CA1334925C - Process for the determination of thyroxine and a suitable standard solution therefor - Google Patents
Process for the determination of thyroxine and a suitable standard solution thereforInfo
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- CA1334925C CA1334925C CA000595751A CA595751A CA1334925C CA 1334925 C CA1334925 C CA 1334925C CA 000595751 A CA000595751 A CA 000595751A CA 595751 A CA595751 A CA 595751A CA 1334925 C CA1334925 C CA 1334925C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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Abstract
For the determination of thyroxine (T4) or triiodothyronine (T3) in serum a standard solution is used for calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine respectively dissolved in a buffer solution.
Description
D e s c r i p t i o n The invention concerns a process for the determination of thyroxine (T4) or triiodothyronine (T3) in serum --using a standard solution as well as this standard solution.
The thyroid hormones occur in blood mainly in protein-bound form. The main carrier protein is the thyroxine-binding globulin, called TBG, on which about 80% of the entire thyroxine and triiodothyronine present in blood are bound. About 5 to 10% of the total thyroxine is bound to albumin and about 10 to 15% of the total thyroxine is bound to pre-albumin. These proteins as a whole are called thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronine are present in blood in the free form, as a rule this corresponds to about 0.03% of T4 and 0.3% of T3 respectively. Only the free hormones are physiologically active, whereas the bound hormones T3 and T4 circulate in blood in a metabolically inert transport form and serve as a buffer to regulate the concentration. It is therefore important to determine both the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood, since these reflect the functional thyroid status independent of alterations in the concentration or saturation of the thyroxine-binding proteins.
Various methods are known for measuring total thyroxine (T4), free thyroxine (FT4) and triiodothyronine (T3). A
difficulty with all of these methods is the provision of suitable standards for calibration. For the calibration, it is necessary to prepare human serum standards with definite thyroxine or triiodothyronine concentrations and with a definite FT4 or FT3 content. Until now human serum has been used as the raw material for this. All thyroxine and triiodothyronine in the free as well as the bound form must first be removed from the human serum and then defined quantities of thyroxine or triiodothyronine must be reintroduced into the human serum so treated. Various methods are described in the literature for this purpose, such as treatment with active charcoal, with ion-exchangers or with carrier-bound anti-T4 or anti-T3-antibodies. These procedures are all very complicated. Apart from this another problem is that each human serum has a different composition and as a consequence the individual batches show large variations and thus the reproducibility leaves much to be desired.
A further disadvantage of standard solutions known up to now for the determination of T3 and T4 is that these solutions, which have the composition of human serum, cannot be stored for long periods because, on the one hand, the proteins which they contain are progressively denatured and, on the other hand, a shift occurs in the adjusted equilibrium betwen bound and free T3 and T4.
Accordingly, it was an object of the present invention to provide a standard solution for a process to determine thyroxine or triiodothyronine which is also stable on longer storage and which can be produced in a simple manner using cheap raw materials.
This object was achieved by a process for the determination of thyroxine or triiodothyronine in serum wherein at least one standard solution is used for calibration containing TBG and thyroxine or _ 3 _ l 33 4 9 25 triiodothyronine respectively dissolved in a buffer solution.
It was established that, in a solution containing TBG
and thyroxine or triiodothyronine dissolved in a buffer system, the equilibrium which establishes between bound and free T4 or T3 is just as in human serum.
Surprisingly, the incomplete equilibration system according to the present invention, in which components of the natural thyroxine-binding proteins are missing, yields even better comparative values than a natural matrix based on human serum which was first made free from and then augmented with thyroid hormone, so that after appropriate calibration such solutions can be used as standard solutions. Thus, in accordance with the present invention a standard solution is provided for a process to determine thyroxine or triiodothyronine which is simple to produce.
Various processes are known for the determination of thyroxine and triiodothyronine. Methods of determination are described for example in Nuc. Compact 16 (1985), 321-327, J. Clin. Immunoassay 7 (1984), 192-205, and J.
Clin. Chem. Clin. Biochem. 22 (1984), 895-904. In particular, immunoassay techniques have been employed for the measurement of thyroid hormones.
In a known process for the measurement of thyroxine or triiodothyronine in serum using an immunoassay technique, labelled T4 or T3 usually compete respectively with T4 or T3 in the sample for a binding partner such as an anti-T4-antibody. By adjustment of the reaction conditions, such as concentration of the individual components, incubation time etc. either the total thyroxlne or triiodothyronine or free thyroxine or - 4 - l 3 3 4 9 2S
triiodothyronine can be determined. For this purpose, the amounts of labelled T4 or T3 bound to the anti-T4 or anti-T3-antibodies and the amounts of unbound labelled T4 or T3 respectively are determined using known methods of detection. The content of T4 or T3 can then be calculated after measurement of the label by relating the measurements to values obtained with known T4 or T3 concentrations. In order to do this, it is necessary to set up a calibration curve in which the measurements (for example the absorbance readings) are plotted against the T4 or T3 concentrations. In the case of thyroxine it is advisable to establish a calibration curve using six thyroxine solutions of different concentrations, since the relationship is not linear and would not allow a two-point standardisation.
