AU622339B2

AU622339B2 - Method and reagent for the determination of antithrombin iii

Info

Publication number: AU622339B2
Application number: AU58885/90A
Authority: AU
Inventors: Andreas Dessauer; Reinhard Herz; Helmut Lill
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1989-07-14
Filing date: 1990-07-11
Publication date: 1992-04-02
Anticipated expiration: 2010-07-11
Also published as: ZA905489B; EP0408075B1; EP0408075A2; EP0408075A3; ES2069635T3; JPH0353898A; ATE119578T1; AU5888590A; DE59008621D1; DD298444A5; DE3923340A1; JPH0734759B2

Abstract

To determine antithrombin III in body fluids by reacting the sample with thrombin and a chromogenic substrate which produces a colour on exposure to thrombin, and measuring the resulting colour, the reaction is carried out in the presence of denaturing agents or of the tetrapeptide Gly-Pro-Arg-Pro, and the substrate used is an oligopeptide whose sensitivity is a factor of 5 to 100 less than that of Tos-Gly-Pro-Arg-pNA.

Description

p i i 622339 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION NAME ADDRESS OF APPLICANT: Boehringer Mannheim GmbH Sandhofer Strasse 112-132 D-6800 Mannheim-Waldhof Federal Republic of Germany NAME(S) OF INVENTOR(S): Andreas DESSAUER Reinhard HERZ Helmut LILL ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Method and reagent for the determination of antithrombin III The following statement is a full description of this invention, including the best method of performing it known to me/us:- "0 o o I 88 0 0 8 O 0 0 2 Description The invention concerns a method for the determination of antithrombin III in body fluids by reacting the sample with thrombin and a chromogenic substrate which forms a colour by the action of thrombin and measurement of the colour formed as well as a reagent which is suitable for this.

Antithrombin III is a factor of the blood coagulation system which has a regulatory role. Blood coagulation is initiated by different proteases interacting in a cascade. The last of the sequential activation steps liberates thrombin which in turn produces fibrin monomers by the cleavage of fibrinogen which agglomerate and form a thrombus. The most important regulator is antithrombin III (AT III) which can form a complex with thrombin and also with other proteases involved in bliood coagulation which blocks the active centre. The amount of antithrombin III in the blood of healthy people is in a relatively narrow range. Reduced amounts of antithrombin III may be caused by a censumptive coagulopathy, a severe liver disease or it may be hereditary. A reduced antithrombin III level is nowadays generally judged to be a thrombosis risk. Therefore, the antithrombin III level is reduced in some cases in an acute thrombosis. The amount of antithrombin III is thus a valuable parameter in clinical diagnosis.

Different procedures are already known for the detection of antithrombin III which are based on immunological methods and also on the use of chomogenic substrates. In the latter case, thrombin is added to the sample which reacts with the antithrombin III present in the sample and is inactivated. Excess thrombin is detected by 300 100 00 0000 0 0 0000 0 0 00 0 0 000 0 000*00 00 0 3 o 00 0 0 0 o*0 00 eo 0 o 0 *o o«o 00 0 «O o |I "0 0 o o i 0 0 0 li." 0 0 *0 0 0 0 0 I so o II a 0 o 11 *'o

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6*6 a4 6+ incubation with a chromogenic substrate which forms a coloured substance by the action of thrombin and measurement of the colour formed, wherein the amount of antithrombin III is inversely proportional to the colour formed. Methods for the determination of antithrombin III are described for example in Bergmeyer, "Methods of Enzymatic Analysis", 3rd edition, Verlag Chemie, Vol. p. 441-448; I. Witt, ed., "Neue Methoden der Gerinnungsanalyse mit chromogenen Substraten", Stormorken, Neue Methoden der Gerinnungsanalyse, pages 119-121; Odegard et al., Haemostasis 7: 202-209 (1978); Fareed et al., in Chromogenic Peptide Substrates (eds.

M.F. Scully and V.V. Kakkar) Churchill Livingstone (1979) 183-191 and Abildgaard et al., Thromb. Res. 11, 549-553 (1977)-.

As a rule the samples are very highly diluted to avoid interferences in the test, since sample colour, turbidity and differences in the pH, as well as salt content of the sample have less influence on the measured result the more the amount of sample can be limited in the test solution. Thus, when carrying out kinetic determinations, in particular on automated analyzers, sample dilutions ranging up to 1:1500 are used. This is, however, impractical for the routine (especially the emergency situation).

It was therefore the object of the invention to improve the known detection methods and to provide a method which can be carried out on automated analyzers, in a short time and without sample dilution and which enables the use of a single standard.

