DRG IGFBP-3 IRMA Growth Factors Instruction Manual

DRG IGFBP-3 IRMA Growth Factors Instruction Manual

DRG Instruments GmbH, Germany

Frauenbergstraße. 18, 35039 Marburg
Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50 Website: www.drg- diagnostics.de
E-mail: drg@drg-diagnostics.de

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081 Phone: 973-564-7555, Fax: 973-564-7556 Website: www.drg- international.com
E-mail: corp@drg-international.com

Please use only the valid version of the Instructions for Use provided with the kit.

1. INTENDED USE

Immunoradiometric assay kit for the in vitro quantitative determination of the Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) in serum.

2. CLINICAL BACKGROUND

The insulin-like growth factor (IGF) system is the primary regulator of normal body growth and regeneration, affecting cell proliferation, differentiation and apoptosis. In addition, the IGF-system appears to modify insulin sensitivity and long-term glucose metabolism. Finally, numerous epidemiological, experimental and clinical data indicate that the IGF-system is also involved in the development of several common cancers as well as frequent diseases such as atherosclerosis and type 2 diabetes mellitus.

The IGF-system consists of a family of closely related peptides, which includes the two primary growth promoting peptides, IGF-I and IGF-II, 6 specific high-affinity IGF-binding proteins (IGFBP-1 to -6), and a large non- IGF-binding glycoprotein, the “acid labile subunit” (ALS).

IGFBP-3 is the most abundant IGF-binding protein, accounting for as much as 75% or more of the circulating IGF-binding capacity in healthy subjects. IGFBP-3 shares functional properties with IGFBP-5 in that both peptides are able to form high molecular weight ternary complexes of ~150 kilo Dalton with ALS and either IGF-I or –II. However, IGFBP-5 circulates in much lower concentrations than IGFBP-3, and in healthy subjects the ternary complexes carry as much as 90% of IGFBP-3 but only about 50% of IGFBP-5.
Originally, the IGFBPs were thought to serve as IGF-carrier proteins, stabilizing plasma IGF levels and controlling the egress of IGF from the circulation to the extra-vascular compartment. Furthermore, it was assumed that IGFBP-complexed IGF was biologically more or less inactive, being deprived its ability to interact with the IGF-I receptor. However, it soon became apparent that in some experimental settings the IGFBPs stimulated rather than inhibited IGF-I mediated actions, and accordingly, the IGFBPs are now often referred to as modulators of IGF-I bioactivity. In addition, the majority of the IGFBPs, and in particular IGFBP-3, exerts IGF-I and IGF-I receptor independent effects, possible involving interactions with specific receptors located at the cell surface and intracellular. For example, IGFBP-3 is nowadays considered to serve as an anti-cancer molecule, apparently protecting against several common cancers, and effects of IGFBP-3 on insulin signaling in cultured adipocytes have also been suggested.

The turnover of the ternary complexes is very slow, and the plasma concentration of IGFBP-3 remains stable throughout the day, being unaffected by short-term nutritional changes. Thus, the level of IGFBP-3 may be determined by one single measurement. GH is the primary regulator of IGFBP-3 as well as of IGF-I and ALS and therefore, all three peptides increase during the pubertal growth spurt, where after levels gradually decline with increasing age. In children, IGFBP-3 has been shown to correlate with the 24-h integrated GH secretion and in particular in children IGFBP-3 may be helpful in the diagnosis of GH deficiency.

3. PRINCIPLES OF THE METHOD

The IGFBP-3 IRMA is an immunoradiometric assay based on coated-tube. Mab1, the capture antibody, is attached to the lower and inner surface of the plastic tube. Calibrators or samples added to the tubes will at first show low affinity for Mab1. Addition of Mab2, the signal antibody labelled with 125I, will complete the system and trigger the immunological reaction. After washing, the remaining radioactivity bound to the tube reflects the antigen concentration.

4. REAGENTS PROVIDED

Note: use the dilution buffer as zero calibrator.

