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EK-1ACE051201 hTSH ELISA KIT (REF: EK-1) / 475 Euro The human thyroid stimulating hormone (hTSH) ELISA system provides a direct quantitative determination of hTSH in human serum. hTSH can be assayed in the range of 0-16 mIU/l using 100 µl serum samples. Each kit contains materials sufficient for 96 determinations permitting the construction of one standard curve and the assay of 39 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 á- and a ß-chain, linked by noncovalent bounds. The primary structure of á subunits of TSH and of the gonadotrophins 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 thyroxin (T4) and by the hypothalamic Thyrotropin-Releasing Hormone (TRH). Thyroid hormones regulate the secretion of TSH by a negative feed-back mechanism. An elevation of T3 or T4 will suppress, and their fall will, in turn, increase the level of TSH 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 remarkable sensitivity of the present hTSH ELISA system makes it particularly suitable for the measurement of subnormal hTSH 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 immunometric assay system. The two antibodies react simultaneously with the antigen present in standards or samples. This reaction leads to the formation of a capture antibody - antigen - signal antibody complex, also referred to as a "sandwich". In the standard solid-phase ELISA system the reaction is carried out in a microtiter plate (12 strips) which acts as the binder of sandwich complex. In the present product standards and samples are incubated with the conjugate which contains horse radish peroxidase (HRPO) labelled antibody at room temperature for 2 hours, then washed repeatedly. After the addition of a ready-to-use tetramethyl-benzidine (TMB)/peroxide substrate the signal is measured in an ELISA photometer at 450 and 405 nm wavelengths. The concentration of antigen is directly proportional to the optical density measured in the wells. The unknown concentration of TSH in patient samples is read off a calibration curve constructed by plotting binding values against a series of calibrators containing known amount of TSH. Contents of the kit 1. 1 vial CONJUGATE (12 ml), ready to use, containing anti- hTSH antibodies in buffer with blue dye and 0,01 % merthiolate and 0,2% chloracetamide (CAA). 2. 6 vials STANDARD, ready to use. 3 ml per vial, containing 0 mIU/l (S0), 1 ml per vial, containing 0,15 (S1), 0,5 (S2), 1 (S3), 2 (S4), 4 (S5), 8 (S6), 16 (S7) mIU/l hTSH (WHO 2nd IRP 80/558) in serum with 0.1% sodium azide. 3. 1 vial CONTROL SERUM, lyophilized. 1.0 ml human serum with 0.1% sodium azide. The concentration of the control serum is specified in the quality certificate enclosed. See Preparation of reagents. 4. 1 vial SUBSTRATE (25 ml) ready to use, in brown plastic bottle. Do not expose to direct light! 5. 1 piece MICROTITER PLATE, ready to use. 12 strips, packed in an air-tight foil. 6. 1 bottle WASH BUFFER CONCENT- RATE (20 ml), 0,01% merthiolate.. See Preparation of reagents. 7. 1 vial STOP REAGENT (6 ml), 1M sulfuric acid. Cover stick Plate map Quality certificate Pack leaflet Materials, tools and equipment required Precision pipettes with disposable tips (50, 100, 200 and 300 µl), distilled water, vortex mixer, shaker, absorbent tissue, disposable plastic reagent-trays (separate for each reagent), ELISA photometer Recommended tools and equipment Repeating pipettes, multi-channel ELISA pipettes, ELISA microplate washer 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 Except the wash buffer concentrate and the control serum, each reagent is supplied in ready to use form. -Add the wash buffer concentrate (20 ml) to 600 ml distilled water to obtain 620 ml wash solution. Upon dilution store at 2-8°C until expiry date. -Add 1 ml distilled water to the lyophilised control serum. Mix gently with shaking or vortexing (foaming should be avoided). Allow the solution to equilibrate at room temparature for at least 20 minutes.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. The reconstituted control serum is stable for 2 weeks at 2-8°C or for 2 months at –18°C. Assay procedure (For a quick guide , refer to Table 1.) 1. Equilibrate reagents and samples to room temperature before use. Homogenize all reagents and samples by gentle mixing to avoid foaming. 2. Label the plate map for duplicates of each standards (S0-S7), control serum (C) and samples (Mx). 3. Pipette 100 µl each of standards, control serum and samples into the appropriate wells. 4. Pipette 100 µl of conjugate solution into each well by the multi-channel pipette. 