Turbo tsh 1

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

1 bottle	TRACER, ready to use 21 ml, containing < 900 kBq ¹²⁵I-signal and capture antibody in buffer with red dye and 0.1% NaN₃.
8 vials	STANDARD, ready to use 1.5 ml per vial, containing 0 (S₀), 0.06 (S_0.06), 0.15 (S_0.15), 0.6 (S_0.6), 2.5 (S_2.5), 15 (S₁₅), 50 (S₅₀) and 100 (S₁₀₀) µIU/ml hTSH (WHO 2^nd IRP 80/558) in serum with 0.1% NaN₃
2 vials	CONTROL SERUM low (CI) and high (CII) 1.5 ml, containing 0.1% NaN₃ The concentration of the control serum is specified in the quality certificate enclosed.
1 bottle	MAGNETIC IMMUNOSORBENT (MIS), ready to use. DO NOT FREEZE! 55 ml, containing paramagnetic particles in buffer with 0.1% NaN₃.
1 bottle	WASH BUFFER CONCENTRATE. 20 ml, containing 0.1% NaN₃. See Preparation of reagents
1 pc	Quality certificate
1 pc	Pack leaflet

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.)

1	Dilute the wash buffer concentrate appropriately.
2	Equilibrate reagents and samples to room temperature before use. (Cold reagents will slow down the reaction and warming up rate will be different from tube to tube.)
3	Label test tubes in duplicate for each standard (0 -100 µIU/ml), control serum and patient samples.
4	Homogenize all reagents and patient samples by gentle mixing. Avoid foaming.
5	Pipette 100 µl of standards, control and patient samples into the bottom of labelled tubes.
6	Pipette 200 µl of tracer into each tube. Total count tubes should be set aside for counting.
7	Incubate tubes for 45 minutes at room temperature (20–28 °C).
8	Vigorously shake and swirl the bottle containing magnetic immunosorbent until homogeneity is achieved. Add 500 µl MIS to each tube. Swirl the bottle of MIS after every 15-20 tubes.
9	Thoroughly vortex mix all tubes, and incubate them for 15 minutes at room temperature.
10	Attach the rack on to the magnetic separator base and ensure that every tube is in contact with the base plate. Let the MIS particles settle for 5 minutes. Do not remove the rack from the separator base after the separation of the solid and liquid phases. Pour off and discard the supernatant. Keeping the separator inverted, place the tubes on a pad of absorbent tissue and allow to drain for 2 minutes.
11	Return the separator to an upright position and add 1.0 ml of washing solution to each tube. For more comfort and precision, it is recommended to use either a repeating pipette (e.g. Eppendorf-pipette) or a dispenser with bottle for the addition of washing solution.
12	Vortex mix each tube thoroughly and repeat Step 10. Intense vortexing is required.
13	Count each tube for at least 60 seconds in a gamma counter.
14	Calculate the TSH concentrations of the samples as described in Calculation of results or use special software.

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

Tube	Total	Standard	Sample	Control
Standard		100
Sample			100
Control				100
Tracer	(200)	200	200	200
Vortex mix Incubate for 45 minutes at room temperature
Magnetic immunosorbent		500	500	500
Vortex mix Incubate for 15 minutes at room temperature
Place the tubes on the magnetic separator for 5 minutes.
Decant the fluid and blot on filter paper
Wash Buffer		1000	1000	1000
Vortex mix
Place the tubes on the magnetic separator for 5 minutes.
Decant the fluid and blot on filter paper
Count radioactivity (60 sec/tube)
Calculate the results

Characterization of the assay for OPTION A

Typical standard curve for the Turbo TSH I-125 IRMA kit (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

	CPM 1	CPM 2	CPM mean	B/T %
Total	411 237	402 625	406 931	-
S₀(NSB)	117	97	107	0.026
S_0.06	297	241	269	0.066
S_0.15	518	572	545	0.134
S_0.6	1 850	1 924	1 887	0.464
S_2.5	7 602	7 736	7 669	1.88
S₁₅	45 678	44 718	45 198	11.11
S₅₀	132 951	131 243	132 097	32.46
S₁₀₀ (B_max)	227 699	229 841	228 770	56.22
CI	1 158	1 046	1 102	0.31
CII	59 101	57 811	58 456	19.4