The process according to the present invention is suitable for determining free thyroxine (FT4), free triiodothyronine (FT3) as well as total thyroxine (T4) and triiodothyronine (T3) in serum.
The thyroxine-binding globulin can be extracted from human sera using known methods. The standard solution according to the present invention preferably contains 5 to 30 ~g/ml TBG. It is particularly preferable to add TBG in a physiological amount which ranges from 9 to 20 ~g/ml TBG according to, for example, the following literature references Lab. med. 6 (1982), 27-29, and J.
Clin. Chem. Clin. Biochem. 23 (1985) 117-127.
When used to determine thyroxine the standard solution contains in addition thyroxine. The preferred amount of thyroxine is from 5 to 500 ng/ml. The addition of physiological quantities is particularly preferred, i.e.
an amount in the range from 5 to 300 ng/ml.
_ 5 _ 1 3 3 4 9 2 5 When used to determine triiodothyronine the standard solution also contains triiodothyronine.
Triiodothyronine is employed in a preferred amount of 5 to 500 ng/ml. The addition of physiological quantities is particularly preferred i.e. in the range from 0.5 to 8 ng/ml.
The standard solution used for the process according to the present invention contains, apart from TBG and thyroxine or triiodothyronine, albumin in the buffer solution which improves its stability. For this purpose it is, however, not necessary to add human serum albumin to the preferred standard solution according to the present invention although this albumin is of course suitable. Equally suitable are the readily available and much more favourable albumins from for example bovine or horse serum. The following albumins are preferred: human serum albumin, bovine serum albumin and horse serum albumin, whereas bovine serum albumin is particularly preferred. The albumin is used in a preferred amount of from 40 to 80 mg/ml solution. Physiological amounts of albumin which range from 40 to 55 mg/ml according to Documenta Geigy, Wissenschaftl. Tabellen, 7. Ausg. 1975, S. 578, are particularly preferred.
To produce the standard solution, TBG and if necessary albumin are dissolved in a buffer solution. All buffers with a pH range from 6 to 8, preferably those with a pH
value from 6.5 to 7.5 are suitable for the buffer system. It is preferable to use GOOD-buffers, for example HEPES- or TRIS-buffer. The buffer concentration is not critical; it is preferable to use a buffer concentration of from 30 to 150 mmol/l. Especially preferred are standard solutions containing 50 to 100 mmol/l buffer.
- 6 _ 1 33 4 9~S
Thyroxine or triiodothyronine are than added to this solution in appropriate concentrations. As a result, an equilibrium is established between protein-bound and free thyroid hormone analogous to the in vivo equilibrium which exists in blood. Because of its composition this solution is stable even over long periods of storage. As it is made up of standardized individual substances it always shows a stable composition and therefore yields reproducible results.
To establish a calibration curve several standard solutions, as a rule four to six, with different thyroid hormone contents are used in order to plot a curve from the measurements thus obtained. In a preferred embodiment of the process according to the present invention, a solution is used as the first standard solution which only contains TBG and if necessary albumin dissolved in a buffer solution but which contains no thyroid hormone. For the remaining calibration, solutions with a defined hormone content are employed.
A further embodiment of the invention is a standard solution for determining thyroxine or triiodothyronine which contains TBG and thyroxine or triiodothyronine respectively dissolved in a buffer system.
The standard solution preferably contains 5 to 30 ~g/ml TBG. The presence of a physiological quantity of TBG in the standard solution, i.e. in the range from 9 to 20 ~g/ml, is especially preferred.
- 7 - l 3 3 4 9 2 5 In a preferred embodiment the standard solution contains in addition albumin for stabilization. The albumin content is preferably from 40 to 80 mg/ml. The employment of physiological amounts of albumin i.e. in the range from 40 to 55 mg/ml albumin is especially preferred.
The standard solution is prepared by dissolving TBG and if necessary albumin in a buffer system and then adding the desired amount of thyroxine or triiodothyronine. The quantity of thyroxine preferably ranges from 5 to 500 ng/ml and it is especially preferable to use physiological quantities of from 5 to 300 ng/ml. If the standard solution is used to determine triiodothyronine then it contains triiodothyronine preferably in a quantity between 0.5 and 12 ng/ml and it is especially preferable to use physiological amounts in the range from 0.5 to 8 ng/ml.
The appropriate pH of the buffer system is between 6 and 8 and it is preferable to use GOOD-buffers.
According to the present invention a standard solution with superior stability is provided which can be prepared from easily obtainable raw materials.