5. SUPPLIES NOT PROVIDED

The following material is required but not provided in the kit:

1. Distilled water
2. Pipettes for delivery of: 10 µL, 50 µL, 100 µL and 1 mL (the use of accurate pipettes with disposable plastic tips is recommended)
3. Plastic tubes for dilution of samples
4. Vortex mixer
5. Tube shaker (400 rpm)
6. Magnetic stirrer
7. 5 mL automatic syringe (Cornwall type) for washing
8. Aspiration system (optional)
9. Any gamma counter capable of measuring 125I may be used (minimal yield 70%).

6. REAGENT PREPARATION

A. Calibrators:
Reconstitute the calibrators with 1.0 mL distilled water.
B. Controls:
Reconstitute the controls with 1.0 mL distilled water.
C. Working Wash solution:
Prepare an adequate volume of Working Wash solution by adding 69 volumes of distilled water to 1 volume of Wash Solution (70x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day.

7. STORAGE AND EXPIRATION DATING OF REAGENTS

• Before opening or reconstitution, all kits components are stable until the expiry date, indicated on the label, if kept at 2 °C to 8 °C.
• The calibrators and controls are very unstable, use them immediately after reconstitution, freeze immediately in aliquots and keep them at -20 °C for maximum 3 months.
• Avoid subsequent freeze-thaw cycles.
• Freshly prepared Working Wash solution should be used on the same day.
• After its first use, tracer is stable until expiry date, if kept in the original well-closed vial at 2 °C to 8 °C.
• Alterations in physical appearance of kit reagents may indicate instability or deterioration.

8. SPECIMEN COLLECTION AND PREPARATION

Serum samples must be kept at 2 °C to 8 °C.
If the test is not run within 24 h., storage at -20 °C is recommended.
Avoid subsequent freeze-thaw cycles.

9. PROCEDURE

9.1 Handling notes

• Do not use the kit or components beyond expiry date.
• Do not mix materials from different kit lots.
• Bring all the reagents to room temperature prior to use.
• Thoroughly mix all reagents and samples by gentle agitation or swirling.
• Use a clean disposable pipette tip for addition of each different reagent and sample in order to avoid cross- contamination. High precision pipettes or automated pipetting equipment will improve the precision.
• Respect the incubation times.
• Prepare a calibration curve for each run, do not use data from previous runs.

9.2 Procedure

1. Label one plain plastic tube for each sample.
2. Dispense 1 mL of Dilution Buffer into each tube.
3. Add 10 μL of sample into these tubes.
4. Label coated tubes in duplicate for each calibrator, sample and control. For the determination of total counts, label 2 normal tubes.
5. Briefly vortex calibrators, controls and pre-diluted samples and dispense 100 μL of each into the respective tubes (use the dilution buffer as zero calibrator).
6. Dispense 50 μL of 125Iodine labelled anti IGFBP-3 into each tube, including the uncoated tubes for total counts.
7. Incubate for 120 minutes at room temperature on a tube shaker (400 rpm).
8. Aspirate (or decant) the content of each tube (except total counts). Be sure that the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.
9. Wash tubes with 2 mL Working Wash solution (except total counts) and aspirate (or decant). Avoid foaming during the addition of the Working Wash solution.
10. Wash tubes again with 2 mL Wash solution (except total counts) and aspirate (or decant).
11. Let the tubes stand upright for two minutes and aspirate the remaining drop of liquid.
12. Count tubes in a gamma counter for 60 seconds.

10. CALCULATION OF RESULTS

1. Calculate the mean of duplicate determinations.
2. On semi logarithmic or linear graph paper plot the c.p.m. (ordinate) for each calibrator against the corresponding concentration of IGFBP-3 (abscissa) and draw a calibration curve through the calibrator points, reject the obvious outliers.
3. Read the concentration for each control and sample by interpolation on the calibration curve.
4. Computer assisted data reduction will simplify these calculations. If automatic result processing is to be used, a 4-parameter logistic function curve fitting is recommended

11. TYPICAL DATA

The following data are for illustration only and should never be used instead of the real time calibration curve.

The calibrators are standardized against the NIBSC/WHO recombinant IGFBP-3, reference reagent coded 93/560.

12 PERFORMANCE AND LIMITATIONS

12.1 Detection limit
Twelve zero calibrators were assayed along with a set of the other calibrators. The detection limit, defined as the apparent concentration of the average count at zero binding plus two standard deviations, was 17.3 ng/mL.

