Description

The anti-hTG [¹²⁵I] RIA system provides a direct quantitative determination of autoantibodies to thyroglobulin in human serum in the range of about 30-3000 IU/ml using 50 µl serum samples. Each kit contains materials sufficient for 100 assay tubes permitting the construction of one standard curve and the assay of 42 unknowns in duplicate.

Introduction

The human thyroglobulin (hTg) is a high molecular weight glycoprotein (605 kDa) found in the thyroid follicular cells. It plays a central role in the uptake, incorporation, and regulated biosynthesis of thyroid hormones, T4 and T3.

Thyroid disorders are, in large part, due to autoimmune origin, and anti-thyroglobulin autoantibodies were the first factor to be discovered. Anti-hTG is found in all thyroid autoimmune diseases (Hashimoto’s thyroiditis, Graves’ diseases), with the highest level observed in Hashimoto’s thyroiditis. Anti-hTG is also characteristic of thyroid cancer, and its determination can be used for the follow up of cancer patients.

The specificity of anti-hTG autoantibodies is complex, and samples from different patients show a variable reactivity towards epitopes present in the hTG molecule.

Principle of method

This determination is based on the competition between biotin labelled polyclonal antibody and antibodies in the sample for the binding to ¹²⁵I-labeled thyroglobulin tracer.

Samples and calibrators are incubated together with biotin labelled anti-TG and ¹²⁵I-TG in the streptavidin coated tubes. After incubation the contents of the tubes are aspirated and the bound activity is measured in a gamma counter.

The concentration of anti-TG is inversely proportional to the radioactivity measured in test tubes. The concentration is read off the calibration curve generated by plotting binding values against a series of calibrators containing known amount of anti-thyroglobulin.

Contents of the kit

Materials, tools and equipment required

Test tube rack
Precision pipettes with disposable tip (50, 100 µl and 2 ml)
Shaker
Plastic foil
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes
Dispenser with 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 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. Samples of a concentration higher than 3000 IU/ml could be diluted with the zero calibrator.

Preparation of reagents, storage

Store the 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.

Add 0.5 ml distilled water to the lyophilized 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 2-8 °C until the expiry date of the kit.

Add the wash buffer concentrate to 200 ml distilled water. The diluted solution can be stored at 2-8 °C until expiry date of the kit.

Samples having anti-TG concentrations above the measuring range can be manually diluted with standard 0. The recommended dilution is 1:10 (20 µl sample and 180 µl S₀).

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 the binding capacity:

Calculate percent binding for each standard, control and sample:

For simplicity, these values are uncorrected for non-specific binding (NSB). This is enabled by low NSB being less than 1% of total count.

Using semi-logarithmic graph paper plot B / B₀ (%) for each standard versus the corresponding concentration of standards. Determine the anti-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 computerised data processing logit-log, or spline fittings can be used.

Table 2. Typical Assay Data

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

Characterization of the assay

Calibration

Standards are calibrated against the international reference standard NIBSC 65/93

Analytical sensitivity

The analytical sensitivity is 13 IU/ml, defined as the concentration corresponding of the mean cpm of zero standard minus its double standard deviation.

Functional sensitivity

This functional assay sensitivity generally represents that concentration which corresponds to the 20% between assay coefficient of variation in the respective precision profiles of the assay in lower concentration range.

The value of functional sensitivity is found to be approx. 30 IU/ml.

Precision and reproducibility

Patient samples were assayed in one run with 17 replicates, and in 8 runs with duplicates to determine the intra-assay and the inter-assay precision, respectively. Values obtained are shown below.

Expected values

Anti-hTG concentrations of 239 female and 236 male blood donors were measured.

For 10 probably not healthy patients (TSH values were beside reference interval) anti-TG values were 395 ± 591 IU/ml.

Excluding these patients from statistical analysis, the following results were obtained:

Pathological value can be assigned to higher than 100 IU/ml in the investigated reference population.
It is recommended that each laboratory establish its own reference intervals.

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

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 65 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
					Antiserum

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