Glucagon-Like Peptide-1 (Active) RIA
This kit is for the quantitative determination of the biologically active form of Glucagon-Like Peptide-1[ i.e.
GLP-1(7-36) amide or GLP-1(7-37)] in plasma and other biological media. The GLP-1 sequence is highly conserved between the species, with no sequence variation occurring at all in mammals. The antibody used in this assay binds specifically to the N-terminus of active GLP-1 and does not react with GLP-1(1-36) amide, GLP-1(1-37), GLP-1(9-36) amide, or GLP-1(9-37). Both the standards and tracer are prepared with GLP-1 (7-36) amide. 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 (including any cross reacting substance) 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 primary 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.
Each kit is sufficient to run 125 tubes and contains the following reagents:
A. GLP-1 (Active) Assay Buffer
0.05M Phosphosaline pH 6.8 containing proprietary protease inhibitors, Tween 20, 0.08% Sodium Azide, and 1% RIA grade BSA
Quantity: 25 ml/vial
Preparation: Ready to use
B. GLP-1 (Active) Antibody
Guinea pig anti-GLP-1(7-36) amide Antibody diluted in assay buffer.
Quantity: 13 ml/vial
Preparation: Ready to use
C. 125 I-Glucagon-Like Peptide 1
125 I-GLP-1(7-36) amide Tracer, HPLC purified (specific activity 636 µCi/µg)
Lyophilized for stability. Freshly iodinated tracer 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 13.5 ml of Assay Buffer immediately before use. Store
hydrated tracer frozen at ≤ -20° C.
D. GLP-1 Standards
GLP-1 (7-36) amide in Assay Buffer at the following concentrations: 10, 20, 50, 100, 200, 500 pM.
Quantity: 1 ml/vial
Preparation: Ready to use
E. Quality Controls 1 & 2
Various peptides including GLP-1 (7-36) amide in QC buffer.
Quantity: 2 ml/vial
Preparation: Ready to use
F. Precipitating Reagent
Goat anti guinea pig IgG serum, 3% PEG in 0.05M Phosphosaline, 0.025M EDTA,
0.08% Sodium Azide, 0.05% Triton X-100
Quantity: 130 ml/vial
Preparation: Ready to use; chill to 4°C.
G. Guinea Pig Carrier
Assay Buffer containing normal guinea pig IgG as a carrier.
Quantity: 2 ml/vial
Preparation: Ready to use
H. Sample Hydrating Solution
Proprietary mixture of protease inhibitors
Quantity: 30 ml/vial
Preparation: Ready to use; chill to 4° C.
Freeze all reagents at ≤ -20°C upon receipt and refreeze after use. Avoid multiple (>5) freeze/thaw cycles, prepare aliquots if necessary. 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 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.
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 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 not be used as GLP-1 sticks to these tubes.)
2. 100 µl pipette with disposable tips
3. 10 µl, 100 µl & 1.0 ml repeating dispenser
4. Refrigerated centrifuge with swinging bucket 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. Centrifugal Vacuum Evaporator (e.g. Savant Speed Vac) or nitrogen gas dry-down apparatus.
10. Microcentrifuge Tubes
11. 95% Ethyl Alcohol
12. Deionized Water
13. DPP-IV Inhibitor (Linco Cat # DPP4 recommended)
14. Ice Bath
15. Microcentrifuge
16. Rubber Stoppers with 18 gauge Needles inserted
VII. SPECIMEN COLLECTION AND STORAGE
1. For plasma collection, collect blood in ice-cooled Vacutainer® EDTA-plasma tubes. Immediately (< 30 seconds) after collection, add appropriate amount of DDP-IV inhibitor according to manufacturer’s directions. Invert tube to mix and store tubes in ice bath. (If using Linco Cat # DPP4, add 10 m l DPP-IV inhibitor per milliliter of blood.) Centrifuge immediately at 1000 xg for 10 minutes in refrigerated centrifuge or place tubes on ice and centrifuge within one hour.
2. Care must be taken when using heparin as an anticoagulant, since an excess will provide falsely high values. 6 Use no more than 10 IU of heparin per ml of blood collected.
3. Specimens can be stored at 4°C if they will be tested within 3 hours of collection. For longer storage, specimens should be stored at -70°C. Avoid multiple (>3) freeze/thaw cycles. Aliquot samples before freezing if necessary.
