Human C-Peptide RIA
Linco’s Human C-Peptide radioimmunoassay kit is for the quantitative determination of Human C-Peptide in serum, plasma, and other tissue culture media. It is a completely homologous assay since the antibody was raised against purified Human C-Peptide and both the tracer and the standard are prepared with Human C-Peptide. This assay has a very low crossreactivity (<4.0%) to Human Proinsulin. 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 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, Inc. Human C-Peptide assay utilizes 125I-labeled Human C-Peptide and a Human C-Peptide antiserum to determine the level of C-Peptide 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, 1% RIA Grade BSA
Quantity: 40 ml/vial
Preparation: Ready to use
B. Human C-Peptide Antibody
Guinea Pig anti-Human C-Peptide Antibody in Assay Buffer
Quantity: 26 ml/vial
Preparation: Ready to use
C. 125 I-Human C-Peptide
125 I-Human C-Peptide Label, HPLC purified (specific activity 658 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 set at room temperature for 30 minutes, with occasional gentle mixing.
D. Label Hydrating Buffer
Assay Buffer containing Normal Guinea Pig Serum as a carrier. Used to hydrate 125I-Human C-Peptide.
Quantity: 27 ml/vial
Preparation: Ready to use
E. Human C-Peptide Standards
Purified Human C-Peptide in Assay Buffer at the following concentrations: 0.1, 0.2, 0.5, 1, 2, 5 ng/ml
Quantity: 2 ml/vial
Preparation: Ready to use
F. Quality Controls 1 & 2
Purified Human C-Peptide in Assay Buffer
Quantity: 1 ml/vial
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 (RSO) 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.2 Use no more than 10 IU heparin per ml of blood collected.
3. Human C-Peptide must be protected from proteolysis during assay procedures and sample storage. Trasylol (Aprotinin) at a concentration of 500 KIU per ml of serum or plasma should be added to samples to protect from proteolysis.
For unprotected samples there is a loss of approximately 25% HCP after 3 months storage at £ -20°C. No loss was observed when unprotected samplew were stored at £ -70°C for up to 12 months.
4. 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.
5. Avoid using samples with gross hemolysis or lipemia.
For optimal results, accurate pipetting and adherence to the protocol are recommended.
A. Day One
1. Pipet 200 µl of Assay Buffer to the Non-Specific Binding (NSB) tubes (3-4) and, 100 µl to Reference (Bo) tubes (5-6).
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 Human C-Peptide 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. Pipet 100 µl of 125I-Human C-Peptide to all tubes. Important: For preparation, see Section III, Part C.
5. Pipet 100 µl of Human C-Peptide antibody to all tubes except Total Count tubes (1-2) and NSB tubes (3-4).
6. Vortex, cover, and incubate overnight (20-24 hours) at 4°C.
B. Day Two
7. Add 1.0 ml of cold (4°C) Precipitating Reagent to all tubes (except Total Count tubes).
8. Vortex and incubate 20 minutes at 4°C.
9. 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 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
10. Immediately decant the supernate of all tubes except Total Count tubes (1-2), drain tubes for at least 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.
11. Count all tubes in a gamma counter for 1 minute. Calculate the ng/ml of Human C-Peptide in unknown samples using automated data reduction procedures.
