Lispro Insulin RIA
Lispro Insulin (Humalogâ ) is a synthetic human insulin analog in which the amino acid sequence of the B-chain at positions 28 (proline) and 29 (lysine) is inverted. The inversion significantly reduces molecular aggregations. Consequently, Lispro Insulin has a higher rate of absorption, higher peak serum levels and a shorter duration of action than regular human insulin following subcutaneous injection. This procedure describes a competitive-binding radioimmunoassay (RIA) for the quantitation of Lispro Insulin in serum, plasma or other biological fluids. The assay is highly specific for Lispro Insulin and has negligible level of cross-reactivity with native human insulin and proinsulin. This kit is for research purposes only.
In this RIA, Lispro Insulin from either standards or unknown samples compete with 125I-Lispro Insulin for binding sites on guinea pig antibody specific to Lispro Insulin during incubation. The antibody-antigen complex is then precipitated with a second antibody against guinea pig IgG in the presence of polyethylene glycol (PEG) and normal guinea pig serum as carrier. After centrifugation, the resulting pellets are counted for radioactivity in a gamma counter. Quantitation of Lispro Insulin in the unknown samples is achieved by interpolation from the standard curve.
Insulin therapy can result in the generation of anti-insulin antibodies in the treated patient or animal. These antibodies to insulin will cause analytical interference in this assay. An inexpensive and easy means to remove insulin antibodies from unknown samples has been described by Arnqvist et al, (Clin. Chem. 33:93-96, 1987) and is outlined in section VIII, page 10.
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, and 1% RIA Grade BSA
Quantity: 40 ml/vial
Preparation: Ready to use
B. Lispro Insulin Antibody
Guinea Pig anti-Lispro Insulin Serum in Assay Buffer
Quantity: 26 ml/vial
Preparation: Ready to use
C. 125I-Lispro Insulin
125I-Lispro Insulin Label, HPLC purified (specific activity 367 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.
D. Label Hydrating Buffer
Assay Buffer containing Normal Guinea Pig IgG as a carrier. Used to hydrate 125I-Lispro Insulin.
Quantity: 27 ml/vial
Preparation: Ready to use
E. Lispro Insulin Standards
Purified Lispro Insulin in Assay Buffer at the following concentrations:
2.5, 5, 10, 20, 50, 100, and 250 m U/ml
Quantity: 2 ml/vial
Preparation: Ready to use.
F. Matrix Solution
For correction of matrix effect in serum and plasma samples. Solution contains 0.08% Sodium Azide
Quantity : 6.5 ml/vial
Preparation : Ready for use.
G. Quality Controls 1 & 2
Lispro Insulin in QC buffer.
Quantity: 2 ml/vial
Preparation: Ready to use.
H. 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, pH 7.4.
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 easy disposal 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
9. Polyethylene glycol, if removal of insulin antibody from samples is desired.
VII. SPECIMEN COLLECTION AND STORAGE
1. To prepare serum, whole blood is directly collected into a Vacutainerâ serum tube that contains no anticoagulant. Let blood clot at room temperature for 30 minutes.
2. Promptly centrifuge the clotted blood at 2000 to 3000xg for 15 minutes at 4° C.
3. Transfer and store the serum in separate tubes. Date and identify each sample.
4. Store serum samples in lab freezer at –20° C.
5. To prepare plasma, blood should be collected into Vacutainerâ EDTA-plasma tubes and centrifuged immediately after collection.
6. Care must be taken when using heparin as an anticoagulant, since an excess will cause falsely high values. (Thorell and Lanner, 1973). Use no more than 10 IU heparin per ml of blood collected.
Helpful Notes:
1. Before attempting the assay, read and familiarize yourself with assay procedures and select the appropriate assay option.
2. Samples are typically assayed in duplicate, although other multiples may be used to obtain a more accurate average value.
3. For optimal results, accurate pipetting of all reagents and strict adherence to the protocol are recommended.
Assay Options:
Assay Option #1: Serum and plasma without endogenous insulin antibodies can be assayed directly by the following procedures without modification of samples. Insulin antibodies are often formed during therapeutic treatment with insulin. Refer to Flow Chart #1 (Lispro Insulin Assay for Unmodified Samples) for a suggested arrangement of samples and sample volume to be assayed. When assaying a large number of samples, additional quality controls may be included near the middle and at the end of the assay.