- 8 - l 3 3 4 9 2 5 The following figures and examples elucidate the inventlon .
Figure 1 shows a calibration curve for different --thyroxine concentrations which were obtained by determining FT4 using a heterogeneous immunoassay.
igure 2 shows a calibration curve for different T4 concentrations which were used for the determination of total thyroxine .
igure 3 shows a calibration curve for different T3 concentrations which can be used for the determination of total triiodothyronine .
xample 1 Solutions containing different thyroxine concentrations were prepared. The solutions had the following composition:
50 mmol/l HEPES-buffer pH 7.0;
60 mg/ml bovine serum albumin;
15 ~g/ml thyroxine-binding globulin (TBG);
increasing quantities of T4 were added to this solution to yield the following concentrations of free thyroxine after equilibrium was established: O; 0.9; 1.5; 2.5;
4.3; 6.5 ng FT4/dl.
In addition, solutions with the same compositions were prepared which, however, contained human serum albumin or horse serum albumin instead of bovine serum albumin.
The following reagents were used to determine the free thyroxine:
Reagent 1: 55 mmol/l barbital buffer, pH 7.8; ---10 mU/ml T4-peroxidase-conjugate;
eagent 2: 1.9 mmol/l ABTS (2,2'-azino-di-[3-ethyl-benz-thiazoline-6-sulphonate]-diammonium salt);
3.2 mmol/l sodium perborate;
100 mmol/l phosphate/citrate buffer, pH 4.4.
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T4-antibodies.
1 ml of Reagent 1 and 20 ~1 of the standard solution were pipetted into these tubes and incubated for 60 min at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 30 min at room temperature. The absorbance was then measured photometrically at 405 nm or 422 nm.
The absorbance readings corresponding to each of the standard concentrations were plotted on graph paper and a curve was then drawn which is shown in Figure 1. From the absorbances of the measured samples, the FT4 concentrations can be read from the calibration curve.
It can be seen that bovine and horse serum albumin yield the same values as human serum albumin.
- lo - 1 334925 ExamPle 2 A standard solution was prepared according to the present invention and stored over a long period under the conditions indicated. Parallel to this, standard solutions were prepared according to conventional methods of the prior art, i.e. solutions of increasing quantities of thyroxine in T4-free human serum, and stored in an analogous fashion. It was then examined how long the standard solutions remained stable.
The solutions prepared according to Example 1 were used as standard solutions according to the present invention and contained respectively O; 0.9; 1.5; 2.5; 4.3; and 6.-5 ng FT4/dl.
Following this, these solutions were lyophilized and stored for three weeks at 35C. After reconstitution they were stored for a further eight weeks at 4C in solution. The solutions were then employed for a determination of FT4 as described in Example 1.
The average recovery of the standard absorbances before and after storage is shown in the following Table I:
T a b 1 e I
Standard Before After storage storage standard solution 100 % 99 %
according to the present invention human serum standard 100 % 89 %
solution As can be seen from these values the standard solution according to the present invention is very much more stable than human serum solutions of the prior art.
Example 3 The recovery of FT4 was examined in 21 human sera. The determination of FT4 was carried out at 20C or 30C as described in Example 1. The same standard solutions were used as in Example 2. These solutions were lyophilized and stored for three weeks at 35C. Afterwards the lyophilizates were reconstituted and stored for a further two weeks at 25C in solution.
As the results of Table II show, recovery is independent of temperature when calibration is performed with the standard solution according to the present invention even after long periods of storage. In contrast, using the more unstable human serum standard solutions different FT4 concentrations were measured at 20C and 30C.
T a b 1 e II
Standard Average recovery of 21 human sera at 30C in comparison to recovery at 20C
Standard solution according to the present invention - 0.3%
human serum standard solution - 19.0%
Example 4 Standard solutions prepared according to Example 1 were used for a T4 test.
The following reagents were used for this:
Reagent 1: 120 mmol/l barbiturate;
18.2 mmol/l phosphate-buffer, pH 8.6;
1.27 mmol/l ANS (8-anilino-1-naphthalene-sulphonic acid);
15 mU/ml T4-peroxidase-conjugate;
Reagent 2: composition as described in Example 1.
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T4-antibodies.
1 ml of Reagent 1 and 20 ~1 of the standard solution were pipetted into these tubes and incubated for 30 min at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 30 min at room temperature. The absorbance was then measured photometrically at 405 nm or 422 nm.
The absorbance readings corresponding to each of the standard concentrations were plotted on graph paper and a curve was then drawn which is shown in Figure 2.
