AU656479B2

AU656479B2 - Method and standard solution for the determination of thyroxine (T4) or triiodothyronine (T3)

Info

Publication number: AU656479B2
Application number: AU44534/93A
Authority: AU
Inventors: Peter Bialk; Nicholas Hoyle; Helmut Lenz
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1992-08-14
Filing date: 1993-08-09
Publication date: 1995-02-02
Anticipated expiration: 2013-08-09
Also published as: CA2103646A1; ES2115703T3; KR940004326A; EP0583717B1; AU4453493A; JP2542787B2; JPH06213898A; DE59308274D1; ZA935914B; IL106634A0; DE4226949A1; EP0583717A1; ATE164229T1

Abstract

In a process for determining thyroxine (T4) or triiodothyronine (T3) in serum, a standard solution which contains bovine thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution is used for calibration.

Description

i-:u i' i :1 Sb 656479

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Boehringer Mannheim GmbH ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

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Method and standard solution for the triiodothyronine

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3 determination The following statement is a full description of this invention, including the best method of performing it known to me/us:of thyroxine

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4P m :?i i. Ir~l 3a- Description oI The invention concerns a method for the determination of thyroxine (T 4 or triiodothyronine (T 3 in serum using a standard solution as well as this standard solution.

The thyroid hormones are present in blood mainly in a protein-bound form. The most important carrier protein for this is thyroxine-binding globulin, which is denoted TBG, to which ca. 80 of the total thyroxine and triiodothyronine present in blood is bound.

Approximately 5 to 10 of the total thyroxine is present bound to albumin and about 10 to 15 of the total thyroxine is present bound to pre-albumin. The totality of these proteins is also referred to as thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronine is present in blood in a free form; usually ca. 0.03 of T 4 or 0.3 of T 3 Only the released hormones are physiologically active whereas the bound hormones T 3 and T4 circulate in the blood as a metabolically inert transport form and serve as a buffer to regulate the level. It is therefore important to determine the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood since this represents the functional thyroid status independent of changes in the concentration or saturation of the thyroxine binding protein.

Various methods are known for the determination of total thyroxine (T 4 free thyroxine (FT 4 and triiodothyronine (T 3 A difficulty in all of these known methods is the provision of a standard which is suitable for the calibration.

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i For the calibration it is necessary to produce human serum standards with particular thyroxine or triiodothyronine concentrations and a defined FT 4 or FT 3 content. Human serum has previously been used as starting material for this. Firstly all the thyroxine and triiodothyronine in a free as well as in a bound form has to be removed from the human serum and then the human serum treated in this way has to again be supplemented with defined amounts of thyroxine or triiodothyronine. Various methods are described in the literature for this purpose such as treatment with active charcoal, with ion exchangers or with carrierbound anti-T 4 or anti-T 3 antibodies. These methods are all very complicated. A further problem is that each human serum has a different co-position thus leading to large variations in the individual lots and as a consequence the reproducibility leaves much to be desired.

A further disadvantage of these known standard solutions for the determination of T 3 and T 4 is that these solutions, which have the composition of human serum, cannot be stored for longer periods since on the one hand the protein which is present is gradually denatured and on the other hand there is a shift in the set equilibrium between bound and free T 3 and T 4 It is known from EP-A 0 337 467 that in order to solve the above-mentioned problems a standard solution can be used for a method to determine thyroxine or triiodothyronine which is still stable after long storage and can be produced in a simple manner using relatively cheap starting materials by using at least one standard solution for the calibration that contains 1.

I r I -3 TBG and thyroxine or triiodothyronine dissolved in a buffer solution.

It has now been found that a further quite considerable improvement of the stability on storage can be achieved if TBG from bovine serum is used in the aforementioned methods.

The invention therefore concerns a method for the determination of thyroxine (T 4 or triiodothyronine (T 3 in serum in which a standard solution is used for the calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution which is characterized in that bovine TBG is used.

Whereas when using the known methods the standard ,solution in liquid reagent form can only just achieve a minimum storage capability of 8 weeks and therefore has to be stored in a lyophilized form, the standard solution used according to the present invention is stable on storage in a liquid form for more than times as long. According to the results obtained thus far the storage stability in a liquid form will attain at least 18 months and after this time period there is still a full binding capacity for T 4 and T 3 whereas the corresponding standard solution with human TBG even when stored below 10 0 C already yields erroneous values which -are 20 too high after 12 weeks. Moreover the bovine serum used to isolate bovine TBG is much more readily obtainable than human serum.