12.2 Specificity
Some potentially interfering hormones have been tested in this assay. At concentrations up to 10 μg/mL, none of the following hormones showed significant interference:

– rhIGF-BP1
– rhIGF-BP2
– rhIGF-BP4
– rhIGF-BP5
– rhIGF-BP6
– rhIGF-I
– rhIGF-II

12.3 Precision

SD: Standard Deviation; CV: Coefficient of variation

12.4 Accuracy

Samples were diluted with Dilution Buffer.

RECOVERY TEST

12.5 Time delay between last calibrator and sample dispensing
As shown hereafter, assay results remain accurate even when a sample is dispensed 30 minutes after the calibrator has been added to the coated tubes.

13. LIMITATIONS

• Specimens from patients who have received preparations of mouse monoclonal antibodies for diagnosis or therapy may contain human anti-mouse antibodies (HAMA). Such specimens may show either falsely elevated or depressed values when tested with assay kits which employ mouse monoclonal antibodies.
• Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays. Patients routinely exposed to animals or animal serum products can be prone to this interference and anomalous values may be observed in case of the presence of heterophelic antibodies. Carefully evaluate the results of patients suspected of having these antibodies. If results are not consistent with other clinical observations, additional information should be required before diagnosis.

14. INTERNAL QUALITY CONTROL

• If the results obtained for Control 1 and/or Control 2 are not within the range specified on the vial label, the results cannot be used unless a satisfactory explanation for the discrepancy has been given.
• If desirable, each laboratory can make its own pools of control samples, which should be kept frozen in aliquots.
• Acceptance criteria for the difference between the duplicate results of the samples should rely on Good Laboratory Practises.

15. REFERENCE INTERVALS

These values are given only for guidance; each laboratory should establish its own normal range of values.

Normal subjects

Remark : the range is based on 2.5 % and 97.5 % percentiles.

16. PRECAUTIONS AND WARNINGS

Safety
For in vitro diagnostic use only.
This kit contains 125I (half-life: 60 days), emitting ionizing X (28 keV) and γ (35.5 keV) radiations.
This radioactive product can be transferred to and used only by authorized persons; purchase, storage, use and exchange of radioactive products are subject to the legislation of the end user’s country. In no case the product must be administered to humans or animals.

All radioactive handling should be executed in a designated area away from regular passage. A logbook for receipt and storage of radioactive materials must be kept in the lab. Laboratory equipment and glassware, which could be contaminated with radioactive substances, should be segregated to prevent cross contamination of different radioisotopes. Any radioactive spills must be cleaned immediately in accordance with the radiation safety procedures. The radioactive waste must be disposed of following the local regulations and guidelines of the authorities holding jurisdiction over the laboratory. Adherence to the basic rules of radiation safety provides adequate protection.

The human blood components included in this kit have been tested by European approved and/or FDA approved methods and found negative for HbsAg, anti-HCV, anti-HIV-1 and 2. No known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections. Therefore, handling of reagents, serum or plasma specimens should be in accordance with the local safety procedures. All animal products and derivatives have been collected from healthy animals. Bovine components originate from countries where BSE has not been reported. Nevertheless, components containing animal substances should be treated as potentially infectious.

Avoid any skin contact with reagents (sodium azide as preservative). Azide in this kit may react with lead and copper in the plumbing and in this way form highly explosive metal azides. During the washing step, flush the drain with a large amount of water to prevent azide build-up.

Do not smoke, drink, eat or apply cosmetics in the working area. Do not pipette by mouth. Use protective clothing and disposable gloves.