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.

	intra-assay
No.	mean (µIU/ml)	CV %
1	0.207	5.8
2	0.737	2.8
3	1.01	3.4
4	1.19	2.6
5	1.89	2.9
6	3.31	1.6
7	6.77	1.2

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.

		inter-assay
sample No.	number of assay runs	mean (µIU/ml)	CV %
1	18	0.049	33.8
2	18	0.079	25.4
3	19	0.090	15.1
4	11	0.095	17.1
5	20	0.124	14.1
6	19	0.143	13.1
7	19	0.196	8.3

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.

sample No.	dilution factor	expected µIU/ml	observed µIU/ml	recovery %
1	1		18.02
1	2.03	8.89	8.62	97.0
1	4.10	4.39	4.38	99.7
1	8.32	2.17	2.14	98.9
2	1		33.51
2	2.00	16.78	16.06	95.7
2	3.98	8.42	7.96	94.5
2	7.97	4.21	3.93	93.5
3	1		34.83
3	2.00	17.43	16.81	96.4
3	3.99	8.74	8.91	102.0
3	7.95	4.38	4.15	94.9
4	1		28.74
4	1.99	14.46	14.31	98.9
4	3.97	7.25	7.53	103.9
4	7.90	3.64	3.85	105.8
5	1		27.02
5	2.00	13.49	12.47	92.5
5	4.03	6.70	6.36	94.9
5	8.05	3.36	3.32	98.8
6	1		44,68
6	2.00	22.37	21.10	94.3
6	3.99	11.21	10.40	92.8
6	7,99	5.59	5.19	92.8
7	1		25.17
7	2.01	12.49	12.10	96.8
7	4.06	6.20	5.96	96.2
7	7.65	3.29	2.90	88.1
8	1		7.61
8	2.00	3.80	3.70	97.1
8	4.03	1.89	1.78	94.2
8	8.11	0.939	0.869	92.6
9	1		109.36
9	2.00	54.6	51.94	95.1
9	4.03	27.12	25.22	93.0
9	8.12	13.47	12.51	92.9
10	1		42.41
10	1.99	21.28	20.63	97.0
10	3.98	10.65	10.25	96.2
10	8.02	5.29	5.25	99.2

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.

sample	base µIU/ml	added µIU/ml	expected µIU/ml	observed µIU/ml	recovery %
1	8.33	17.27	25.61	25.48	99.3
2	7.36	18.72	26.07	25.39	96.3
3	8.34	18.57	26.90	26.66	98.7
4	8.98	16.39	25.37	25.00	97.7
5	5.18	18.01	23.19	20.84	86.9
6	5.38	15.83	21.20	20.54	95.8
7	10.22	15.79	26.02	26.22	101.3
8	14.44	13.42	27.86	27.46	97.0
9	10.02	13.57	23.59	22.82	94.3
10	0.750	14.35	15.10	14.61	96.6
11	1.46	14.71	16.17	15.90	98.2
12	0.921	14.39	15.31	14.44	94.0
13	0.011	2.66	2.67	2.80	104.9
14	0.013	2.70	2.72	2.95	108.7
15	0.029	2.73	2.76	3.03	109.9
16	0.058	2.65	2.71	3.20	118.5
17	0.102	3.62	3.72	4.05	108.9
18	0.053	2.74	2.79	2.93	105.0