4 This object is achieved by a method for the determination of antithrombin III in body fluids by reacting the sample with thrombin and a chromogenic substrate which forms a colour by the action of thrombin, and measurement of the colour formed, which is characterized in that the reaction is carried out in the presence of denaturing agents or of the tetrapeptide Gly-Pro-Arg-Pro and in which an oligopeptide substrate is used as the substrate which has a sensitivity to thrombin which is a factor of 5 to 100 lower compared with Tos-Gly-Pro-Arg-pNA.

0 0 Surprisingly, it was possible to determine antithrombin III exactly and reproducibly by the addition of 0 o .o o denaturing agents or a tetrapeptide and by use of a .0 ,o relatively insensitive oligopeptide substrate for o° thrombin, whereby long test runs can be performed a. without restrictions on automated analyzers.

oe 4 In order to determine antithrombin III in a body fluid, a sample solution is mixed with thrombin in excess (with respect to antithrombin III). In this process, complexes are formed from antithrombin III and thrombin. Thrombin complexed in this way no longer has any protease activity. A substrate is added to the solution which is cleaved by thrombin and thereby forms a colour. Only the thrombin which is in excess and is not complexed reacts i with the substrate. The measured signal is inversely 4 proportional to the antithrombin III concentration of the sample. Heparin, which accelerates the reaction between antithrombin III and thrombin, is preferably added to carry out the method.

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5 ft I ft. 9r 9 9 I 4 44 ft. 9 9 I Of There are a multitude of substrates which are suitable for this method of which Tos-Gly-Pro-Arg-pNA-acetate (ChromozymR TH) is used most often. This reagent is highly sensitive.

According to the present invention, a denaturing agent or the tetrapeptide Gly-Pro-Arg-Pro is added in addition in the first step. It was found that the initial thrombin value did not remain constant in long test series when using undiluted samples. This is rectified by addition of the components mentioned above.

Substances are suitable as denaturing agents which are known in this field. Urea or guanidinium hydrochloride are preferably used. The denaturing agent is preferably used in a concentration of 0.1 to 1 mol/1 in the test or 1 to 6 mol/l in the substrate solution. If the tetrapeptide is used as the component, this is then added in a concentration in the range between 0.04 to 1 mg/ml in the test.

The denaturing agent or the tetrapeptide can either already be added to the reagent solution or they can also be added when the sample solution is incubated with the reagent solution. If urea is used as the denaturing ageni' then it i3 advantageous to first add the urea together with the substrate, since urea has an unfavourable influence on the stability of thrombin.

A further key feature of the method according to the present invention is the use of a chromogenic oligopeptide substrate for thrombin whose sensitivity, however, is a factor 5 to 100 lower compared with Chromozym® TH. All substrate which fulfil this criterium are suitable such as e.g.

'ftE ft 4 ft .9 i 1 *f I I ~----IYddC--4Y_ 6 0o 0 G 0 a 0 0 00 i oo ooo *o o o o o o 0 00 *00 oO 0 a 0 0 0 CBZ-Val-Gly-Arg-pNA; H-D-Pro-Phe-Arg-pNA; H-D-Val-Leu-Arg-pNA; Bz-Val-Leu-Arg-pNA; Bz-Leu-Leu-Arg-pNA; Bz-Phe-Leu-Arg-pNA as well as Bz-Leu-Ile-Arg-pNA.

(CBZ: carbobenzoxy, Bz: benzoyl, D-Pro: D-proline, D- Val: D-valine, pNA: p-nitroaniline).

All these exemplary compounds mentioned above have p-nitroaniline as a chromophore which is cleaved off by thrombin. Other usual chromophores are equally suitable such as e.g. 5-amino-2-nitrobenzoic acid or methoxynitroaniline, which can be present in the substrate instead of pNA-. A peptide of the formula R-OCO-Gly-Pro- Arg-pNA is preferably used as the substrate in which R denotes an alkyl residue with 1 to 3 C atoms and is preferably a methyl residue.

Substrates which have a sensitivity which is reduced by a factor 10 to 30 compared to ChromozymD TH are particularly preferably used.

The concentration of the substrate as it is present in the test solution depends on its Michaelis constant.

Substrate concentrations are suitable which are in a range from 2 to 20 times the respective Michaelis constants.