The superior stability of the standard solution according to the present invention can be seen in

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4 k Table 1. From this it is apparent that with human TBG (H-TBG) the analyte stability decreases rapidly so that erroneously high values are found which already after 3 months are 20 higher 120 of the initial value) than with a fresh solution. In contrast using the standard solution according to the present invention no change whatsoever can be observed even after 12 months i.e. the analytical values are unchanged and correct at 100 The stated contents in percent relate to the measured recovery of analyte compared to a fresh product. Here it should be noted that analyte bound to TBG, i.e. T 4 or T 3 is not detected at all and an increase in the value for T 4 or T 3 means that the binding to TBG has deteriorated thus yielding false values compared to fresh natural TBG. The limit which is still just acceptable is a change of 5 Various methods are known for determining thyroxine and triiodothyronine. Methods of determination are for *0 example described in Nuc. Compact 16 (1985), 321-327, J.

Clin. Immunoassay Z (1984), 192-205, and J. Clin. Chem.

Clin. Biochem. 22 (1984), 895-904. Determination methods based on immunoassays are used especially for determining thyroid hormones.

In a known method for the determination of thyroxine or triiodothyronine in serum based on an immunoassay Stechnique labelled T 4 or T 3 and T 4 or T 3 from the sample S usually compete for a binding partner such as for example an anti-T 4 antibody. By adjusting the reaction conditions such as the concentration of the individual components, incubation period etc., it is possible co either determine the total thyroxine or total triiodothyronine or the free thyroxine or free triiodothyronine. For this the amount of labelled T 3 or 5

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4 bound to the anti-T 4 or anti-T 3 antibody or non-bound labelled T 3 or T 4 is determined via known detection reactions. After measuring the label the content of T 3 or T can then be calculated by comparing the value obtained to values obtained with known T3 or T 4 concentrations. For this it is necessary to establish a calibration curve in which the measurement signal (e.g.

the absorbance value) is plotted against the T3 or T 4 concentration. In the case of thyroxine it is expedient to establish a calibration curve from six thyroxine solutions with different concentrations since there is no linear relationship which would allow a two-point standard measurement.

The method according to the present invention is suitable for the determination of free thyroxine (FT 4 S free triiodothyronine (FT 3 as well as of total thyroxine (T 4 and thyroxine (T 3 in serum.

The thyroxine-binding bovine globulin can be isolated from bovine sera in a known manner. The standard solution used according to the present invention S' preferably contains 5 to 30 Ag/ml bovine TBG. It is I particularly preferred to use bovine TBG in a physiological amount which is in the range from 9 to 20 Ag/ml TBG which for example can be gathered from the 'literature references Lab. Med. 6 (1982), 27-29, and J.

4 Clin. Chem. Clin. Biochem. 23 (1985) 117-127.

If the standard solution is to be used to determine thyroxine it additionally contains thyroxine. The thyroxine is preferably present in an amount of 5 to 500 ng/ml. It is particularly preferable to add a physiological amount of thyroxine to the standard V S 300 ng/ml.

If the standard solution is to be used to determine triiodothyronine it additionally contains triiodothyronine. The triiodothyronine is preferably used in an amount of 0.5 to 12 ng/ml. It is particularly preferable to add triiodothyronine in a physiological amount i.e. in the range of 0.5 to 8 ng/ml.

The standard solution used for the method according to the present invention preferably also contains albumin which improves the stability of the solution in addition to bovine TBG and thyroxine or triiodothyronine in buffer solution. In this connection it is not necessary to add human serum albumin, which is of course suitable, to the standard solution preferred according to the o present invention. Equally suitable are the universally and easily obtainable and much more preferable albumins, h ifor example from bovine or horse serum. Human serum albumin, bovine serum albumin or horse serum albumin and in particular bovine serum albumin is preferably used as Salbumin. The albumin is preferably used in an amount of to 80 mg/ml solution. It is particularly preferable to use albumin in a physiological amount which is in a range from 50 to 70 mg/ml.

SIn order to produce the standard solution, bovine TBG and, if desired, albumin are dissolved in a buffer solution. All buffers which have a pH range of 6.0 to are suitable as the buffer system, preferably those with a pH value of 6.5 to 7.5. GOOD buffers are preferably used, for example HEPES or TRIS buffer. The buffer concentration is not critical, the buffer is preferably used at a concentration of 30 to 150 mmol/l.

i. 1 I -7 to 100 mmol/l buffer.

Subsequently the thyroxine or triiodothyronine in appropriate concentrations is added to this solution. In this process an equilibrium is established between protein bound and free thyroid hormones which is similar to the equilibrium which is present in vivo in blood.

This solution is also stable over long periods of storage because of its composition. Since it is produced from standardized individual substances it always has a uniform composition and therefore yields reproducible values.

In order to establish a calibration curve, several standard solutions as a rule four to six are used with different contents of thyroid hormones so that a curve can be drawn from the values obtained. In a preferred embodiment of the method according to the present invention a solution is used as the first standard solution which only contains bovine TBG and, if desired, albumin in a buffer solution but contains no thyroid hormone. Solutions with defined hormone contents are then used for the further calibration.

The invention also concerns a standard solution for the determination of thyroxine or triiodothyronine which contains bovine TBG and thyroxine or triiodothyronine dissolved in a buffer system.