OPTION B - The Hyper Sensitive Method

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

1	Dilute the wash buffer concentrate appropriately.
2	Equilibrate reagents and samples to room temperature before use. (Cold reagents will slow down the reaction and warming up rate will be different from tube to tube.)
3	Label test tubes in duplicate for each standard (0 -100 µIU/ml), control serum and patient samples.
4	Homogenize all reagents and patient samples by gentle mixing. Avoid foaming.
5	Pipette 200 µl of standards, control and patient samples into the bottom of labelled tubes.
6	Pipette 200 µl of tracer into each tube. Total count tubes should be set aside for counting.
7	Incubate tubes for 45 minutes at room temperature (20–28 °C).
8	Vigorously shake and swirl the bottle containing magnetic immunosorbent until homogeneity is achieved. Add 500 µl MIS to each tube. Swirl the bottle of MIS after every 15-20 tubes.
9	Thoroughly vortex mix all tubes. and incubate them for 15 minutes at room temperature.
10	Attach the rack on to the magnetic separator base and ensure that every tube is in contact with the base plate. Let the MIS particles settle for 5 minutes. Do not remove the rack from the separator base after the separation of the solid and liquid phases. Pour off and discard the supernatant. Keeping the separator inverted. place the tubes on a pad of absorbent tissue and allow to drain for 2 minutes.
11	Return the separator to an upright position and add 1.0 ml of washing solution to each tube. For more comfort and precision. it is recommended to use either a repeating pipette (e.g. Eppendorf pipette) or a dispenser with bottle for the addition of washing solution.
12	Vortex mix each tube thoroughly and repeat Step 10. Intense vortexing is required.
13	Count each tube for at least 60 seconds in a gamma counter.
14	Calculate the TSH concentration of the samples as described in Calculation of results or use special software.

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

Tube	Total	Standard	Sample	Control
Standard		200
Sample			200
Control				200
Tracer	(200)	200	200	200
Vortex mix Incubate for 45 minutes at room temperature
Magnetic immunosorbent		500	500	500
Vortex mix Incubate for 15 minutes at room temperature
Place the tubes on the magnetic separator for 5 minutes.
Decant the fluid and blot on filter paper
Wash Buffer		1000	1000	1000
Vortex mix.
Place the tubes on the magnetic separator for 5 minutes.
Decant the fluid and blot on filter paper
Count radioactivity (60 sec/tube)
Calculate the results.

Characterization of the assay for OPTION B

Typical standard curve for the Turbo TSH I-125 IRMA kit (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

	CPM 1	CPM 2	CPM mean	B/T %
Total	408 066	405 954	407 010	-
S₀(NSB)	54	90	72	0.018
S_0.06	478	522	500	0.123
S_0.15	895	1 037	966	0.237
S_0.6	3 864	3 642	3 753	0.922
S_2.5	15 321	14 229	14 775	3.63
S₁₅	82 328	84 720	83 524	20.52
S₅₀	218 273	215 871	217 072	53.33
S₁₀₀ (B_max)	310 516	308 014	309 265	75.99
CI	1 906	2 088	1 997	0.30
CII	121 274	119 754	120 514	19.3

Sensitivity

The value of analytical sensitivity is 0.008 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: 0.05 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 8/1.

	intra-assay
sample No.	mean (µIU/ml)	CV %
1	0.040	39.6
2	0.062	18.5
3	0.082	13.4
4	0.101	13.7
5	0.128	16.1
6	0.135	10.3
7	0.199	3.8
8	0.289	7.4
9	0.609	2.2
10	0.710	2.4
11	0.964	1.9
12	1.17	2.8
13	1.80	2.1
14	3.17	1.6
15	6.39	2.9
16	19.47	1.8
17	22.12	1.5

		inter-assay
sample No.	number of assay runs	mean (µIU/ml)	CV %
1	19	0.036	28.5
2	19	0.061	16.7
3	19	0.080	9.1
4	14	0.094	7.1
5	20	0.118	7.1
6	20	0.136	5.7
7	21	0.196	6.9
8	21	0.284	4.5
9	21	0.607	2.6
10	20	0.735	3.5
11	20	0.988	3.6
12	21	1.20	3.4
13	21	1.85	3.5
14	21	3.14	2.5
15	21	6.34	2.7
16	21	19.87	2.7
17	21	22.89	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 9.

