Description

The insulin [¹²⁵I] assay system provides the quantitative determination of insulin in human serum. Insulin can be assayed in the range 0-400 µIU/ml (0-14 ng/ml). Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and assay of 42 unknowns and 1 control in duplicate.

Introduction

The insulin is a light polypeptide hormone with molecular weight 6000. It is synthesized in the beta cells of the pancreas from the precursor proinsulin consisting of 86 amino acids. Proinsulin enzymatically splits into insulin and C-peptide that are stored in the pancreas and release from there in equimolar quantitiest into the blood system. Insulin consists of two polypeptide chains: A (21 amino acids) and B (30 amino acids) connected to each other by two disulphide bridges. While in the amino acid sequence of C-peptide great differences can be observed in the case of various mammalis, in insulin these differences are insignificant: e.g. porcine and bovine insulin only differ from human insulin in one and three amino acids, respectively.

Insulin is an important metabolic hormone that has several direct and indirect effects on the organism. Its general influence is that it stimulates the synthesis and accumulation of macromolecules playing role in energy supply and in the regulation of metabolic processes. Insulin increases the rate of glucose transport trough the cell membranes, helps the admission of other monosacharides, amino acids, potassium and magnesium ions into the cells.

Insulin promotes the utilization and oxidation of glucose, glycogenesis, lipogenesis, as well as the formation of ATP, DNA and RNA. Insulin stimulates these processes in the muscles, the liver and fatty tissues, blood cells and the brain, reabsorption in renal tubules and on the intestinal mucosa.

The symptoms of diabetes mellitus can be attributed to the inappropriate insulin response to glucose concentration. While in the case of unambiguous diabetes reduced insulin response is observed, in various early stages of diabetes the insulin level of the patients may be normal or even high, and increase of various degrees can be found in stimulation tests. The fasting hyperglycaemia of not overweight patients is usually accompanied by normal circulatory insulin level, while in obese patients this level is high, in proportion of overweight.

Principle of the method

This assay is based on the competition between unlabelled insulin and a fixed quantity of ¹²⁵I-labelled insulin for a limited number of binding sites on insulin specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. Upon addition of magnetizable immunosorbent the antigen-antibody complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

Materials, tools and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Precision pipettes (50 µl, 100 µl and 500 µl)
Vortex mixer
Magnetic separator, or, alternatively, centrifuge
Gamma counter

Recommended tools and equipment

Orbital shaker
Repeating pipettes

Specimen collection and storage

Serum samples can be prepared according to common procedures used routinely in clinical laboratory practice. Samples can be stored at 2-8 °C if the assay is carried out within 48 hours, otherwise aliquots should be prepared and stored deep frozen (-20 °C). Frozen samples should be thawed and thoroughly mixed before assaying. Repeated freezing and thawing should be avoided. Do not use lipemic, hemolyzed or turbid specimens.

Preparation of reagents, storage

Store the reagents between 2-8 °C after opening. At this temperature each reagent (except reconstituted standard and control) is stable until expiry date. The actual expiry date is given on the package label and in the quality certificate.

Add 0.5 ml distilled water to the lyophilised standard and control serum, and mix gently with shaking or vortexing (foaming should be avoided). Ensure that complete dissolution is achieved, and allow the solution to equilibrate at room temperature for at least 20 minutes. For repeated use the rest of reagent can be stored at -20 °C for two months.

CAUTION! Equilibrate all reagents and serum samples to room temperature. Mix all reagents and samples thoroughly before use. Avoid excessive foaming.

Assay procedure

(For a quick guide refer to Table 1)

Table 1. Assay Protocol, Pipetting Guide (all volumes in microliters)

Calculation of results

Calculate binding capacity:

Calculate the percent binding for each standard, control and sample:

These values are uncorrected for non-specific binding (NSB). This is enabled by low NSB being less than 2% of total count.

Using semi-logarithmic graph paper plot B / B₀% for each standard versus the corresponding concentration of standards.

Determine the insulin concentration of the unknown samples by interpolation from the standard curve. Out of fitting programs applied for computerized data processing logit-log, or spline fittings can be used.

Table 2. Typical Assay Data

Insulin concentration µIU/ml

Figure 1.
A typical standard curve
(Do not use to calculate sample values)

Characterization of the assay

Assay parameters

Calibration

Standards are calibrated against the international reference standard NIBSC 66/304.

Specificity

Cross reactivity was defined by weight at the 50% displacement level in per cent.

Analytical sensitivity

5 µIU/ml, defined as the concentration corresponding to the mean cpm of zero standard minus its double standard deviation..

Precision and reproducibility

Patient samples were assayed in one run with 20 replicates and in 10 runs with duplicates to determine intra-assay and inter-assay precision, respectively. Values obtained are shown below.

Recovery

Recovery was defined as the measured increase expressed as percent of expected increase upon spiking serum samples with known amount of insulin. Values for 7 serum pools spiked with insulin at 3 levels were as follows:

Expected values

It is recommended that each laboratory establish its own reference intervals. As a guide, 5 - 35 µIU/ml was obtained from normal patients on an empty stomach.

The results obtained should only be interpreted in the context of the overall clinical picture. None of the in vitro diagnostic kits can be used as the one and only proof of any disease or disorder.

Conversion of SI units can be performed according to the following formula:

1 µIU/ml = 5.99 pmol/l
1 ng/ml = 28.7 µIU/ml

Additional information

Components from various lots or from kits of different manufacturers should not be mixed or interchanged.

Precautions and warnings

Radioactivity

This product contains radioactive material. It is the responsibility of the user to ensure that local regulations or code of practice related to the handling of radioactive materials are satisfied.

Biohazard

Human blood products used in the kit have been obtained from healthy human donors. They were tested individually by using approved methods (EIA, enzyme immunoassay), and were found to be negative, for the presence of both Human Immunodeficiency Virus antibody (Anti-HIV-1) and Hepatitis B surface Antigen (HBsAg).

Care should always be taken when handling human specimens to be tested with diagnostic kits. Even if the subject has been tested, no method can offer complete assurance that Hepatitis B Virus, Human Immunodeficiency Virus (HIV-1), or other infectious agents are absent. Human blood samples should therefore be handled as potentially infectious materials.

Chemical hazard

Components contain sodium azide as an antimicrobial agent. Dispose of waste by flushing with copious amount of water to avoid build-up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 80 mg.