4. Avoid using samples with gross hemolysis or lipemia.
For optimal results, accurate pipetting and adherence to the protocol are recommended.
Sample Extraction Procedure
Note: Due to the low circulating levels of active GLP-1 in plasma, a minimum of 600 µl plasma sample is required for duplicate assay tubes (see Flow Chart).
Keep all assay and extraction reagents (except alcohol) at 4° C before use. Samples must be thawed in a manner to maintain plasma in a cold (4° C) environment at all times. During extraction procedures, samples may be stored in an ice water bath or at 4° C for not longer than 3 hours.
Sample Extraction with Alcohol
Note: Samples are typically extracted and assayed in duplicate although other multiples may be used. Singlets may be used if sample volume is very limited, although this is not recommended.
1. Label microfuge tubes (1.5 ml size) and arrange in an ice bath. Add 1.1 ml of 95% ethyl alcohol in each tube.
2. Add 300 m l plasma sample to each tube. Do not extract less than 300 m l sample volume. Cap tubes tightly, invert tubes and vortex well immediately after the plasma sample has been added.
3. Incubate tubes in an ice bath for 30 minutes.
4. Invert tubes to mix, then centrifuge at 10,000 rpm for 10 minutes in a microfuge.
5. Decant supernatants into glass assay tubes (Borosilicate glass 12x75mm). Stopper the tubes with a rubber stopper with an 18 gauge needle inserted.
6. Place tubes into a Speed VacÒ (centrifugal vacuum evaporator) for dry down. Dry the tubes for 2 hours on medium heat (45° C) then switch to 6 hours at ambient temperature (total of 8 hours). Check all tubes for complete dryness. Remove dried tubes, continue drying partially dried tubes in 30-minute increments until complete. Other methods to dry plasma extract may be used (i.e. nitrogen stream without heat). However, these methods must be validated prior to routine use.
7. Rehydrate samples with 300 m l of cold Sample Hydrating Solution. Incubate 30 minutes on ice or at 4° C. Vortex gently until sample has dissolved into solution (Note: most samples will exhibit some turbidity). The samples are now ready to be assayed directly in these tubes.
Assay Method
Notes:
1. Borosilicate glass tubes (12 x 75 mm) are required for this procedure.
2. Refer to the Assay Procedure Flow Chart for a suggested arrangement of tubes. Quality Control (QC) samples should be included in every assay prior to sample analysis and at the end of all assays
Day One
1. Pipette 400 µl of Assay Buffer in the Total Tubes (1-2) and Non-Specific Binding (NSB) tubes (3-4). Pipette 300 µl of Assay Buffer in the Reference (Bo) tubes (5-6). Pipette 200 µl of Assay Buffer into the standard curve and QC tubes (7-22).
2. Pipette 100 µl of Standards and Quality Controls (Low & High) in duplicate to tubes 7-22.
3. Organize hydrated sample tubes. Pipette 300 µl of each sample in duplicate.
4. Pipette 100 µl of GLP-1 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.
Day Two
6. Hydrate the 125I-GLP-1 tracer with 13.5 ml of Assay Buffer. Gently mix. Pipette 100 µl of 125I-GLP-1 to all tubes. Freeze any unused tracer for future use.
7. Vortex, cover and incubate overnight (22-24 hours) at 4°C.
Day Three
8. Add 10 m l of Carrier IgG to all tubes except Total Count tubes (1-2).
9. Add 1.0 ml of cold Precipitating Reagent to all tubes except Total Count tubes (1-2).
10. Vortex and incubate 20 minutes at 4°C.
11. 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
12. 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.
Calculate results according to Sections IX.
