GHRELIN (Active) RIA
Linco's Ghrelin (Active) Radioimmunoassay (RIA) Kit utilizes an antibody, which is specific for the biologically active form of ghrelin with the octanoyl group on Serine 3. Sensitivity of 7.8 pg/ml can easily be achieved when using a 100µl serum or plasma sample in a two-day, disequilibrium assay (400 µl Total Volume). 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, Inc. Ghrelin (Active) assay utilizes 125I-labeled Ghrelin and a Ghrelin antiserum to determine the level of active Ghrelin in serum, plasma or tissue culture media by the double antibody/PEG technique.
Each kit is sufficient to run 125 tubes and contains the following reagents.
A. Ghrelin (Active) Assay Buffer
0.05M Phosphate, 0.025M EDTA, 0.08% Sodium Azide and 0.1% Gelatin, pH 6.85
Quantity: 20 ml/vial
Preparation: Ready to use
B. Ghrelin (Active) Antibody
Guinea Pig anti-Ghrelin Serum in Assay Buffer
Quantity: 13 ml/vial
Preparation: Ready to use
C. 125I-Ghrelin
125I-Ghrelin Label, HPLC purified (specific activity 302 µCi/µg)
Lyophilized for stability. Freshly iodinated label contains <1.5 µCi (56 kBq), calibrated to the 1st Monday of each month.
Quantity: 13.5 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. Freeze remaining label for future use.
D. Ghrelin (Active) Label Hydrating Buffer
Assay Buffer containing 0.025% Triton-X 100 and Normal Guinea Pig IgG as a carrier. Used to hydrate 125I-Ghrelin
Quantity: 13.5 ml/vial
Preparation: Ready to use
E. Ghrelin (Active) Standard (lyophilized)
Lyophilized standard containing Ghrelin in sodium phosphate buffer containing a non-mercury preservative.
Preparation: Contents Lyophilized. Reconstitute with 2 mL distilled or deionized water. The actual concentration of Ghrelin present in the vial will be lot-dependent. Please refer to the analysis sheet for exact Ghrelin concentration present in a specific lot.
F. Ghrelin (Active) Quality Controls 1 and 2 (lyophilized)
One vial each, lyophilized, containing Ghrelin at two different levels.
Preparation: Contents Lyophilized. Reconstitute with 1 mL distilled or deionized water.
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: 130 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. Unused reconstituted last Standard and Quality Controls should be aliquotted and stored at £ -20°C.
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 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 (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 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. 100 µl pipette with disposable tips
3. 10 µl, 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 is not recommended.)
5. Absorbent paper
6. Vortex mixer
7. Refrigerator
8. Gamma Counter
9. 1N HCl (recommended in SPECIMEN COLLECTION AND STORAGE section)
10. Phenylmethylsulfonyl fluoride (PMSF) (recommended in SPECIMEN COLLECTION AND STORAGE section) can be dissolved in 100% methanol or isopropanol.
VII. SPECIMEN COLLECTION AND STORAGE
The active form of the Ghrelin molecule is very unstable and labile in serum/plasma due to the nature of the octanoyl group on serine-3.
Samples should be processed as quickly as possible and kept on ice to retard the breakdown of active Ghrelin. We recommend acidification of the plasma with 50 µl of 1 N HCl and addition of 10 µl of Phenylmethylsulfonyl fluoride (PMSF) per one ml of plasma. Addition of acid may cause a precipitation of some serum proteins but does not affect the assay. This precipitation may be removed by centrifugation if desired.
Note: It is essential to prepare fresh solution of PMSF in 100% methanol or isopropanol at a concentration of 10 mg/ml before addition to serum/plasma.
1. A maximum of 100 µl per assay tube of serum or plasma (plasma is preferred) should 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 excess will provide falsely high values. Use no more than 10 IU heparin per ml of blood collected.
3. Specimens can be stored at 4ºC if they will be tested within 4 hours. For longer storage, specimens should be aliquot and stored at £ - 20ºC or below. Multiple freeze/thaw cycles should be avoided since each freeze/thaw cycle will reduce results.
4. Avoid using samples with gross hemolysis or lipemia.
VIII. STANDARD AND QUALITY CONTROLS PREPARATION
Active Ghrelin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Active Ghrelin Standard with 2 mL distilled or deionized water to give a concentration prescribed in the analysis sheet. Invert and mix gently, let sit for 5 minutes or until completely dissolved then mix well.
2. Label eight tubes 1, 2, 3, 4, 5, 6, 7 and 8. Add 0.5 mL Assay Buffer to each of the eight tubes. Prepare serial dilutions by adding 0.5 mL of the reconstituted standard to tube 1, mix well and transfer 0.5 mL of tube 1 to tube 2, mix well and transfer 0.5 mL of tube 2 to tube 3, mix well and transfer 0.5 mL of tube 3 to tube 4, mix well and transfer 0.5 mL of tube 4 to tube 5, mix well and transfer 0.5 mL of tube 5 to tube 6, mix well and transfer 0.5 mL of tube 6 to tube 7, mix well and transfer 0.5 mL of tube 7 to tube 8 and mix well.
Note: Do not use a Repeater pipette. Change tip for every dilution. Wet tip with Standard before dispensing. Unused portions of the reconstituted standard should be aliquotted and stored at £ -20°C. Avoid multiple freeze/thaw cycles.