- 13 - l 3 3 4 9 2 5 ExamPle 5 Solutions containing different triiodothyronine concentrations were prepared. The solutions had the following composition:
50 mmol/l HEPES-buffer, pH 7.0 60 mg/ml bovine serum albumin 15 ~g/ml thyroxine-binding globulin (TBG) Increasing quantities of T3 were added. Standard solutions prepared in this way were employed in a T3-test. For this the following reagents were used (concentrations of the ready-to-use solutions):
eagent 1: 120 mmol/l barbiturate;
18.2 mmol/l phosphate-buffer, pH 8.6;
1.27 mmol/l ANS
(8-anilino-1-naphthalene-sulphonic acid);
12 mU/ml T3-peroxidase-conjugate.
eagent 2: lOOmmol/l phosphate-citrate-buffer, pH 5.0;
1.47 mmol/l sodium perborate;
9.1 mmol/l ABTSR (2,2'-azino-di-[3-ethyl-benz-thiazoline-6-sulphonate]-diammonium salt).
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T3-antibodies.
1 ml of Reagent 1 and 100 ~l of the standard solution were pipetted into these tubes and incubated for 2 hours at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 60 min at room temperature. The 1 3349~5 absorbance was then measured photometrically at 405 nm or 422 nm. The results are shown in Figure 3.
The thyroid hormones occur in blood mainly in protein-bound form. The main carrier protein is the thyroxine-binding globulin, called TBG, on which about 80% of the entire thyroxine and triiodothyronine present in blood are bound. About 5 to 10% of the total thyroxine is bound to albumin and about 10 to 15% of the total thyroxine is bound to pre-albumin. These proteins as a whole are called thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronine are present in blood in the free form, as a rule this corresponds to about 0.03% of T4 and 0.3% of T3 respectively. Only the free hormones are physiologically active, whereas the bound hormones T3 and T4 circulate in blood in a metabolically inert transport form and serve as a buffer to regulate the concentration. It is therefore important to determine both the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood, since these reflect the functional thyroid status independent of alterations in the concentration or saturation of the thyroxine-binding proteins.
Various methods are known for measuring total thyroxine (T4), free thyroxine (FT4) and triiodothyronine (T3). A
difficulty with all of these methods is the provision of suitable standards for calibration. For the calibration, it is necessary to prepare human serum standards with definite thyroxine or triiodothyronine concentrations and with a definite FT4 or FT3 content. Until now human serum has been used as the raw material for this. All thyroxine and triiodothyronine in the free as well as the bound form must first be removed from the human serum and then defined quantities of thyroxine or triiodothyronine must be reintroduced into the human serum so treated. Various methods are described in the literature for this purpose, such as treatment with active charcoal, with ion-exchangers or with carrier-bound anti-T4 or anti-T3-antibodies. These procedures are all very complicated. Apart from this another problem is that each human serum has a different composition and as a consequence the individual batches show large variations and thus the reproducibility leaves much to be desired.
A further disadvantage of standard solutions known up to now for the determination of T3 and T4 is that these solutions, which have the composition of human serum, cannot be stored for long periods because, on the one hand, the proteins which they contain are progressively denatured and, on the other hand, a shift occurs in the adjusted equilibrium betwen bound and free T3 and T4.
Accordingly, it was an object of the present invention to provide a standard solution for a process to determine thyroxine or triiodothyronine which is also stable on longer storage and which can be produced in a simple manner using cheap raw materials.
This object was achieved by a process for the determination of thyroxine or triiodothyronine in serum wherein at least one standard solution is used for calibration containing TBG and thyroxine or _ 3 _ l 33 4 9 25 triiodothyronine respectively dissolved in a buffer solution.
It was established that, in a solution containing TBG
and thyroxine or triiodothyronine dissolved in a buffer system, the equilibrium which establishes between bound and free T4 or T3 is just as in human serum.
Surprisingly, the incomplete equilibration system according to the present invention, in which components of the natural thyroxine-binding proteins are missing, yields even better comparative values than a natural matrix based on human serum which was first made free from and then augmented with thyroid hormone, so that after appropriate calibration such solutions can be used as standard solutions. Thus, in accordance with the present invention a standard solution is provided for a process to determine thyroxine or triiodothyronine which is simple to produce.
Various processes are known for the determination of thyroxine and triiodothyronine. Methods of determination are described for example in Nuc. Compact 16 (1985), 321-327, J. Clin. Immunoassay 7 (1984), 192-205, and J.
Clin. Chem. Clin. Biochem. 22 (1984), 895-904. In particular, immunoassay techniques have been employed for the measurement of thyroid hormones.
