

    Day Two

  6. Pipet 100 µl of 125I-Human Leptin tracer to all tubes.
  7. Vortex, cover, and incubate overnight (22-24 hours) at room temperature.

    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, at 4°C, for 20 minutes at 2,000-3,000 xg. Note: If less than 2,000 xg is used, 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 = revolutions per minute
  11. Immediately decant supernatant from all centrifuged 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 of Sensitive Human Leptin in unknown samples using automated data reduction procedures.