Day One
1. Pipette 300 m l Assay Buffer into the Non-Specific Binding (NSB) tubes (#3,4) and 100 m l Assay Buffer into the Reference tubes (#5,6) and sample tubes (#25 through the end).
2. Pipette sequentially 100 m l Standards and Quality Controls in duplicate into tubes #7 to 24.
3. Pipette sequentially 100 m l samples in duplicate into tubes #25 to the end. If sample volume is less than 100 m l, see notes in the Flow Chart.
4. Pipette sequentially 100 m l Matrix Solution-PS into tubes #5 to 24. See notes in the Flow Chart.
5. Pipette 100 m l hydrated Lispro Insulin label into all tubes.
6. Pipette 100 m l Lispro Insulin antibody into all tubes except Total Count tubes (#1,2) and NSB tubes (#3,4).
7. Vortex, cover tubes and incubate for 20-24 hours at room temperature.
Day Two
8. Add 1.0 ml Precipitating Reagent to all tubes except Total Count tubes (#1,2).
9. Vortex and incubate 20 minutes at 4oC.
10. Centrifuge tubes at 3000 xg at 4oC for 25 minutes.
11. Immediately decant supernatant and drain the tubes for 20 to 30 seconds.
12. Count pellets in a gamma counter following the manufacturer’s instructions.
13. Calculate results according to Section IX.
Assay Option #2: When a sample of serum or plasma contains insulin antibodies, the latter have to be removed from the sample prior to the assay. The antibodies can be extracted with polyethylene glycol (PEG) according the following procedures. However, the introduction of PEG to the sample necessitates similar treatment of calibration standards and quality controls with PEG.
Extraction of Calibration Standards, QCs, and Serum or Plasma Samples by PEG
Refer to Flow Chart #2 for a suggested extraction protocol to generate enough material for an assay of duplicate sampling tubes. For assays of samplings higher than duplicates, increase all components proportionally for the extraction. The PEG is not provided in this kit, but can be purchased from other sources. Borosilicate tubes of 12 x 75 mm size can be used as extraction tubes.
1. Dissolve 25 grams of PEG [molecular weight 7000~9000] with 0.9% NaCl solution to a final volume of 100 ml. The 25% PEG is stored at 4 oC and should be used within 2 weeks after preparation.
2. Add 300 m l Assay Buffer to extraction tube #1 and 175 m l to extraction tubes #10 to the end.
3. Add 175 m l of Lispro Insulin Standards, from 5 m U/ml to 250 m U/ml, to extraction tubes #2 to 7, respectively.
4. Add 175 m l QC Low and QC High to extraction tube #8 and 9, respectively.
5. Add 175 m l serum or plasma samples to extraction tubes #10 to the end.
6. Add 300 m l Matrix Solution-PS to extraction tube #1 and 175 m l to extraction tubes #2 to 9.
7. Add 600 m l 25% PEG solution to extraction tube #1 and 350 m l to extraction tubes #2 to the end.
8. Vortex and incubate all extraction tubes at 4 oC for 25 minutes.
9. Centrifuge all extraction tubes in a swinging bucket rotor at 4oC with 2000 to 3000 xg for 25 minutes.
10. Carefully transfer the supernatants to another set of clean empty tubes and discard the pellets. Store supernatants at 4oC until the time of assay.
Assay Lispro Insulin content in the supernatants according to the following protocol. Refer to Flow Chart #3 (Lispro Insulin Assay for PEG Extracted Samples) for a suggested arrangement of samples. When assaying a large number of samples, additional Quality Controls may be included near the middle and at the end of the assay. To accommodate higher amount of QCs required, extract proportionally more QCs with PEG prior to the assay.
Day One
1. Pipette 100 m l Assay Buffer into the Non-Specific Binding (NSB) assay tubes (#3,4).
2. Pipette 200 m l supernatant from extraction tube #1 into NSB assay tubes (#3,4) and into the reference (Bo) assay tubes (#5,6).
3. Pipette 200 m l supernatants in duplicates from extraction tubes #2-9 (PEG extracted Lispro Insulin Standards and QCs) into assay tubes #7-22.