The thrombin concentration used for the method can, according to the present invention, be increased up to times, preferably 7 to 15 times the thrombin concentration used according to the state of the art; this corresponds to up to 630 U thrombin/l (international enzyme units of thrombin with Tos-Gly- 000oo0 0 9 i aoo 0 0 o 0 0 00 0 I 0 4 060w 1* 4 041 0 44O 3 00 Op0 44 eo 9 7 Pro-Arg-pNA as substrate at 25 0 C) test solution. Within this range, but however, not with less thrombin, a linear calibration curve can be obtained with the method according to the present invention so that a kinetic determination can be carried out over a wide range and, above all, a convenient test procedure with only one standard on automated analyzers is possible. If it is not linear over the measurement range then several standards have to be used which hinders the rapid application of the test, which is for example essential for the emergency situation.

The amount of sample depends on the measurement range of the test and on the technical feasibility. The lower limit is 1 Al for automated analyzers. The upper limit depends on the type of analyzer and is not usually more than 5 il. If the amount of sample is larger then, if the other test conditions remain the same, a smaller measurement range is available and, in addition, interferences by formation of fibrin occur to an increasing extent when the amount of sample is larger.

On the other hand, the actual measurement signal is reduced when using smaller amounts of sample which, together with the increasing inaccuracy when pipetting very small volumes, leads to a worsening of the test precision.

A further object of the invention is a reagent for the determination of antithrombin III which contains thrombin, a denaturing agent or the tetrapeptide Gly- Pro-Arg-Pro as well as a chromogenic substrate which forms a colour by the action of thrombin and which is a factor of 5 to 100-fold less sensitive with respect to thrombin compared to Chromozyn TH.

oo 440 oD 0 4 40 .4 0 4 00~r j. j i 8 '4 4 4 *wr 9 o 44 4 4 P 44 4 4 40 4 0 4, 44 4 In a preferred embodiment the reagent also contains heparin.

The invention is elucidated by the following Examples: E x a m p 1 e 1 (comparison) (Method according to the state of the art, Bergmeyer) 1 part thrombin (500 U/1) is mixed with 20 parts Tris/HCl buffer, pH 8.1 containing at the same time heparin in a sufficient amount (2 USP units/ml). To measure the initial thrombin value, 0.10 ml physiological saline solution and at the start of the enzyme/substrate reaction 0.200 ml thrombin substrate solution (Chromozym® TH: Tos-Gly-Pro-Arg-pNA, 0.16 mmol/l in the test mixture) were added to 2 ml of this thrombin reagent. In the test mixture thus defined, reaction rates (AA/min) were achieved at 405 nm of 0.220 at 250C and 0.400 at 370C.

Exampl e 2 Antithrombin III was determined in sample solutions according to the method described in Example 1 in which, however, Chromozym TH was replaced by the substrate according to the present invention.

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49 4, 44 4 44 31:1 e 4 9 Manual test without sample pre-dilution: 0.01 ml undiluted sample (corresponding to 6.6 pl sample per ml test volume) 1.25 ml thrombin reagent (308 U/1 thrombin in the test, 14.74-fold compared to the state of the art)

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"O ao ft 0 Q eoo 9 Q ff aO e e B 0 5, 5, at o 00 d *«0o a Q a 0.25 ml substrate (0.298 mmol/l methyl-OCO-Gly-Pro-Arg- OrpNAA 0.5 mol/1 urea in the test).

Tests on automated analyzers: Hitachi 704/705: 0.003 ml undiluted sample (corresponding to 7.1 gl sample per ml test volume) 0.350 ml thrombin reagent .(308 U/1 thrombin and 248 mg/1 Gly-Pro-Arg-Pro in the test) 0.070 ml substrate (0.298 mmol/l MeOCO-Gly-Pro-Arg-pNA in the test.

Hitachi 717: 0.002 ml undiluted sample (corresponding to 6.6 ul sample per ml test volume) 0.250 ml thrombin reagent (308 U/l thrombin and 248 mg/1 Gly-Pro-Arg-Pro in the test) 0.050 ml substrate (0.298 mmol/1 MeOCO-Gly-Pro-Arg-pNA in the test) Table 1 Comparison of the initial thrombin values (without sample) under the test conditions chosen here using Chromozynm TH and MeOCO-Gly-Pro-Arg-pNA:

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A

10 MeOCO-Gly..... ChromozymR TH S(AA/min) A/min) 4 t

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Ii 1 4 f it 4 t 44 4 rW 44~ 4 444, Hitachi 717, 370C 0.400 5.700 Hitachi 717, 25 0 C 0.200 2.900 Hitachi 704, 37 0 C 0.300 4.300 Hitachi 704, 25 0 C 0.150 2.100 The measurement limit of kinetic enzyme/substrate reactions is ca 1.00 L A/min for the Hitachi instruments. The other known analyzers also run into analytical limits which are not feasible at these high values for A A/min. The same, of course, applies to the manual test in which, under the conditions described above without sample predilution and using Chromozym TH as substrate, AA-values of 3.3 at 25 0 C and of 6.0 at 37 0 C would occur.