In a known process for the measurement of thyroxine or triiodothyronine in serum using an immunoassay technique, labelled T4 or T3 usually compete respectively with T4 or T3 in the sample for a binding partner such as an anti-T4-antibody. By adjustment of the reaction conditions, such as concentration of the individual components, incubation time etc. either the total thyroxlne or triiodothyronine or free thyroxine or - 4 - l 3 3 4 9 2S
triiodothyronine can be determined. For this purpose, the amounts of labelled T4 or T3 bound to the anti-T4 or anti-T3-antibodies and the amounts of unbound labelled T4 or T3 respectively are determined using known methods of detection. The content of T4 or T3 can then be calculated after measurement of the label by relating the measurements to values obtained with known T4 or T3 concentrations. In order to do this, it is necessary to set up a calibration curve in which the measurements (for example the absorbance readings) are plotted against the T4 or T3 concentrations. In the case of thyroxine it is advisable to establish a calibration curve using six thyroxine solutions of different concentrations, since the relationship is not linear and would not allow a two-point standardisation.
The process according to the present invention is suitable for determining free thyroxine (FT4), free triiodothyronine (FT3) as well as total thyroxine (T4) and triiodothyronine (T3) in serum.
The thyroxine-binding globulin can be extracted from human sera using known methods. The standard solution according to the present invention preferably contains 5 to 30 ~g/ml TBG. It is particularly preferable to add TBG in a physiological amount which ranges from 9 to 20 ~g/ml TBG according to, for example, the following literature references Lab. med. 6 (1982), 27-29, and J.
Clin. Chem. Clin. Biochem. 23 (1985) 117-127.
When used to determine thyroxine the standard solution contains in addition thyroxine. The preferred amount of thyroxine is from 5 to 500 ng/ml. The addition of physiological quantities is particularly preferred, i.e.
an amount in the range from 5 to 300 ng/ml.
_ 5 _ 1 3 3 4 9 2 5 When used to determine triiodothyronine the standard solution also contains triiodothyronine.
Triiodothyronine is employed in a preferred amount of 5 to 500 ng/ml. The addition of physiological quantities is particularly preferred i.e. in the range from 0.5 to 8 ng/ml.
The standard solution used for the process according to the present invention contains, apart from TBG and thyroxine or triiodothyronine, albumin in the buffer solution which improves its stability. For this purpose it is, however, not necessary to add human serum albumin to the preferred standard solution according to the present invention although this albumin is of course suitable. Equally suitable are the readily available and much more favourable albumins from for example bovine or horse serum. The following albumins are preferred: human serum albumin, bovine serum albumin and horse serum albumin, whereas bovine serum albumin is particularly preferred. The albumin is used in a preferred amount of from 40 to 80 mg/ml solution. Physiological amounts of albumin which range from 40 to 55 mg/ml according to Documenta Geigy, Wissenschaftl. Tabellen, 7. Ausg. 1975, S. 578, are particularly preferred.
To produce the standard solution, TBG and if necessary albumin are dissolved in a buffer solution. All buffers with a pH range from 6 to 8, preferably those with a pH
value from 6.5 to 7.5 are suitable for the buffer system. It is preferable to use GOOD-buffers, for example HEPES- or TRIS-buffer. The buffer concentration is not critical; it is preferable to use a buffer concentration of from 30 to 150 mmol/l. Especially preferred are standard solutions containing 50 to 100 mmol/l buffer.
- 6 _ 1 33 4 9~S
Thyroxine or triiodothyronine are than added to this solution in appropriate concentrations. As a result, an equilibrium is established between protein-bound and free thyroid hormone analogous to the in vivo equilibrium which exists in blood. Because of its composition this solution is stable even over long periods of storage. As it is made up of standardized individual substances it always shows a stable composition and therefore yields reproducible results.
To establish a calibration curve several standard solutions, as a rule four to six, with different thyroid hormone contents are used in order to plot a curve from the measurements thus obtained. In a preferred embodiment of the process according to the present invention, a solution is used as the first standard solution which only contains TBG and if necessary albumin dissolved in a buffer solution but which contains no thyroid hormone. For the remaining calibration, solutions with a defined hormone content are employed.
A further embodiment of the invention is a standard solution for determining thyroxine or triiodothyronine which contains TBG and thyroxine or triiodothyronine respectively dissolved in a buffer system.
The standard solution preferably contains 5 to 30 ~g/ml TBG. The presence of a physiological quantity of TBG in the standard solution, i.e. in the range from 9 to 20 ~g/ml, is especially preferred.
- 7 - l 3 3 4 9 2 5 In a preferred embodiment the standard solution contains in addition albumin for stabilization. The albumin content is preferably from 40 to 80 mg/ml. The employment of physiological amounts of albumin i.e. in the range from 40 to 55 mg/ml albumin is especially preferred.
The standard solution is prepared by dissolving TBG and if necessary albumin in a buffer system and then adding the desired amount of thyroxine or triiodothyronine. The quantity of thyroxine preferably ranges from 5 to 500 ng/ml and it is especially preferable to use physiological quantities of from 5 to 300 ng/ml. If the standard solution is used to determine triiodothyronine then it contains triiodothyronine preferably in a quantity between 0.5 and 12 ng/ml and it is especially preferable to use physiological amounts in the range from 0.5 to 8 ng/ml.
