BL-28-CT: 100 determinations

Insulin, a polypeptide hormone with e molecular weight of 5800, is secreted by the beta cells of the islets of Langerhans from the pancreas. Insulin possesses a wide spectrum of biological actions. It stimulates cellular glucose uptake, glucose oxydation, glycogenesis, proteogenesis and the formation of DNA and RNA. Insulin plays e key role in the regulation of plasma glucose level (hepatic output inhibition, stimulation of peripheral glucose utilisation). The resulting hypoglycemic effects of insulin are contrebalanced by hormones with hyperglycemic effects (glucagon, growth hormone, cortisol, epinephrine). Insulin secretion is mainly controlled by the plasma glucose levels : hyperglycemia induces a prompt and important increase in circulating insulin levels. Neural influences, as well as various metabolic and hormonal factors (amino acids, glucagon, gastro intestinal hormone) also participate to the control of insulin secretion. Type 1 (insulin dependent « juvenile ») diabetes is due to a destruction of the beta cells, with a consequence of absolute lack of insulin. In Type II (non-insulino dependent : « maturity onset ») diabetes, insulin resistance may play an important role ; however after several years of evolution, beta-cells failure may occur, leading to a relative insulinopenia requiring, in some cases, insulin administartion. Insulin resistance is associated with high circulation levels of the hormone. The most common case of insulin resistance is represented by obesity. Various endocrinipathies (acromegaly, Cushing syndrome) as well as rare cases of insulin receptor defects or cases with anti-insulin receptorvdefects or cases with anti-insulin receptor antibodies are associated with glucose intolerance or even diabetes due to insulin resistance. The determination of plasma insulin levels is an important parameter in the diagnostis of hypoglycemia. Insulin levels are high in case of insulinoma (beta-cell tumor). Functional postprandial hypoglycemia may also be associated with inappropriate insulin release to carbohydrate intake. Insulin levels are determined either in the fasting state or during dynamic test :

stimulation test : carbohydrate rich meal, oral glucose tolerance test (OGTT), arginin infusion, tolbutamide or other sulfonylureas administration

inhibition test : fasting, somatostatine infusion.

The Bio-Line Insulin-Irma is an immunoradiometric assay based on coated-tube separation in which several monoclonal antibodies directed against distinct epitopes of Insulin have been used.. Mabs1, the capture antibodies, are attached to the lower and inner surface of the plastic tube. Standards or samples added to the tubes will at first show low affinity for Mabs1. 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. The use of several distinct Mabs avoids hyperspecificity, common to two-site IRMA, as well as need of a shaker or incubation at 37°C.

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:

1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

2. Do not pipet by mouth reagents containing radioactive materials.

3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

4. Work with radioactive materials should be performed in a designed area.

5. Radioactive materials should be stored in an acceptable location.

6. A log should be kept for receipt and disposal of radioactive materials.

7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

            8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies                 holding jurisdiction over the laboratory.

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other infectious agents.

Kit contains sufficient reagents for 100 determinations.

1. Human serum with sodium merthiolate and azide based standards & control: 8 lyophilised vials to reconstitute with each 0.5ml of distilled water except zero with 2.0 ml.

See exact values on vial labels.

2. ¹²⁵I-Monoclonal-anti-insulin tracer: 1 vial (red solution) containing 5.5 ml. Activity < 10µCi or 350 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-insulin monoclonal antibodies.

4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be diluted into 700 ml distilled water and stored at 4° C.

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

1. Distilled water

2. Pipettes for delivery of: 50 µl, 500 µl and 2 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3. 5 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

5. Gamma counter, set for ¹²⁵I counting and Vortex mixer and magnetic stirrer.

 

Serum or plasma : Serum and plasma must be kept at 2 – 8°C.

If the test is not run within 24 hours, storage at –20°C is recommended and will not result in loss of immuno-reactivity for at least 6 months.

Serum, heparinized or EDTA plasma provide similar results.

Do not use haemolysed samples.

Do not use the kit or components beyond expiration date. Do not mix materials from differents 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 standard curve for each run, do not use data from previous runs.

1. Label coated tubes in duplicate for each standard, sample, control. For determination of total counts, label 2 normal tubes.

2. Vortex mix briefly standards, samples, controls and dispense 50 µl of each into the respective tubes.

            3. Dispense 50 µl of tracer into each tube, including the ones for Total count.

4. Shake the rack containing the tubes gently by hand.

5. Incubate for 2h at room temperature.

6. 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.

7. Wash the tubes with 2 ml Wash Solution (except total counts). Avoid foaming during the addition of the Wash Solution.

Aspirate (or decant) the content of each tube (except total counts).

Wash again tubes with 2 ml Wash solution (exept total counts) and aspirate or decant.

After the last washing let the tubes standing upright for 2 minutes and aspirate the remaining drop of liquid.

            11. Count the tubes in a gamma counter for 60 seconds.

On semilogarithmic or linear graph paper plot the c.p.m. (ordinate) for each standard against the corresponding concentration of INS (abscissa) and draw a standard curve through the standard points, reject the obvious outliers. Read the concentration for each control and sample by interpolation on the standard curve. Computer assisted data reduction will simplify these calculations.

B/BO x 100 = Counts (Std or sample) / Counts (Zero Standard) x 100

Insulin concentrations for subjects with normal oram glucose tolerance test range form 2 to 25 µIU/ml.

A. Minimum Detectable Concentration

The MDC is estimated at 0.1 µIU/ml and is defined as the concentration of Insulin which corresponds to a cpm level 2 standard deviations above the mean of 20 replicates of the zero standard.

C. Speciflcity

Cross-reactive hormones were added to a high value standard (100µIU/ml or 4 ng/ml). The apparent INS response was measured.

As shown hereafter, animal insulins (exept rat insulin) cross-react whereas human, pork and beef proinsulins present no cross-reaction.

        RECOVERY TEST

        DILUTION TEST

1. FLIER J.S., KAHN C.R. and ROTH J. (1979)Receptors, antireceptor antibodies and mechanisms of insulin resistance.N. Engl. J. Med. 300 ; 8 :413-419

2. FRIER B.M., ASHBY J.P., NAIRN I.M. and BAIRS J.D. (1981)Plasma insulin, C-peptide and glucagon concentrations in patients with insulin- ependent diabetes treated with chlorpropamide.Diab. Metab. 7 ; 1 :45-49

3. JUDSEWITHSCH R.G., PFEIFER M.A., BEST J.D., BEARD J.C., HALTER J.B. and PORTE D.Jr. (1982)Chronic chlorpropamide therapy of non-insulino-dependent diabetes augments basal and stimulated insulin secretion by increasing islet sensitivity to glucose.J. Clin. End. And Metab. 55 ; 2 :321-328

4. KOSAKA K., HAGURA R. and KUZUYA T. (1977)Insulin responses in equivocal and definite diabetes, with special reference to subjects who had mild glucose intolerance but later developed definite diabetes.Diabetes 26 ; 10 :944-952

5. STARR J.I., MAKO M.E., JUHN D. and RUBENSTEIN A.H. (1978)Measurement of serum proinsulin-like material : cross-reactivity of porcine in the insulin radioimmunoassay.J. Lab. Clin. Med. 91 ; 4 :691-692

6. TEMPLE R.C., CARRINGTON C.A., LUZIO S.D, OWENS D.R., SCHNEIDER A.E. SOBEY W.J., HALES C.N. (1989)Insulin deficiency in non-insulino-dependent diabetes.The Lancet, Feb. 11 :293-295