5. Cover the plate by the enclosed foil and incubate for 2 hours at room temperature (18 - 25°C) with shaking at 300 – 400 RPM (IKA – VIBRAX – VXR). 6. Take the plate out of the machine. Remove the cover and pour the liquid directly over the lab sink. Holding in the upside down position place the plate immediately on an absorbent tissue. Pay special attention to crossing-over between wells due to droplets backflow. 7. Add 300 µl wash buffer into each well, then decant (tap and blot) or aspirate. Repeat this step 5 times. An automatic or manual plate washer can be used. Follow manufacturer’s instruction for proper usage. 8. Pour the substrate into its plastic tray, and pipette 200 µl to each well with the aid of the multi -channel pipette. Place the plate into the dark for 30 minutes. (If less than the whole volume is used in one assay, do not pipette directly from the bottle, and never fill the unused reagent back into its original bottle). 9. Pipette 50 µl stop reagent into each well, and shake gently for 30 seconds. 10. Measure in the ELISA photometer at 450nm (0 mIU/l-4 mIU/l) and 405nm (0 mIU/l -16 mIU/l) within 20 minutes after adding the stop solution. 11. Calculate the concentrations of the samples as described in Calculation of results. Table 1. Assay Protocol, Pipetting Guide (all volumes in microlitres)
Calculation of results The calculation is illustrated using representative data. Data obtained should be similar to those shown in Table 2 - 3. Manual calculation Calculate the average OD for each pair of duplicates. Draw the standard curve on a lin-lin graph paper by plotting calculated OD of each standard level (ordinate) against the respective concentration (abscissa). Obtain sample values by interpolation of sample OD values on the standard curve. Data evaluation using normalized binding For computerised calculations and/or quality assessment normalised specific binding values, rather than OD values are used. Specific binding values can be calculated for each standard and sample according to the following equation: 450 nm: S1-5/ C / Mx (OD) B/Bmax (%) = ____________________ x 100 S5 (OD) 405 nm: S2-7 / C / Mx (OD) B/Bmax (%) = ____________________ x 100 S7 (OD) Table 2. Typical assay data (450nm)
0,00,51,01,52,02,53,001234hTSH concentration (mIU/l)OD 450 nm Figure 1: A typical standard curve (Do not use to calculate unknown samples!) Table 2. Typical assay data (405 nm)
0,00,51,01,52,02,50246810121416hTSH concentration (mIU/l)OD 405 nm Characterization of assay Typical assay parameters Bo/Bmax < 5 % Sensitivity Analytical sensitivity or detection limit On the basis of results of 16 replicate determinations of the zero standard, the minimum TSH concentration detectable by the present method is 0.020 mIU/l (450 nm). At 405 nm the analytical sensitivity is 0.053 mIU/l. The detection limit is defined as the value deviating by 2 SD from that of the zero standard. Functional sensitivity 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 0.07 mIU/l. (at 450 nm). Hook effect There is no high dose " hook effect " up to the TSH concentration of 60000 mIU/l . Specificity The following hormones were tested: Hormone Apparent TSH value mIU/l HCG 1000 mIU/ml 1,294 mIU/l LH 150 mIU/ml 0,159 mIU/l FSH 150 mIU/ml 0,005 mIU/l Precision 7 patient samples were assayed in 15 replicates to determine intra-assay precision. Values obtained are shown below. Sample Number of replicates Mean value CV% 1 15 0.122 11.8 2 15 0.596 4.2 3 15 1.011 5.9 4 15 1.949 2.3 5 15 4.102 4.7 6 15 6.340 1.7 7 15 8.033 4.6 Reproducibility To determine inter-assay precision 9 patient samples were measured in duplicates in 15 independent assays by 2 operators using different kit batches. Values obtained are shown below. Sample Number of runs Mean value CV% 1 15 0.048 31.2 2 15 0.113 16.4 3 15 0.163 10.7 4 15 0.396 7.0 5 15 0.579 5.6 6 15 1.039 8.4 7 15 3.153 8.0 8 15 5.530 11.2 9 15 6.426 5.6 Recovery Recovery was defined as the measured increase expressed as per cent of expected increase upon spiking serum samples with known amount of TSH. 94,2-112,1% was obtained for 5 serum pools. Dilution test (linearity) A serial dilution (1:2 – 1:16) of 5 individual serum samples was carried out with the zero-standard. The recovery was: 83,6-105,4% Expected Values Expected euthyroid range is 0.27 mIU/l - 3.75 mIU/l. It is recommended that each laboratory determine a reference range for euthyroids for its own patient population, since this may vary in different laboratories or regions | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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