The standard solution preferably contains 5 to 30 Ag/ml bovine TBG. It is particularly preferred that a physiological amount of bovine TBG, i.e. 9 to 20 gg/ml, is present in the standard solution.

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I 1 i 8 In a preferred embodiment the standard solution additionally contains albumin for stabilization. In this case the albumin is preferably present in an amount of to 80 mg/ml. It is particularly preferable to use physiological amounts of albumin i.e. in the range of to 70 mg/ml albumin.

The standard solution is produced by dissolving bovine TBG and, if desired, albumin in a buffer system and adding the amount of thyroxine or triiodothyronine desired in each case. In this connection the amount of thyroxine is preferably in the range of 5 to 500 ng/ml and particularly preferably in the physiological range of 5 to 300 ng/ml. If the standard solution is used to determine triiodothyronine it then contains triiodothyronine preferably in an amount of 0.5 to 12 ng/ml and particularly preferably in a physiological amount in the range of 0.5 to 8 ng/ml.

The buffer system preferably has a pH in the range to 8.0 and is preferably a GOOD buffer.

According to the invention a standard solution with a superior stability is provided which can be produced in a simple manner from easily obtainable starting materials.

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,i! 9 -9- Example 1 Isolation of bovine TBG Bovine plasma is brought to a concentration of 2 mol/1 ammonium sulfate, the supernatant is decanted off diluted to 1 mol/l ammonium sulfate and applied to a chromatography column (Sepharose to which T 4 is bound).

It is washed in succession with: 1. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 2. 50 mmol/1 Tris pH 7.5, 750 mmol/l sodium chloride 3. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride 4. 50 mmol/l Tris pH 7.5, 100 mmol/l sodium chloride mmol/l 8-anilino-naphthaline sulfonic acid.

The eluate is concentrated in an osmotic cell and 8anilino-naphthaline sulfonic acid is removed by e dialysing for two days against 5 mmol/l sodium phosphate pH 7.5, 10 mmol/1 sodium chloride.

Example 2 Stability of T 3

/T

4 calibrators S Composition of the calibrators: 6 bovine serum albumin, mmol/ 1 Hepes buffer, pH 7.4 g/ml bovine TBG

FT

3 content: 0, 2, 7, 15, 30 pg/ml or FT 4 content: 1, 10, 20, 40, 80 pg/ml if 10 The following Table shows the stability of T3/T 4 calibrators.

Table 1: Storage period Recoveryl) with at 4 80C an analyte content (months) of 2 30 pg/ml

T

3 without TBG

T

3 with human TBG

T

3 with bovine TBG 118 130 115 130 138 100 97 1) The recovery shows the measurement value found for T 3 in relation to the measurement value for T 3 at the beginning of storage (100 ii fjr r

Claims

1. Method for the production of a standard solution for calibrating a standard curve in a method for the determination of thyroxine or triiodothyronine in serum comprising the steps of dissolving bovine TBG in a de.ined concentration in a buffer solution, adding a defined amount of thyroxine or triiodothyronine.

2. Method according to claim 1, wherein 5 to 30 pg/ml bovine TBG are dissolved in the buffer solution. r r I r ~I

3. Method according to claims 1 ng/ml thyroxine are added.

4. Method according to claims 1 /m\ ng triiodothyronine are added.

5. Method according to claims 1 added to the solution.

6. Method according to claim 5, ablumin are added.

7. Method according to claims 5 serum albumin is used as albumin. or 2, wherein 5 to 500 or 2, wherein 0.5 to 12 to 4 wherein albumin is wherein 40 to 80 mg/ml to 6, wherein bovine J 14 .4

8. Standard solution for calibrating a standard curve in a method for the determination of thyroxine or triiodothyronine in serum comprising bovine TBG in a defined concentration and thyroxine or triiodothyronine in a defined concentration dissolved in a buffer solution.

9. Standard solution according to claim 8 further comprising albumin in a defined concentration. 940922,p:\oper\jmw,cl44534-93.265,11 Ir ;i I r, Ohl ltL LI: h 'I -12- Standard solution according to claims 8 or 9 produced by the methods oi claims 1 to 7.

11. Method for the determination of thyroxine or triiodothyronine in serum wherein a standard solution according to claims 8 to 10 is used for calibrating a standard curve.

12. A method according to claim 1 or claim 11 or a standard solution according to claim 8 when ed. in 14 i-nventie-n substantially as hereinbefore described in any one of the Examples. r oo s D r I o~r o r r o s DATED this 26th day of SEPTEMBER 1994 Boehringer Mannheim GmbH By DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) >1 I 1ii 0i~i 940926,p:\operjmwc144534-93.265,12 r:.l I: 1 IL r Abstract A standard solution containing bovine thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine dissolved in a buffer solution is used for calibration in a method for the determination of thyroxine (T 4 or triiodothyronine (T 3 in serum. rr r r r r I oo 1i S L