The appropriate pH of the buffer system is between 6 and 8 and it is preferable to use GOOD-buffers.
According to the present invention a standard solution with superior stability is provided which can be prepared from easily obtainable raw materials.
- 8 - l 3 3 4 9 2 5 The following figures and examples elucidate the inventlon .
Figure 1 shows a calibration curve for different --thyroxine concentrations which were obtained by determining FT4 using a heterogeneous immunoassay.
igure 2 shows a calibration curve for different T4 concentrations which were used for the determination of total thyroxine .
igure 3 shows a calibration curve for different T3 concentrations which can be used for the determination of total triiodothyronine .
xample 1 Solutions containing different thyroxine concentrations were prepared. The solutions had the following composition:
50 mmol/l HEPES-buffer pH 7.0;
60 mg/ml bovine serum albumin;
15 ~g/ml thyroxine-binding globulin (TBG);
increasing quantities of T4 were added to this solution to yield the following concentrations of free thyroxine after equilibrium was established: O; 0.9; 1.5; 2.5;
4.3; 6.5 ng FT4/dl.
In addition, solutions with the same compositions were prepared which, however, contained human serum albumin or horse serum albumin instead of bovine serum albumin.
The following reagents were used to determine the free thyroxine:
Reagent 1: 55 mmol/l barbital buffer, pH 7.8; ---10 mU/ml T4-peroxidase-conjugate;
eagent 2: 1.9 mmol/l ABTS (2,2'-azino-di-[3-ethyl-benz-thiazoline-6-sulphonate]-diammonium salt);
3.2 mmol/l sodium perborate;
100 mmol/l phosphate/citrate buffer, pH 4.4.
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T4-antibodies.
1 ml of Reagent 1 and 20 ~1 of the standard solution were pipetted into these tubes and incubated for 60 min at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 30 min at room temperature. The absorbance was then measured photometrically at 405 nm or 422 nm.
The absorbance readings corresponding to each of the standard concentrations were plotted on graph paper and a curve was then drawn which is shown in Figure 1. From the absorbances of the measured samples, the FT4 concentrations can be read from the calibration curve.
It can be seen that bovine and horse serum albumin yield the same values as human serum albumin.
- lo - 1 334925 ExamPle 2 A standard solution was prepared according to the present invention and stored over a long period under the conditions indicated. Parallel to this, standard solutions were prepared according to conventional methods of the prior art, i.e. solutions of increasing quantities of thyroxine in T4-free human serum, and stored in an analogous fashion. It was then examined how long the standard solutions remained stable.
The solutions prepared according to Example 1 were used as standard solutions according to the present invention and contained respectively O; 0.9; 1.5; 2.5; 4.3; and 6.-5 ng FT4/dl.
Following this, these solutions were lyophilized and stored for three weeks at 35C. After reconstitution they were stored for a further eight weeks at 4C in solution. The solutions were then employed for a determination of FT4 as described in Example 1.
The average recovery of the standard absorbances before and after storage is shown in the following Table I:
T a b 1 e I
Standard Before After storage storage standard solution 100 % 99 %
according to the present invention human serum standard 100 % 89 %
solution As can be seen from these values the standard solution according to the present invention is very much more stable than human serum solutions of the prior art.
Example 3 The recovery of FT4 was examined in 21 human sera. The determination of FT4 was carried out at 20C or 30C as described in Example 1. The same standard solutions were used as in Example 2. These solutions were lyophilized and stored for three weeks at 35C. Afterwards the lyophilizates were reconstituted and stored for a further two weeks at 25C in solution.
As the results of Table II show, recovery is independent of temperature when calibration is performed with the standard solution according to the present invention even after long periods of storage. In contrast, using the more unstable human serum standard solutions different FT4 concentrations were measured at 20C and 30C.
T a b 1 e II
Standard Average recovery of 21 human sera at 30C in comparison to recovery at 20C
Standard solution according to the present invention - 0.3%
human serum standard solution - 19.0%
Example 4 Standard solutions prepared according to Example 1 were used for a T4 test.
The following reagents were used for this:
Reagent 1: 120 mmol/l barbiturate;
18.2 mmol/l phosphate-buffer, pH 8.6;
1.27 mmol/l ANS (8-anilino-1-naphthalene-sulphonic acid);
15 mU/ml T4-peroxidase-conjugate;
Reagent 2: composition as described in Example 1.
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T4-antibodies.
1 ml of Reagent 1 and 20 ~1 of the standard solution were pipetted into these tubes and incubated for 30 min at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 30 min at room temperature. The absorbance was then measured photometrically at 405 nm or 422 nm.
The absorbance readings corresponding to each of the standard concentrations were plotted on graph paper and a curve was then drawn which is shown in Figure 2.