Assay Procedure Flow Chart
|
Day One |
Day Two |
Day Three |
|||||||
|
Set-up |
Step 1 |
Step 2&3 |
Step 4 |
Step 5 |
Step 6 |
Step 7 |
Step 8 |
Step 9 |
Steps 10, 11, & 12 |
|
Tube Number |
Add Buffer |
Add Standard/QC Sample |
Add |
Vortex, Cover, and Incubate 20-24 hrs at 4° C |
Add I-125 GLP-1 Tracer |
Vortex, Cover and Incubate 22-24 hrs at 4° C |
Add Carrier IgG |
Add Precipitating Reagent |
Incubate 20 min. at 4° C, Centrifuge, Decant and Count |
|
1,2 |
400 m l |
- |
- |
100 m l |
- |
- |
|||
|
3,4 |
400 m l |
- |
- |
100 m l |
10m l |
1.0 ml |
|||
|
5,6 |
300 m l |
- |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
7,8 |
200 m l |
100 m l of 10 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
9,10 |
200 m l |
100 m l of 20 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
11,12 |
200 m l |
100 m l of 50 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
13,14 |
200 m l |
100 m l of 100 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
15,16 |
200 m l |
100 m l of 200 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
17,18 |
200 m l |
100 m l of 500 pM |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
19,20 |
200 m l |
100 m l of QC low |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
21,22 |
200 m l |
100 m l of QC high |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
|
23-n |
- |
300 m l of unknown |
100 m l |
100 m l |
10m l |
1.0 ml |
|||
A. Explanation
The calculations for GLP-1 (Active) can be automatically performed by most gamma counters possessing data reduction capabilities or by independent treatment of the raw data using a commercially available software package. Choose weighted 4-parameter or weighted log/logit for the mathematical treatment of the data.
NOTE: Be certain the procedure used subtracts the NSB counts from each average count, except Total Counts, prior to final data reduction. Due to use of 300m l of extracted sample, computer generated data must be divided by 3 to accommodate for the concentration factor.
B. Manual Calculation
1. Average duplicate counts for Total Count tubes (1-2), NSB tubes (3-4), Total Binding tubes (reference, Bo) (5-6), and all duplicate tubes for standards and samples to the end of the assay.
2. Subtract the average NSB counts from each average count (except for Total Counts). These counts are used in the following calculations.
3. Calculate the percentage of tracer bound [(Total Binding Counts/Total Counts) X 100]. This should be 30-40%.
4. Calculate the percentage of total binding (%B/Bo) for each standard and sample
%B/Bo = (Sample or Standard/Total Binding ) X 100
5. Plot the % B/Bo for each standard on the y-axis and the known concentration of the standard on the x-axis using log-log graph paper.
6. Construct the reference curve by joining the points with a smooth curve.
7. Determine the pM of GLP-1 in the unknown samples (unknowns and controls) by interpolation of the reference curve.
NOTE: Due to use of 300m l of extracted sample, manual or computer generated data must be divided by 3 to accommodate for the concentration factor. When sample volumes assayed differ from 300 µl, an appropriate mathematical adjustment must be made to accommodate for the dilution factor.
Acceptance Criteria
1. The run will be considered accepted when all Quality Control Values fall within the calculated Quality Control Range; if any QC’s fall outside the control range, review results with the supervisor.
2. If the difference between duplicate results of a sample is >10% CV, repeat the sample.
3. The limit of sensitivity for the assay is 3 pM (300 µl sample size).
4. The limit of linearity for the assay is 167 pM (300 µl sample size). Any result greater than 167 pM should be repeated on dilution using Assay Buffer as a diluent.
XI. Assay Characteristics
A. Sensitivity
The lowest level of GLP-1 (Active) that can be detected by this assay is 3 pM when using a 300 µl extracted sample size.
B. Performance
The following parameters of assay performance, expressed as mean ± SD, are derived from the results of 9 independent assays.
| ED(80) = | 27 ± 3 pM |
| ED(50) = | 85 ± 6 pM |
| ED(20) = | 253 ± 21 pM |
C. Specificity
The specificity (also known as selectivity) of an analytical test is its ability to selectively measure the analyte in the presence of other like components in the sample matrix.
| GLP-1 (7-36) (Human) |
100% |
| GLP-1 (7-37) (Human) |
100% |
| GLP-1 (9-36) (Human) |
<1% |
| GLP-2 (Human) |
ND |
| Glucagon (Human) |
16% |
| Insulin (Human) |
ND |
| GIP (Human) |
ND |
| Secretin (Human) |
ND |
| VIP (Human, Porcine, Rat) |
ND |
ND-not detectable
D. Precision
Within and Between Assay Variation
|
Sample Number |
Mean pM |
Within % CV |
Between % CV |
|
1 |
14 |
30.3 |
34 |
|
2 |
24 |
21.2 |
34 |
|
3 |
40 |
27.9 |
12 |
Within and between assay variation was performed on three human plasma samples containing varying concentrations of GLP-1. Data shown are from five duplicate determinations for within and ten duplicate determinations for between.
