Description - 100 test kit

The hTSH [¹²⁵I] IRMA system provides a direct quantitative in vitro determination of human thyroid stimulating hormone (hTSH) in human serum. hTSH can be assayed in the range 0-100 µIU/ml using 100 or 200 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.

Introduction

The thyroid stimulating hormone (thyrotropin or TSH) is a glycoprotein with a molecular weight of 28000, secreted by the adenohypophysis. Like other glycoprotein hormones (FSH, LH and HCG), TSH contains two different subunits, an alpha- and a ß-chain, linked by noncovalent bounds. The primary structure of alpha subunits of TSH and of the gonadotropins is the same, whilst their ß subunits are different. The ß subunits are responsible for the immunological and biological specificity of these hormones.

The synthesis and the release of TSH are controlled by the circulatory level of thyroid hormones; triiodothyronine (T3) and thyroxine (T4) and by the hypothalamic thyrotropin releasing hormone (TRH). Thyroid hormones regulate the secretion of TSH by a negative feedback mechanism. An elevation of T3 or T4 will suppress, and their fall will, in turn, increase the level of thyroid stimulating hormone in serum. The increased concentration of TSH in the serum is the earliest and best indicator of primary hypothyroidism.

The determination of TSH by immunoassay methods plays a crucial role in the diagnosis of thyroid disorders and in the evaluation of the functional integrity of the hypothalamic-pituitary axis.

The outstanding sensitivity of the present hTSH IRMA system makes it particularly suitable for the measurement of subnormal thyroid stimulating hormone levels, a key to both the diagnosis and treatment follow up of hyperthyroid patients.

Principle of method

The technology uses two high affinity monoclonal 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 hTSH molecule which is different from that recognised by the unlabelled 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”.

Standards and samples are incubated with a mixture of the antibodies at room temperature. At the end of a 45 minutes incubation period (no need for a shaker), magnetic immunosorbent (MIS) is added in excess. MIS particles selectively bind the hTSH – signal antibody – capture antibody complex 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 thyroid stimulating hormone, the unknown concentration of hTSH in patient samples can determined.

Contents of the kit

Materials, tools and equipment required

Distilled water
Round bottom polystyrene or polypropylene test tubes, about 12 x 75 mm.
Precision pipettes (100, 200, 500 and 1000 µl)
Vortex mixer
Rack and magnetic separator
Gamma counter
Absorbent tissue

Recommended tools and equipment

Repeating pipettes (e.g. Eppendorf)
Dispenser with 1-L reservoir (instead of the 1 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.

Preparation of reagents, storage

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

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.

The Way of Use

The assay can be used in different procedures. There are two options for running the assay. The procedures are labelled as “A” & “B”. Patient sample values and expected values are the same for both procedures.

OPTION “A”: The Basic Procedure

It is very economical on sample consumption. Only 100 µl sample volume is needed. The sensitivity attainable is 0.015 µIU/ml. At the expiry date of the kit the analytical sensitivity is 0.04 µIU/ml. When the kit has less then 3 weeks to its expiration standard 0.06 µIU/ml can be omitted. However if sample consumption is not critical we recommend you OPTION B.

Standard solution 50 µIU/ml can also be omitted if the curve-fitting algorithm of the gamma counter gives similar results with or without this point.

OPTION “B”: The Hyper Sensitive Method

It works in the same way as OPTION A except the sample volume, which is 200 µl. The sensitivity attainable is 0.008 µIU/ml. At the expiry date of the kit the analytical sensitivity is 0.03 µIU/ml.

OPTION A - The Basic Procedure

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

Table 1. Assay Protocol, Pipetting Guide - for OPTION A
All volumes are in microliters (µl).

Characterization of the assay for OPTION A

Figure 1.
OPTION A Typical standard curve corresponding to Table 2.
(Do not use to calculate sample values!)

Table 2. Typical assay data for OPTION A

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 0.015 µIU/ml measured using fresh tracer.

The functional sensitivity is a measure of the hTSH concentration that is significantly different from zero as determined by the inter-assay precision profile (22% CV).

The value of functional sensitivity is: 0.09 µIU/ml.

Hook effect

There is no high dose hook effect up to an hTSH concentration 500 µIU/ml.

Precision

The within-assay precision was determined with 15 replicates within a single run, the between-assay precision was estimated in 15 independent runs carried out in duplicates. CV values are summarized below:

Table 3/1.

The between-assay (inter-assay) precision was determined using pooled human serum samples in independent assay runs. The number of measurements on a sample was a function of sample volume available. Three different operators took part in the investigation process and four different tracer batches were used at different ages of the reagents. Four different lots of MIS were used to determine the inter-assay precision profile. Results are presented below.

Table 3/2.

Linearity – dilution test

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

Table 4.

Recovery – addition test

Individual human serum samples were spiked with known amount of an elevated stock sample. Recovery % is to be interpreted as = (observed-base)/added*100. The results are summarised below.

Table 5.

OPTION  B - The Hyper Sensitive Method

(For a quick guide, refer to Table 6.)

Table 6. Assay Protocol, Pipetting Guide for OPTION B
All volumes are in microliters (µl).

Characterization of the assay for OPTION B

Figure 2.
OPTION B Typical standard curve corresponding to Table 7.
(Do not use to calculate sample values!)

Table 7. Typical assay data for OPTION B

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 0.008 µIU/ml measur