- 13 - l 3 3 4 9 2 5 ExamPle 5 Solutions containing different triiodothyronine concentrations were prepared. The solutions had the following composition:
50 mmol/l HEPES-buffer, pH 7.0 60 mg/ml bovine serum albumin 15 ~g/ml thyroxine-binding globulin (TBG) Increasing quantities of T3 were added. Standard solutions prepared in this way were employed in a T3-test. For this the following reagents were used (concentrations of the ready-to-use solutions):
eagent 1: 120 mmol/l barbiturate;
18.2 mmol/l phosphate-buffer, pH 8.6;
1.27 mmol/l ANS
(8-anilino-1-naphthalene-sulphonic acid);
12 mU/ml T3-peroxidase-conjugate.
eagent 2: lOOmmol/l phosphate-citrate-buffer, pH 5.0;
1.47 mmol/l sodium perborate;
9.1 mmol/l ABTSR (2,2'-azino-di-[3-ethyl-benz-thiazoline-6-sulphonate]-diammonium salt).
As reaction vessels polystyrol tubes were used which were coated with polyclonal anti-T3-antibodies.
1 ml of Reagent 1 and 100 ~l of the standard solution were pipetted into these tubes and incubated for 2 hours at room temperature. The tubes were then aspirated and washed with tap water. Afterwards 1 ml of Reagent 2 was added and incubated for 60 min at room temperature. The 1 3349~5 absorbance was then measured photometrically at 405 nm or 422 nm. The results are shown in Figure 3.
Claims (40)
1. Process for the determination of thyroxine (T4) or triiodothyronine (T3) in serum, wherein a first standard solution is used for calibration which contains thyroxine-binding globulin (TBG) and thyroxine or triiodothyronine respectively dissolved in a buffer solution.
2. Process as claimed in claim 1, which comprises a second standard solution which contains TBG
dissolved in a buffer solution but no thyroxine or triiodothyronine.
dissolved in a buffer solution but no thyroxine or triiodothyronine.
3. Process as claimed in claim 1, wherein albumin is added to the standard solution.
4. Process as claimed in claim 2, wherein albumin is added to both standard solution.
5. Process as claimed in claim 3, wherein the albumin used is bovine serum albumin.
6. Process as claimed in claim 3, wherein a standard solution is used which contains 40 to 80 mg/ml albumin.
7. Process as claimed in claim 4, wherein the albumin used is bovine serum albumin.
8. Process as claimed in claim 6, wherein the standard solution used contains 40 to 55 mg/ml albumin.
9. Process as claimed in claim 7, wherein the standard solution used contains 40 to 55 mg/ml albumin.
10. Process as claimed in claim 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein a standard solution is used which contains 5 to 30 µg/ml TBG.
11. Process as claimed in claim 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein a standard solution is used which contains 9 to 20 µg/ml TBG.
12. Process as claimed in claim 1, 3, 4, 5, 6, 7, 8 or 9, wherein a standard solution is used which contains 5 to 500 ng/ml thyroxine.
13. Process as claimed in claim 10, wherein a standard solution is used which contains 5 to 500 ng/ml thyroxine.
14. Process as claimed in claim 11, wherein a standard solution is used which contains 5 to 500 ng/ml thyroxine.
15. Process as claimed in claim 12, wherein the standard solution contains 5 to 300 ng/ml thyroxine.
16. Process as claimed in claim 13 or 14, wherein the standard solution contains 5 to 300 ng/ml thyroxine.
17. Process as claimed in claim 1, 3, 4, 5, 6, 7, 8 or 9, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
18. Process as claimed in claim 10, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
19. Process as claimed in claim 11, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
20. Process as claimed in claim 12, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
21. Process as claimed in claim 17, wherein the standard solution contains 0.5 to 8 ng/ml triiodothyronine.
22. Process as claimed in claim 18 or 19, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
23. Process as claimed in claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 18, 19, 20 or 21, wherein a GOOD-buffer or another buffer with a pH range from 6 to 8 is used as the buffer-system.
24. Process as claimed in claim 10, wherein a GOOD-buffer or another buffer with a pH range from 6 to 8 is used as the buffer-system.
25. Process as claimed in claim 11, wherein a GOOD-buffer or another buffer with a pH range from 6 to 8 is used as the buffer-system.
26. Process as claimed in claim 12, wherein a GOOD-buffer or another buffer with a pH range from 6 to 8 is used as the buffer-system.
27. Process as claimed in claim 17, wherein a standard solution is used which contains 0.5 to 12 ng/ml triiodothyronine.
28. Process as claimed in claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 18, 19, 20, 21, 24, 25 or 26 wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
29. Process as claimed in claim 10, wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
30. Process as claimed in claim 11, wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
31. Process as claimed in claim 12, wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
32. Process as claimed in claim 17, wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
33. Process as claimed in claim 23, wherein a buffer solution is used which contains 30 to 150 mmol/l buffer.
34. Standard solution for the determination of thyroxine or triiodothyronine consisting of albumin, TBG and thyroxine or triiodothyronine dissolved in a buffer solution.