E. Recovery
Extraction Recoveries of QC Samples
|
Sample |
Unextracted Value pM |
Extracted Value pM |
% Recovery |
|
QC 101 |
31 |
17 |
57 |
|
QC 201 |
342 |
255 |
78 |
Quality Control Low & High measured before and after extraction procedure.
Spike and Recovery of GLP-1 (Active) in Human Plasma
|
Human Plasma Sample I |
GLP-1(7-36 amide) Added pM |
Observed pM |
Expected pM |
% Recovery |
|
1 |
0 |
6 |
- |
- |
|
2 |
25 |
14 |
31 |
45 |
|
3 |
50 |
24 |
56 |
43 |
|
4 |
100 |
40 |
106 |
38 |
|
Human Plasma Sample II |
GLP-1(7-36 amide) Added pM |
Observed pM |
Expected pM |
% Recovery |
|
1 |
0 |
4 |
- |
- |
|
2 |
25 |
12 |
29 |
41 |
|
3 |
50 |
21 |
54 |
39 |
|
4 |
100 |
39 |
104 |
38 |
Good Laboratory Practice (GLP) requires that Quality Control (QC) specimens be run with each standard curve to check the assay performance. Two levels of controls are provided for this purpose. These and any other control materials should be assayed repeatedly to establish mean values and acceptable ranges. Each individual laboratory is responsible for defining their system for quality control decisions and is also responsible for making this system a written part of their laboratory manual. The ranges for Quality Control 1 and 2 are provided on the card insert or can be located at the Linco Research website www.lincoresearch.com.
Recommended batch analysis decision using two controls (Westgard Rules):4
1. When both controls are within ±2 SD. Decision: Approve batch and release analyte results.
2. When one control is outside ±2 SD and the second control is within ±2 SD.
Decision: Hold results, check with supervisor. If no obvious source of error is identified by the below mentioned check of systems, the supervisor may decide to release the results.
Technician check of systems:
1. Check for calculation errors
2. Repeat standards and controls
3. Check reagent solutions
4. Check instrument
| Reagents | Cat. # |
| 125I-Glucagon-Like Peptide 1 (<1.5 uCi, <56 kBq) | 9035-HK |
| GLP-1 Standards (1 ml each) | 8035-K |
| GLP-1 (Active) Antibody (13 ml) | 1035-HK |
| Precipitating Reagent (130 ml) | PR-UVHK |
| QC 1 & 2 (2 ml each) | 6016-K |
| GLP-1 (Active) Assay Buffer (25 ml) | AB-GLPHK |
| Guinea Pig Carrier (2 ml) | GPC-HK |
| Sample Hydrating Solution (30 ml) | SHS-GLPHK |
XIV. REFERENCES
1. Nathan DM, Schreiber E, Fogel H, Mojsov S, Hebener JF. Insulinotropic Action of Glucagon-like peptide-1 (7-37) in Diabetic and Nondiabetic Subjects. Diabetes Care 15: 270-276, 1992
2. Morgan, C.R. and Lazarow, A. Immunoassay of Insulin: Two antibody system. Plasma insulin levels in normal, subdiabetic and diabetic rats. Diabetes 12:115-126, 1963
3. Feldman, H. and Rodbard, D. "Mathematical Theory of Radioimmunoassay", in:W.D. Odell and Doughaday, W.H. (Ed.) Principles of Competitive Protein-Binding Assays Philadelphia: J.B. Leppincott Company; pp 158-203, 1971
4. Westgard, J.O., et. al. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem 27:493-501, 1981
5. Holst JJ, Cathrin Orskov, Bolette Hartmann, Carolyn F. Deacon: Postranslational processing of proglucagon and postsecretory fate of proglucagon products; in Fehmann HC, Goke B (eds) : The Insulinotropic Gut Hormone Glucagon-Like Peptide-1. Front Diabetes. Basel, Karger, 1997, vol 13, pp 24-4