GHRELIN (Total) RIA
Linco's Ghrelin (Total) Radioimmunoassay (RIA) Kit utilizes an antibody which is specific for total ghrelin and does not require the presence of the octonyl group on Serine 3. Sensitivity of 93 pg/ml can easily be achieved when using a 100 µl 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 Ghrelin (Total) assay utilizes 125I-labeled Ghrelin and a Ghrelin antiserum to determine the level of Total 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 (Total) Assay Buffer
0.01M Phosphate, 0.01M EDTA, 0.08% Sodium Azide and 0.1% Gelatin, pH 8.5
Quantity: 20 ml/vial
Preparation: Ready to use
B. Ghrelin (Total) Antibody
Rabbit 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. On the day the tracer is added to the assay, hydrate with entire contents of Label Hydrating Buffer. Allow to set at room temperature for 30 minutes, with occasional gentle mixing. Immediately freeze remaining label for future use.
D. Ghrelin (Total) Label Hydrating Buffer
Assay Buffer containing 0.025% Triton-X 100 and Normal Rabbit IgG as a carrier. Used to hydrate 125I-Ghrelin
Quantity: 13.5 ml/vial
Preparation: Ready to use
E. Ghrelin (Total) 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 (Total) 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-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
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 thereof 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. 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 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 (plasma is preferred) 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 4ºC if they will be tested within 4 hours. For longer storage, specimens should be aliquoted and stored at £ - 20ºC or below. Multiple freeze/thaw cycles should be avoided since each freeze/thaw may reduce results.
4. Avoid using samples with gross hemolysis or lipemia.
VIII. STANDARD AND QUALITY CONTROLS PREPARATION
Total Ghrelin Standard Preparation
1. Use care in opening the lyophilized Standard vial. Using an Eppendorf pipette, reconstitute the Total 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 six tubes 1, 2, 3, 4, 5, and 6. Add 0.5 mL Assay Buffer to each of the six 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 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.
| Volume of Deionized Water to Add |
Volume of Standard to Add |
Standard Concentration pg/mL |
|
| 2 mL | 0 | X (Refer to analysis sheet for exact concentration) |
|
Tube # |
Volume of Assay Buffer to Add |
Volume of Standard to Add |
Standard Concentration pg/mL |
| 1 | 0.5 mL | 0.5 mL of reconstituted standard |
X/2 |
| 2 | 0.5 mL | 0.5 mL of tube 1 | X/4 |
| 3 | 0.5 mL | 0.5 mL of tube 2 | X/8 |
| 4 | 0.5 mL | 0.5 mL of tube 3 | X/16 |
| 5 | 0.5 mL | 0.5 mL of tube 4 | X/32 |
| 6 | 0.5 mL | 0.5 mL of tube 5 | X/64 |
Total Ghrelin Quality Control 1 and 2 Preparation
1. Use care in opening the lyophilized Quality Control vials. Using an Eppendorf pipette, reconstitute each of the Total Ghrelin Quality Control 1 and Quality Control 2 with 1 mL distilled or deionized water. Invert and mix gently, let sit for 5 minutes then mix well.
Note: For exact concentration of Quality Control 1 and 2, refer to Analysis Sheet. Unused portions of the reconstituted Quality Controls should be stored at £ -20°C. Avoid multiple freeze/thaw cycles.
For optimal results, accurate pipetting and adherence to the protocol are recommended.
Day One
1. Pipette 300 µl of Assay Buffer to the Non-Specific Binding (NSB) tubes (3-4). Pipette 200 µl of Assay Buffer in the Reference (Bo) tubes (5-6). Pipette 100 µl of Assay Buffer to tubes seven through the end of the assay.
2. Pipette 100 µl of Standards and Quality Controls in duplicate (see assay flow chart).
3. Pipette 100 µl of each sample in duplicate. (NOTE: Smaller volumes of sample may be used when Ghrelin 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 Assay Buffer). Refer to Section IX for calculation modification.
4. Pipette 100 µl of Ghrelin 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 4ºC.
Day Two
6. Hydrate the 125I-Ghrelin tracer with 13.5 ml of Label Hydrating Buffer. Gently mix. Pipette 100 µl of 125I-Ghrelin to all tubes.
7. Vortex, cover and incubate overnight (22-24 hours) at 4ºC.
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), blot excess liquid from lip of tubes and count pellet using the gamma counter according to the manufacturer's instructions.