Assay Procedure Flow Chart
|
Day One |
Day Two |
|||||||
|
Set-up |
Step 1 |
Steps 2 & 3 |
Step 4 |
Step 5 |
Step 6 |
Step 7 |
Step 8 |
Steps 9-11 |
|
Tube Number |
Add |
Add Standard / QC Sample |
Add I-125 Human C-Peptide Tracer |
Add Human C-Peptide Antibody |
Vortex, Cover, and Incubate 20-24 hrs at 4° C |
Add Precipitating Reagent |
Vortex, cover, and Incubate 20 min. at 4° C |
Centrifuge at 4° C for 20 min., Decant, and Count |
|
1,2 |
- |
- |
100 m l |
- |
- |
|||
|
3,4 |
200 m l |
- |
100 m l |
- |
1.0 ml |
|||
|
5,6 |
100 m l |
- |
100 m l |
100 m l |
1.0 ml |
|||
|
7,8 |
- |
100 m l of 0.1 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
9,10 |
- |
100 m l of 0.2 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
11,12 |
- |
100 m l of 0.5 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
13,14 |
- |
100 m l of 1.0 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
15,16 |
- |
100 m l of 2.0 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
17,18 |
- |
100 m l of 5.0 ng/ml |
100 m l |
100 m l |
1.0 ml |
|||
|
19,20 |
- |
100 m l of QC 1 |
100 m l |
100 m l |
1.0 ml |
|||
|
21,22 |
- |
100 m l of QC 2 |
100 m l |
100 m l |
1.0 ml |
|||
|
23-24 |
- |
100 m l of unknown |
100 m l |
100 m l |
1.0 ml |
|||
|
25,n |
- |
100 m l of unknown |
100 m l |
100 m l |
1.0 ml |
|||
A. Explanation
The calculations for Human C-Peptide 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.]
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 35-50%.
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 ng/ml of Human C-Peptide in the unknown samples (unknowns and controls) by interpolation of the reference curve.
NOTE: When sample volumes assayed differ from 100 µl, an appropriate mathematical adjustment must be made to accommodate for the dilution factor (e.g. if 50 µl of sample is used, then calculated data must be multiplied by 2).
A. 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 Human C-Peptide assay is 0.1 ng/ml (100 µl sample size).
4. The limit of linearity for the Human C-Peptide assay is 5.0 ng/ml (100 µl sample size). Any result greater than 5.0 ng/ml should be repeated on dilution using Assay Buffer as a diluent.
Normal fasting range:
0.5-1.5 ng/ml
A. Sensitivity
The lowest level of C-Peptide that can be detected by this assay is 0.1 ng/ml when using a 100 µl sample size.
B. Performance
The following parameters of assay performance are expressed as Mean + Standard Deviation.
ED80 = 0.3 ± 0.1 ng/ml
ED50 = 1.2 ± 0.2 ng/ml
ED20 = 4.9 ± 0.5 ng/ml
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.
| Human C-Peptide |
100% |
| Monkey C-Peptide |
90% |
| Human Proinsulin |
<4% |
| Human Insulin |
ND |
| Glucagon |
ND |
| Somatostatin |
ND |
| Pancreatic Polypeptide |
ND |
| Rat C-Peptide |
ND |
| Porcine C-Peptide |
ND |
ND - not detectable
D. Precision
Within and Between Assay Variation
|
Sample |
Mean |
Within |
Between |
|
1 |
0.4 |
3.4 |
9.3 |
|
2 |
0.9 |
4.4 |
4.4 |
|
3 |
1.8 |
6.4 |
4.2 |
|
4 |
2.1 |
4.2 |
3.2 |
|
5 |
2.8 |
4.5 |
2.4 |
Within and between assay variation was performed on five human serum samples containing varying concentrations of Human C-Peptide. Data (mean and % CV) shown are from five duplicate determinations of each serum sample in five separate assays.
E. Recovery
Spike and Recovery of C-Peptide in Human Serum
| Sample No. |
Human C-Peptide |
Observed ng/ml |
Expected ng/ml |
% Recovery |
| 1 | 0 | 0.39 | - | - |
| 2 | 0.25 | 0.64 | 0.64 | 100 |
| 3 | 0.50 | 0.90 | 0.89 | 101 |
| 4 | 1.00 | 1.37 | 1.39 | 99 |
| 5 | 2.50 | 2.76 | 2.89 | 96 |
Varying concentrations of Human C-Peptide were added to five human serum samples and the Human C-Peptide content was determined by RIA. Mean of the observed levels from five duplicate determinations in five separate assays are shown. Percent recovery was calculated on the observed vs. expected.