900 0 0 09. 00 0 0 0 0 0 0 0.0 000 0* 4 0 00 o 040 0 0 0000 0000 0 00 0 0 0 088 00 0 0 0 00 00, 000 0 0 0 0 t 0 0 0 0 0 0 000 0 0 ~0 000 0 00000 00 00 0 0 0 00 00000 0 0 0 0000000000 0 0 0 0 0 Table IT Test instrument: Hitachi 717 Test mixture: 0.002 ml undiluted sample 0.250 ml thrombin reagent; 14.74-fold in comparison with 308 U/L in the test) 0.050 ml substrate the state of the art substrate sensitivity of Chromozymn' TH) thrombin 7.5-fold (156.8 U/1) concentration in 14 .74-fold (308 U/1) the test (627 U/i) 250C 37 0 C 25 0 C 37 0 C 250 C 37 0

C

1% 0.014 0.028 0.029 0.'057 0.058 0.114 2% 0.029 0.057 0.058 0.114 0.116 0.228 7% 0.100 0.200 0.200 0.400 0.400 0.800 e. g. Me-0C0-Gly-Pro-Arg-pNA 17 0.246 0.484 0.4S3 0.969 0.986 1.938 e.g. i-Prop-OCO-Gly-Pro-Arg-pNA 0.280 0.560 0.580 1.140 1.160 2.280 -11 12 It becomes clear that when using the substrates with a higher sensitivity (17 or 20 of Chromozym® TH), not all test versions 37°C or higher thrombin j! concentrations) can be carried out with very good results; the scope within which the method according to the present invention can be carried out advantageously without sample dilutions is apparent.

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Claims

1. Method for the determination of antithrombin III in body fluids by reacting the sample with thrombin and a chromogenic substrate which forms a colour by the action of thrombin and measurement of the colour formed, w h e r e i n the reaction is carried out in the presence of denaturing agents or of the tetrapeptide Gly-Pro-Arg-Pro and wherein an oligopeptide substrate is used as the substrate which has a sensitivity to thrombin which is a factor of 5 to 100 lower compared with Tos-Gly-Pro- Arg-pNA.

2. Method as claimed in claim 1, w h e r e i n or guanidinium hydrochloride is used as the denaturing agent. irea 4, *r~ 4O 4 4 4 i

3. Method as claimed in claim 2, w h e r e i n the denaturing agent is used in a concentration of 0.1 to 1 mol/l in the test.

4. Method as claimed in claim 1, w h e r e i n the tetrapeptide is used in a concentration of 0.04 to 1 mg/ml in the test.

Method as claimed in one of the previous claims, w h e r e i n a peptide having the formula p 4 IILIIIP-rr~a~_lSIPS I~ E(SIPI iTiill3iiLE-_i~iE=SiC~S ~FU IECIP 14 R-OCO-Gly-Pro-Arg-pNA in which R is an alkyl residue with 1 to 3 C atoms is used as the oligopeptide substrate.

6. Method as claimed in one of the previous claims, w h e r e i n an oligopeptide substrate is used which has a sensitivity which is a factor 10 to lower compared with Tos-Gly-Pro-Arg-pNA.

7. Reagent for the determination of antithrombin III, w h e r e i n it contains thrombin, a denaturing agent or the tetrapeptide Gly-Pro-Arg-Pro and an oligopeptide substrate as substrate which forms a colour by the action of thrombin and which has a to 100-fold lower sensitivity compared with Tos- Gly-Pro-Arg-pNA. r. I II .t I p a I 4 0 I lee "I A4 0 0 S I1 4 .444 oI 4 0 .0 0I 4.r $4 444 F. Li lE;

8. A method as claimed in claim or reagent according to claim 7, substantially as hereinbefore described with reference to the examples. DATED this 17th day of January, 1992 BOEHRINGER MANNHEIM GmbH by its Patent Attorneys DAVIES COLLISON CAVE I I t *4t I It 0 S S I 4* *0 00 0 00 0 00 00 0 0 00 0 00 00 0 0 400 4 I. 4 4* o I, 0 00 4* 4 0 0000 0 0044 400040 0 00 -I 0 10 0 C 920117,PASDAT.12Z5W85-90.rsP,15