Description

The hThyroglobulin [¹²⁵I] IRMA system provides a direct quantitative in vitro determination of human thyroglobulin (hTg) in human serum. hTg can be assayed in the range 0-250 ng/ml using 100 µl serum sample. Each kit contains materials sufficient for 100 assay tubes permitting the construction of one standard curve and the assay of 40 unknowns in duplicate (20 sample with recovery test in duplicate).

Introduction

Thyroglobulin is a iodoglycoprotein consisting of heterogeneous molecules, the composition of which is in part depending on the degree of iodination. The prevailing molecular form is 660 kDa (dimeric form, the two subunits, linked by noncovalent bounds), but both larger and smaller molecular forms exist in the thyroid gland. Tg is the site of synthesis and storage of thyroid hormone. produced by the thyroid gland. Tg is synthesized and stored in thyroid follicles and some of the nonenzimatically digested protein is released into the circulation upon stimulation with thyrotropin (TSH) together with thyroxine (T₄) and triiodothyronine (T₃).

The determination of Tg by immunoassay methods plays a crucial role in the diagnosis of thyroid disorders, such as Grave’s multinodular goiter, benign thyroid adenoma, thyroiditis (acute phase), and differentiated carcinoma, and is a useful tool in screening population at risk for thyroid carcinoma after previous irradiation.

The sensitivity of the present hTg IRMA system makes it suitable for the measurement of subnormal hTg levels, which is an early and reliable marker of tumor recurrence.

Principle of method

The technology uses two high affinity monoclonal antibodies in an immunoradiometric assay (IRMA) system.

The ¹²⁵I labelled signal-antibody binds to an epitope of the Tg molecule spatially different from that recognised by the biotin-capture-antibody. The two antibodies react simultaneously with the antigen present in standards or samples, which leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a “sandwich”.

During an overnight incubation period the immuno-complex is immobilized to the reactive surface of streptavidin coated test tubes. Reaction mixture is then discarded, test tubes washed exhaustively, and the radioactivity is measured in a gamma counter.

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 hTg, the unknown concentration of hTg in patient samples can determined.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tips (100, 200 and 2000 µl)
Distilled water
Vortex mixer
Shaker
Plastic foil
Adsorbent tissue
Gamma counter

Recommended tools and equipment

Repeating pipettes (e.g., Eppendorf)
Dispenser with 1-L 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 thyroglobulin 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 700 ml distilled water to obtain 720 ml wash solution. Upon dilution store at 2-8 °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.

Recovery test

Anti-Tg antibodies or unspecific effects in a patient’s serum can interfere with serum thyroglobulin measurement, which leads to underestimation of the Tg concentration in IRMA system. Interference can be detected by using recovery test. The recovery test should be carried out as described in the Assay procedure.

The concentration of the recovery sample (approximately 500ng Tg/ml) should be checked with serum diluent (recovery reference tubes (D_R)).

Recovery (in %) in the serum sample:

Recoveries between 70% and 130% are considered valid. Levels of <70% or >130% are due to interference and the Tg level of the relevant original sample is considered invalid.

Assay procedure

(For a quick guide, refer to Table 1.)

Note for the washing step: Decantation is the most critical step of the assay procedure. Pay a special attention not to contaminate the outer surface of tubes, when turning the test tube-rack upside down. Even a small contamination may introduce a high, unidentified background resulting in a substantial over-estimation of concentration. The error associated may become particularly high in the low range of concentration, which is of vital importance for the reliable determination of subnormal Tg-values. On the same reason, regular checking of the instrument background is indispensable. This is particularly important, when multi-channel counters are used. Make ensure that background values and variation between individual channels are within the range of acceptance as specified in counter's service book.

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

Calculation of results

The calculation is illustrated using typical data. The assay data collected 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:

For simplicity, these values are uncorrected for non-specific binding (NSB). This is enabled by low NSB being less than 3% of total count.

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

Determine the Tg 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.

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

Table 2. Typical assay data

Characterization of the assay

Typical assay parameters

Sensitivity

The analytical sensitivity or minimum detectable limit is calculated by the interpolation of the mean counts of zero standard plus 2 standard deviation from the standard curve. Determination was carried out using 20 replicates of zero standard response.

The value of analytical sensitivity is 0.022 ng/ml measured using fresh tracer. The declared analytical sensitivity is better then 0.1 ng/ml in any time before expiry.

Hook effect

There is no high dose hook effect up to an hTg concentration 20000 ng/ml.

Linearity – dilution test

Three individual human serum samples were diluted with the zero standard of the kit. The diluted samples were measured according to kit protocol.

Recovery – addition test

49 individual human serum samples were spiked with known concentration serum based stock solution made from BCR certification reference preparation 457 in different amount. The results are summarised below.

Intra-interassay

Expected Values

Expected normal value range is 2 ng/ml - 70 ng/ml.

It is recommended that each laboratory determine a reference range for healthy persons for its own patient population, since this may vary in different laboratories or regions.

Procedural notes

1) Source of error! Reactive test tubes packed in plastic boxes are not marked individually. Care should be taken of not mixing them with common test tubes. To minimize this risk, never take more tubes than needed out of plastic box, and put those left after work back to the box. It is recommended to label assay tubes by a marker pen.

2) Source of error! To ensure the efficient rotation, tubes should be firmed tightly inside the test tube rack. Never use a rack type with open hole. An uneven or incomplete shaking may result in a poor assay performance.

3) Addition of wash buffer. For the addition of wash buffer the use of a common laboratory dispenser equipped with a 1-L 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		Coated tube
	Biological risk		Temperature limitation Store between 2-8 °C		Tracer
	Consult instructions for use		Catalogue number		Wash buffer
			Serum diluent		Recovery serum