F. Linearity
Effect of Serum Dilution
|
Sample |
Volume |
Observed |
Expected |
% Of Expected |
|
1 |
100 µl | 2.8 | 2.8 | 100 |
| 75 µl | 2.8 | 100 | ||
| 50 µl | 2.7 | 99 | ||
| 25 µl | 2.5 | 92 | ||
|
2 |
100 µl | 2.1 | 2.1 | 100 |
| 75 µl | 2.0 | 98 | ||
| 50 µl | 2.0 | 97 | ||
| 25 µl | 1.9 | 89 | ||
|
3 |
100 µl | 1.8 | 1.8 | 100 |
| 75 µl | 1.7 | 96 | ||
| 50 µl | 1.7 | 94 | ||
| 25 µl | 1.5 | 83 | ||
|
4 |
100 µl | 1.8 | 1.8 | 100 |
| 75 µl | 1.8 | 99 | ||
| 50 µl | 1.8 | 99 | ||
| 25 µl | 1.6 | 90 | ||
Aliquots of pooled human serum containing varying concentrations of Human C-Peptide were analyzed in the volumes indicated. Dilution factors of 1, 1.33, 2 and 4 representing 100 µl, 75 µl, 50 µl and 25 µl, respectively, were applied in calculating observed concentrations. Mean Human C-Peptide levels and percent of expected for five separate assays are shown.
G. Example of Assay Results
This data is presented as an example only and should not be used in lieu of a standard curve prepared with each assay.
|
Tube # |
ID |
CPM |
Ave CPM |
Ave Net CPM |
% B/Bo |
ng/ml |
|
1 |
Totals |
16751 |
||||
|
2 |
² |
17014 |
16883 |
- |
- |
- |
|
3 |
NSB |
378 |
||||
|
4 |
² |
388 |
383 |
- |
- |
- |
|
5 |
Bo |
8815 |
||||
|
6 |
² |
8692 |
8754 |
8371 |
- |
- |
| Standards | ||||||
|
7 |
0.1 ng/ml |
8037 |
||||
|
8 |
8079 |
8058 |
7675 |
91.7 |
- |
|
|
9 |
0.2 ng/ml |
6977 |
||||
|
10 |
6609 |
6793 |
6410 |
76.6 |
- |
|
|
11 |
0.5 ng/ml |
6128 |
||||
|
12 |
5692 |
5910 |
5527 |
66.0 |
- |
|
|
13 |
1 ng/ml |
4458 |
||||
|
14 |
4392 |
4425 |
4042 |
48.3 |
- |
|
|
15 |
2 ng/ml |
3219 |
||||
|
16 |
3069 |
3143 |
2760 |
33.0 |
- |
|
|
17 |
5 ng/ml |
1788 |
||||
|
18 |
1772 |
1780 |
1397 |
16.7 |
- |
|
| Controls/Unknown | ||||||
|
19 |
QC 1 |
5962 |
||||
|
20 |
5928 |
5945 |
5562 |
66.4 |
.45 |
|
|
21 |
QC 2 |
2664 |
||||
|
22 |
2672 |
2668 |
2285 |
27.3 |
2.63 |
|
|
23-n |
unknown |
|||||
QUALITY CONTROLS
Good Laboratory Practice (GLP) requires that Quality Control 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 Rule)4:
1. When both controls are within ±2 SD. Decision: Approve batch and release analytical 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-Human C-Peptide (<3 uCi, 111 kBq) | 9020 |
| Label Hydrating Buffer (27ml) | LHB-P |
| Human C-Peptide Standards (2 ml each) | 8020-K |
| Human C-Peptide Antibody (26 ml) | 1020-K |
| Precipitating Reagent (260 ml) | PR-UV |
| QC 1&2 (1 ml each) | 6000-K |
| Assay Buffer (40 ml) | AB-P |
1. 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.
2. Thorell, J.I. Scand. J. Clin. Lab. Invest. 31:187, 1973.
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.