4. Pipette 200 m l supernatants in duplicates from extraction tubes #10 and up (PEG extracted samples) into assay tubes #23 to the end. If sample supernatant needs to be diluted, see notes in the flow chart.
5. Pipette 100 m l hydrated Lispro Insulin label into all assay tubes.
6. Pipette 100 m l Lispro Insulin antibody into assay tubes #5 to the end.
7. Vortex, cover tubes and incubate for 20-24 hours at room temperature.
Day Two
8. Add 1.0 ml Precipitating Reagent to all tubes except Total Count tubes (#1,2)
9. Vortex and incubate 20 minutes at 4oC.
10. Centrifuge tubes at 3000 xg at 4oC for 25 minutes.
11. Immediately decant supernatant and drain the tubes for 20 to 30 seconds.
12. Count pellets in a gamma counter following the manufacturer’s instructions.
13. Calculate results according to Section IX.
Flow Chart #1: Lispro Insulin Assay for Unmodified Samples
[Note: This method is optimized for quantification of Lispro Insulin in unmodified human serum or plasma. To measure Lispro Insulin in serum or plasma samples other than human source, it may be necessary to adjust the amount of Matrix Solution-PS used in the assay. In addition, all sample volumes within an assay should be kept constant. If sample volume is less than 100 m l, choose one of the following alternatives in setting up the assay: 1) make up the deficiency in sample volume from 100 m l with pooled human serum or plasma from healthy non-diabetic subjects with no recent history of Lispro Insulin injection; or 2) use Matrix Solution-PS in tubes 5-24 in a quantity identical to the sample volume used in tubes 25-n and make up the volume deficiency from 100 m l in both with Assay Buffer. The first alternative has to be selected if different sample volumes are to be used among the unknowns within an assay. In both cases, the results obtained should be corrected for the appropriate dilution factor in the assay.]
Flow Chart #2: Polyethylene Glycol Extraction of Standards, QCs, and Serum or Plasma Samples
1. Mix the following components in 12 x 75 mm borosilicate tubes according to the chart below: [Note: Because of the high viscosity of PEG solution, precision in pipetting the 25% PEG solution and PEG extracted samples is crucial to the success of this assay]
|
Extraction |
Assay Buffer |
Standards, QCs, Samples |
Matrix Solution-PS |
25% PEG in |
|
1 |
300 m l |
---- |
300 m l |
600 m l |
|
2 |
---- |
175 m l of 5 m U/ml |
175 m l |
350 m l |
|
3 |
---- |
175 m l of 10 m U/ml |
175 m l |
350 m l |
|
4 |
---- |
175 m l of 20 m U/ml |
175 m l |
350 m l |
|
5 |
---- |
175 m l of 50 m U/ml |
175 m l |
350 m l |
|
6 |
---- |
175 m l of 100 m U/ml |
175 m l |
350 m l |
|
7 |
---- |
175 m l of 250 m U/ml |
175 m l |
350 m l |
|
8 |
---- |
175 m l of QC, Low |
175 m l |
350 m l |
|
9 |
---- |
175 m l of QC, High |
175 m l |
350 m l |
|
10 |
175 m l |
175 m l of Sample # 1 |
---- |
350 m l |
|
11-n |
175 m l |
175 m l of Sample # 2 - end |
---- |
350 m l |
2. Mix well by vortex. Incubate at 4oC for 25 minutes. Centrifuge in a swinging bucket rotor at 4oC and 2000 to 3000 xg for 25 minutes.
3. Carefully transfer the supernatants to clean empty tubes or vials with ID numbers and discard pellets. Assay supernatants per assay instructions.
Flow Chart #3: Lispro Insulin Assay for PEG Extracted Samples
[Note: This method is optimized for quantification of Lispro Insulin in human serum or plasma after PEG extraction. To measure Lispro Insulin in serum or plasma samples other than human source, it may be necessary to adjust the amount of Matrix Solution-PS used in the assay. If PEG extracted samples need to be diluted, use a diluent prepared as follows. To one volume of pooled serum or plasma from healthy non-diabetic subjects add one volume of Assay Buffer and two volumes of 25% PEG dissolved in 0.9% NaCl. Mix well and incubate at 4oC for 20 min. Centrifuge the mixture at 4oC and 3000 xg for 20 min. Collect the supernatant and use as sample diluent.]