35. Standard solution as claimed in claim 34, wherein said solution contains 5 to 30 µg/ml TBG and 30 to 150 mmol/l buffer.
36. Standard solution as claimed in claim 35, wherein said solution contains 40 to 80 mg/ml albumin.
37. Standard solution as claimed in claim 34, 35 or 36, wherein said solution contains 5 to 500 ng/ml thyroxine.
38. Standard solution as claimed in claim 37, wherein said solution contains 5 to 300 ng/ml thyroxine.
39. Standard solution as claimed in claim 35, 37 or 38, wherein said solution contains 0.5 to 12 ng/ml triiodothyronine.
40. Standard solution as claimed in claim 39, wherein said solution contains 0.5 to 8 ng/ml triiodothyronine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3812609A DE3812609A1 (en) | 1988-04-15 | 1988-04-15 | METHOD FOR DETERMINING THYROXIN AND STANDARD SOLUTION SUITABLE FOR THIS |
| DEP3812609.5 | 1988-04-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1334925C true CA1334925C (en) | 1995-03-28 |
Family
ID=6352084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000595751A Expired - Fee Related CA1334925C (en) | 1988-04-15 | 1989-04-05 | Process for the determination of thyroxine and a suitable standard solution therefor |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0337467B1 (en) |
| JP (1) | JPH079431B2 (en) |
| AT (1) | ATE108559T1 (en) |
| CA (1) | CA1334925C (en) |
| DE (2) | DE3812609A1 (en) |
| ES (1) | ES2057000T3 (en) |
| ZA (1) | ZA892702B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4226949A1 (en) * | 1992-08-14 | 1994-02-17 | Boehringer Mannheim Gmbh | Method and standard solution for the determination of thyroxine (T¶4¶) or triiodothyronine (T¶3¶) |
| US5342788A (en) * | 1988-04-15 | 1994-08-30 | Boehringer Mannheim Gmbh | Method and standard solution for the determination of thyroxine (T4) or triiodothyronine (T3) |
| JP2622653B2 (en) * | 1992-12-18 | 1997-06-18 | 三洋化成工業株式会社 | Thyroid hormone aqueous solution |
| US5795789A (en) * | 1997-06-04 | 1998-08-18 | Dade Behring Inc. | Standard solution for the determination of thyroid function |
| WO1998056719A2 (en) * | 1997-06-13 | 1998-12-17 | Medical Analysis Inc. | Control standards for clinical chemistry assays |
| CA2432523C (en) * | 2000-12-21 | 2014-06-03 | Resuscitek, Inc. | Compositions of stable t3 and methods of use thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4052504A (en) * | 1976-06-30 | 1977-10-04 | Corning Glass Works | Assay for thyroxine binding globulin |
| US4157895A (en) * | 1977-10-07 | 1979-06-12 | Nuclear International Corporation | RIA reagents and processes |
| JPS61225654A (en) * | 1985-03-29 | 1986-10-07 | Eiken Kagaku Kk | Measurement of isolated type thyroxin in blood |
| US4824777A (en) * | 1987-07-08 | 1989-04-25 | Ciba Corning Diagnostics Corp. | Method for determining thyroxine uptake |
| JPH01232263A (en) * | 1988-03-14 | 1989-09-18 | Tosoh Corp | Immunoassay of triiodothyronine ingestion rate |
-
1988
- 1988-04-15 DE DE3812609A patent/DE3812609A1/en not_active Withdrawn
-
1989
- 1989-04-05 CA CA000595751A patent/CA1334925C/en not_active Expired - Fee Related
- 1989-04-13 ES ES89106644T patent/ES2057000T3/en not_active Expired - Lifetime
- 1989-04-13 DE DE58908025T patent/DE58908025D1/en not_active Expired - Fee Related
- 1989-04-13 AT AT89106644T patent/ATE108559T1/en not_active IP Right Cessation
- 1989-04-13 EP EP89106644A patent/EP0337467B1/en not_active Expired - Lifetime
- 1989-04-13 ZA ZA892702A patent/ZA892702B/en unknown
- 1989-04-14 JP JP1093282A patent/JPH079431B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| EP0337467A3 (en) | 1990-09-05 |
| ATE108559T1 (en) | 1994-07-15 |
| JPH01305359A (en) | 1989-12-08 |
| DE58908025D1 (en) | 1994-08-18 |
| DE3812609A1 (en) | 1989-11-09 |
| ES2057000T3 (en) | 1994-10-16 |
| EP0337467A2 (en) | 1989-10-18 |
| JPH079431B2 (en) | 1995-02-01 |
| ZA892702B (en) | 1989-12-27 |
| EP0337467B1 (en) | 1994-07-13 |
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