17. SUMMARY OF THE PROTOCOL

18. BIBLIOGRAPHY / LITERATUR

1. LEROITH D., BONDY C., YAKAR S., LIU JL., BUTLER A. The somatomedin hypothesis : 2001. Endocr Rev 2001; 22:53-74.
2. POLLAK MN., SCHERNHAMMER ES., HANKINSON SE. Insulin-like growth factors and neoplasia. Nat Rev Cancer 2004; 4:505-518.
3. YUEN K., FRYSTYK J., UMPLEBY M., FRYKLUND L., DUNGER D. Changes in free rather than total insulin-like growth factor-I enhance insulin sensitivity and suppress endogenous peak growth hormone (GH) release following short-term low dose GH administration in young healthy adults. J Clin Endocrinol Metab 2004; 89:3956-3964.
4. KHANDWALA HM., McCUTCHEON IE., FLYVBJERG A., FRIEND KE. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev 2000; 21:215-244.
5. RENEHAN AG., ZWAHLEN M., MINDER PC., O’DWYSER ST., SHALET PS., EGGER PM. Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk : systematic review and meta-regression analysis. The Lancet 2004; 363:1346-1353.
6. JUUL A., SCHEIKE T., DAVIDSEN M., GYLLENBORG J., JORGENSEN T. Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease : a population-based case-control study. Circulation 2002; 106:939-944.
7. SANDHU MS., HEALD AH., GIBSON JM., CRUICKSHANK JK., DUNGER DB., WAREHAM NJ. Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance : a prospective observational study. The Lancet 2002; 359:1740-1745.
8. VAESSEN N., HEUTINK P., JANSSEN JA., WITTEMAN JC., TESTERS L., HOFMAN A., LAMBERTS SW., OOSTRA BA., POLS HA., VAN DUIJN CM. A polymorphism in the gene for IGF-I : functional properties and risk for type 2 diabetes and myocardial infarction. Diabetes 2001; 50:637-642.
9. JUUL A. Serum levels of insulin-like growth factor-I and its binding proteins in health and disease. Growth Horm IGF Res 2003; 13:113-170
10. FIRTH SM., BAXTER RC. Cellular actions of the insulin-like growth factor binding proteins Endocr Rev 2002; 23:824-854.
11. BAXTER RC., MEKA S., FIRTH SM. Molecular distribution of IGF binding protein-5 in human serum. J Clin Endocrinol Metab 2002; 87:271-276.
12. RICORT JM. Insulin-like growth factor binding protein (IGFBP) signaling. Growth Horm IGF Res 2004; 14:277-286.
13. JONES JI., CLEMMONS DR. Insulin-like growth factors and their binding proteins : biological actions. Endocr Rev 1995; 16:3-34.
14. ALI O., COHEN P., LEE KW. Epidemiology and biology of insulin-like growth factor binding protein-3 (IGFBP-3) as an anti-cancer molecule. Horm Metab Res 2003; 35:726-733.
15. CHAN SS., TWIGG SM., FIRTH SM., BAXTER RC. Insulin-like growth factor binding protein-3 leads to insulin resistance in adipocytes. J Clin Endocrinol Metab 2005; 90:6588-6595.
16. JUUL A., MAIN K., BLUM WF., LINDHOLM J., RANKE MB., SKAKKEBAEK NE. The ration between serum levels of insulin-like growth factor (IGF)-I and the IGF binding proteins (IGFBP-1, 2 and 3) decreases with age in healty adults and is increased in acromegalic patients. Clin Endocrinol (Oxf) 1994; 41:85-93.
17. JUUL A., MAIN K., BLUM WF., LINDHOLM J., RANKE MB., SKAKKEBAEK NE. The ration between serum levels of insulin-like growth factor (IGF)-I and the IGF binding proteins (IGFBP-1, 2 and 3) decreases with age in healty adults and is increased in acromegalic patients. Clin Endocrinol (Oxf) 1994; 41:85-93.
18. BLUM WF., ALBERTSSON-WIKLAND K., ROSBERG S., RANKE MB. Serum levels of insulin-like growth factor I (IGF-I) and IGF binding protein 3 reflect spontaneous growth hormone secretion. J Clin Endocrinol Metab 1994; 76:1610-1616.
19. FRYSTYK J., IVARSEN P., SKJAERBAEK C., FLYVBJERG A., PEDERSEN EB., ORSKOV H. Serum-free insulin-like growth factor I correlates with clearance in patients with chronic renal failure. Kidney Int 1999; 56:2076-2084.
20. FRYSTYK J. In Endocrinology and Metabolism – Clinics of North America 2005 : Endocrinology of aging, Chapter XI : Aging somatotropic axis mechanisms and implications of IGFBP adaptation.

SYMBOLS USED

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References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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