sample No.	dilution factor	expected µIU/ml	observed µIU/ml	recovery %
1	1		12.31
1	2.02	6.11	5.89	96.4
1	4.08	3.02	2.91	96.5
1	8.23	1.50	1.47	98.0
2	1		25.17
2	2.02	12.48	12.24	98.1
2	4.05	6.22	6.07	97.7
2	8.14	3.09	3.01	97.4
3	1		32.81
3	1.99	16.47	15.68	95.2
3	3.97	8.26	7.83	94.9
3	7.92	4.14	3.97	95.8
4	1		12.92
4	1.99	6.48	6.73	103.8
4	3.97	3.25	3.37	103.7
4	7.96	1.62	1.69	104.2
5	1		30.55
5	2.03	15.06	14.48	96.1
5	4.12	7.42	6.54	88.1
5	8.33	3.67	3.29	89.7
6	1		1.43
6	2.03	0.705	0.687	97.5
6	4.10	0.349	0.341	97.8
6	8.33	0.172	0.158	92.0
7	1		22.99
7	2.00	11.46	11.74	102.4
7	4.04	5.69	5.87	103.1
7	8.12	2.83	2.91	102.8
8	1		0.867
8	2.02	0.429	0.411	95.9
8	4.09	0.212	0.195	92.0
8	8.25	0.105	0.092	87.5
9	1		1.88
9	2.00	0.938	0.930	99.1
9	4.00	0.470	0.472	100.5
9	8.00	0.235	0.233	99.3
10	1		14.13
10	2.02	7.01	7.08	101.0
10	4.05	3.49	3.57	102.3
10	8.18	1.73	1.69	98.0

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 summarized below.

Table 10.

sample	basic µIU/ml	added µIU/ml	expected µIU/ml	observed µIU/ml	recovery %
1	0.916	14.80	15.72	13.75	86.7
2	0.613	14.59	15.21	13.50	88.3
3	1.02	14.69	15.71	13.87	87.5
4	0.809	14.77	15.58	14.18	90.5
5	1.86	14.44	16.30	15.48	94.4
6	0.215	2.90	3.11	3.40	110.0
7	0.006	2.77	2.78	2.95	106.3
8	0.007	2.82	2.83	3.04	107.6
9	0.025	2.85	2.88	3.09	107.4
10	0.032	2.77	2.80	3.34	119.4
11	0.126	3.78	3.90	4.23	108.5
12	0.076	2.86	2.93	3.09	105.5
13	0.024	3.24	3.26	3.35	102.7
14	0.062	2.82	2.89	3.39	117.9
15	0.011	2.87	2.88	3.05	106.0
16	0.027	3.11	3.14	3.28	104.6
17	0.001	3.56	3.56	3.80	106.9
18	0.042	2.78	2.82	2.99	106.1
19	0.007	3.51	3.52	3.64	103.5
20	0.004	2.78	2.78	2.94	105.8
21	0.003	3.27	3.27	3.50	106.9
22	0.011	2.76	2.77	3.02	108.9
23	0.916	14.80	15.72	13.75	86.7

Option independent data

Specificity

No cross reactivity with hFSH and hTSH can be detected in normal physiological concentrations. 2 000 mIU/ml hCG gives an apparent 3.5 µIU/ml increase in hTSH concentration.

Expected Values

Expected euthyroid range is 0.25 µIU/ml - 3.55 µIU/ml.

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.

Calculation of results

The calculation is illustrated using representative data. The assay data collected should be similar to those shown in Table 2 / 7.

Calculate the average count per minute (cpm) for each pair of assay tubes. Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation:

	Standard [Control, Sample] (cpm)
B / T (%) =	————————————	x 100
	T (cpm)

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

Determine the hTSH concentration of the controls & unknown samples by interpolation from the standard curve. Automated data processing systems are also applicable.

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 operating instructions	Catalogue number	Wash buffer

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Turbo TSH (thyroid stimulating hormone) IRMA test