Description

The calcitonin [¹²⁵I] IRMA system provides a direct quantitative determination of human calcitonin in human serum. Calcitonin can be assayed in the range of 0-2000 pg/ml using 200 µl serum samples. Each kit contains materials sufficient for 50 assay tubes permitting the construction of one standard curve and the assay of 16 unknowns in duplicate.

Introduction

Calcitonin (MW 3.4 kDa) is primarily secreted by the parafollicular C-cells of the thyroid gland. The mature peptide hormone comprises 32 amino acid residues. Calcitonin exerts its biological effect by acting on its target organs: bone, kidney and the gastrointestinal tract. The physiological role of calcitonin in bone metabolism is not fully understood and is still under investigation. It is well established that abnormally elevated levels of Calcitonin are characteristic of thyroid C-cell hyperplasia and medullary thyroid carcinoma (MTC). MTC represents 5-10% of all thyroid cancer and exists in either familial or sporadic form. Calcitonin IRMA is recommended for diagnosis and follow up of MTC and for diagnosis of preclinical cases of the familial forms of MTC. The circulating concentrations of calcitonin are low, normal values are less then 15 pg/ml, and 10 pg/ml, for males and females, respectively.

In the blood the apparent calcitonin-like immunoreactivity is contributed by various calcitonin-related species, including the monomeric, dimeric and polymeric forms, as well as fragments and precursors of the parent hormone.

Principle of method

The technology uses a high affinity monoclonal and polyclonal antibodies in an immunoradiometric assay (IRMA) system. It offers an increased level of sensitivity and specificity compared with conventional RIA methods.

The ¹²⁵I labelled signal-antibody binds to an epitope of the calcitonin molecule which is different from that recognised by the unlabelled capture-antibody. In the first step, the signal antibodies react with the antigen present in standards or samples after which, in the second step, the reaction with the capture antibody leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”.

Standards and samples are incubated with the signal antibodies at 2-8 °C temperature. At the end of an overnight incubation period, capture antibodies are pipetted into the reaction mixture with a two hours incubation time at room temperature. Afterwards, magnetic immunosorbent (MIS) is added in excess. MIS particles bind the calcitonin—signal antibody—-capture antibody complex selectively and settle out in a magnetic field. A wash step is critical to reducing non-specific binding to a minimum for increased low end precision.

The concentration of antigen is directly proportional to the radioactivity measured in test tubes. By constructing a calibration curve plotting binding values against a series of calibrators containing known amount of calcitonin, the unknown concentration of calcitonin in patient samples can determined.

Contents of the kit

Test tube rack
Precision pipettes with disposable tip (50, 100, 200, and 1000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)
Dispenser with 300 ml reservoir (instead of the 2 ml pipette)

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 24 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. Samples with a calcitonin concentration higher than that of the most concentrated standard should be diluted and reassayed.

Preparation of reagents, storage

Add the wash buffer concentrate (20 ml) to 200 ml distilled water to obtain 220 ml wash solution. Upon dilution store at 2-8 °C until expiry date.

Add 1000 µl distilled water to the standards and lyophilized control serum. 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. Store at -20 °C until expiry date.

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

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)

Table 2. Typical Assay Data

Figure 1
A typical standard curve
(Do not use to calculate unknown samples)

Calculation of results

The calculation is illustrated using representative data. Data obtained should be similar to those shown in Table 2.

Calculate the average CPM for each pair of assay tubes. Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

Using semi-logarithmic graph paper plot B/T (%) for each standard versus the corresponding concentration of calcitonin.

Determine the calcitonin concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Out of fitting programs applied for computerized data processing logit-log, or spline fittings can be used. Automated data processing systems are also available.

Characterization of the assay

Assay parameters

Sensitivity

A detection limit of 2 pg/ml has been obtained by assaying 20 replicates of the zero standard. The sensitivity is defined as the concentration corresponding to the sum of the mean cpm and its double standard deviation.

Hook effect

There is no high dose "hook effect" up to a calcitonin concentration of 100000 pg/ml.

Specificity

The monoclonal and the polyclonal antibodies used in this IRMA kit are specific for hcalcitonin. No cross reactivity with eel, salmon, porcine and eel analogue calcitonin can be detected up to 200000 pg/ml concentrations.

Precision

4 patient samples were assayed in 15 replicates to determine intra-assay precision. Values obtained are shown below.

Reproducibility

To determine inter-assay precision 4 patient samples were measured in duplicates in 20 independent assays by 2 operators using different kit batches. 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 hcalcitonin.The average percent recovery for 4 serum pools spiked with calcitonin at 3 levels was: 93.0 ± 9.9 (mean ± SD).

Expected values

Healthy adults: 0 - 10 pg/ml

It is recommended that each laboratory determine a reference range for its own patient population.

Procedural notes

Addition of wash buffer. For the addition of wash buffer the use of a common laboratory dispenser equipped with a 300 ml glass bottle, and a flexible outlet tubing end is recommended. In lack of this tool a large volume syringe attached to a repeating pipette can be used.

Additional information

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

Precaution

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 74 mg.

	Use by		In vitro diagnostic medical device		Control
	Batch code		Manufacturer		Standard
	Caution, consult accompanying documents		Radioactive material		Magnetic immunosorbent
	Biological risk		Temperature limitation Store between 2-8 °C		Tracer
	Consult instructions for use		Catalogue number		Wash buffer
			Serum diluent		Antiserum