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Publication numberUS20020012958 A1
Publication typeApplication
Application numberUS 09/078,760
Publication dateJan 31, 2002
Filing dateMay 15, 1998
Priority dateMay 17, 1997
Also published asCA2237817A1, CA2237817C, DE19720853A1, EP0878551A2, EP0878551A3, EP0878551B1, US6376209
Publication number078760, 09078760, US 2002/0012958 A1, US 2002/012958 A1, US 20020012958 A1, US 20020012958A1, US 2002012958 A1, US 2002012958A1, US-A1-20020012958, US-A1-2002012958, US2002/0012958A1, US2002/012958A1, US20020012958 A1, US20020012958A1, US2002012958 A1, US2002012958A1
InventorsThomas Wissel, Hermann Keuper, Hubert Nettelhoff, Heinz-Georg Kandel, Reiner Muth, Michael Kraus
Original AssigneeDade Behring Marburg Gmbh
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Increasing the fvii sensitivity of a thromboplastin reagent
US 20020012958 A1
Abstract
The present invention relates to a process for increasing the FVII sensitivity of a thromboplastin reagent by means of heat treatment.
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Claims(22)
1. A process for increasing the FVII sensitivity of thromboplastin reagents, which comprises selectively inhibiting the residual FVII/FVIIa activity in the reagent.
2. The process as claimed in claim 1, wherein a solution of these reagents is heated to greater than 65 C.
3. The process as claimed in claim 2, wherein a solution of tissue thromboplastin of human origin is heated.
4. The process as claimed in claim 2, wherein a solution of a recombinant tissue thromboplastin is heated.
5. The process as claimed in claim 2, wherein a solution of a biologically active variant of a tissue thromboplastin is employed.
6. The process as claimed in one of claims 2 to 5, wherein heating is carried out continuously and for a short time in a heat transfer apparatus having indirect heating.
7. The process as claimed in one of claims 2 to 6, wherein the heating temperature is between +65 C. and +160 C., preferably between +80 C. and +140 C.
8. The process as claimed in one of claims 2 to 7, wherein the heating or cooling time is less than 30 seconds.
9. The process as claimed in claim 2, wherein the holding time is between 0.1 and 30 seconds, preferably between 0.5 and 20 seconds.
10. The process as claimed in claim 1, wherein protease inhibitors are added to the reagent.
11. The process as claimed in claim 1, wherein anti-FVII antibodies are added to the reagent.
12. The process as claimed in claim 1, wherein oxidizing agents are added to the reagent.
13. The process as claimed in claim 12, wherein the oxidation is carried out in the presence of metals.
14. The process as claimed in claims 10-13, wherein a solution of tissue thromboplastin of human origin is employed as the thromboplastin reagent.
15. The process as claimed in claims 10-14, wherein a solution of a recombinant tissue thromboplastin is employed.
16. The process as claimed in claims 10-13, wherein a solution of a biologically active variant of a tissue thromboplastin is employed.
17. The process as claimed in claim 10, wherein phenylmethylsulfonyl fluoride (PMSF), p-aminoethylbenzenesulfonyl fluoride (AEBSF), 4-aminophenylmethanesulfonyl fluoride (p-APMSF) or organofluorophosphates, such as diisopropyl fluorophosphate (DFP), or combinations of these inhibitors, are employed as protease inhibitors.
18. The process as claimed in claim 11, wherein a polyclonal anti-FVII antibody is employed as antibody.
19. The process as claimed in claim 12, wherein chloramine T, hydrogen peroxide, ascorbic acid, or their combinations, are employed as oxidizing agent.
20. The process as claimed in claim 13, wherein the oxidation is carried out in the presence of the metals iron, copper or zinc, or their mixtures.
21. The preparation of an anti-FVII antibody, wherein the antibody inhibits the coagulation-inducing activity of the tissue factor/FVIIa complex.
22. The preparation of a polyclonal anti-FVII antibody, wherein the antibody inhibits the coagulation-inducing activity of the tissue factor/FVIIa complex.
Description

[0001] The present invention relates to a process for increasing the FVII sensitivity of a thromboplastin reagent by means of heat treatment.

[0002] Quick's thromboplastin time (PT) is employed as a test for screening for a deficiency in coagulation factors in patient blood. The PT is also used to monitor therapy with oral anticoagulants. The preferred PT for normal blood donors is 10-14 seconds. The preferred PT for factor VII-deficient plasmas should be more than 60 seconds. The FVII sensitivity is therefore defined as the ratio of the coagulation time of the FVII-deficient plasma and the coagulation time for normal blood donors.

[0003] The invention describes a process for increasing the factor VII sensitivity of thromboplastin reagents by increasing the PT for factor VII-deficient plasmas without significantly altering the PT for normal plasmas.

[0004] Active thromboplastin induces coagulation in plasma and is composed of a lipid component and a protein component. The protein, i.e. tissue factor, is membrane-bound and is found in many different tissues. The binding between the protein and the lipid is based on hydrophobic interactions and is Ca2+-independent. The protein moiety is composed of a glycoprotein having a molecular weight of 43-53 kDa. One molecule of tissue factor is able to bind to one molecule of FVII or FVIIa. The binding of FVII/FVIIa to tissue factor is Ca2+-dependent. The complex composed of lipid, tissue factor and FVIIa cleaves FX to form FXa and thereby finally elicits coagulation by means of activating prothrombin.

[0005] The onset of coagulation in a plasma at from 10 to 14 sec after adding a thromboplastin reagent indicates that the coagulation system is intact. An increase in the coagulation time points to an impairment. Impairments can occur as a result of the concentrations of one or more coagulation factors being too low. Thromboplastin reagents of varying origin frequently differ in their sensitivity for indicating the deficiency of particular factors. This can sometimes be due to the carry-over of small quantities of the factors to be determined into the reagent. Common methods for specifically removing small quantities of protein are, inter alia, immunoadsorption or, in the case of the vitamin K-dependent coagulation factors such as FII, VII, IX and X, barium sulfate adsorption (WO 90/05740). In the case of reagents of human origin, it is frequently only the sensitivity with regard to FVII which is inadequate.

[0006] Proteases, i.e. factors VIIa, IXa, Xa, XIa and XIIa, and plasma kallikrein and thrombin, are essential factors of the coagulation cascade.

[0007] Disadvantages of the previously described methods are, firstly, that FVII binds to tissue factor and the yield of tissue factor which is achieved by such an adsorption method is therefore reduced and, secondly, that both methods demand a substantial input of equipment and time, particularly on a production scale.

[0008] In the present invention, it is demonstrated that it is surprisingly possible, under defined conditions, to selectively inhibit the residual FVIIa activity of the reagent and consequently to arrive at the desired result in a simple manner without impairing the tissue factor or other factors of the coagulation cascade. The ratio, which is termed FVII sensitivity, of the coagulation time of the FVII-deficient plasma to the coagulation time of a normal plasma is preferably >4, particularly preferably >6 and very particularly preferably >10.

[0009] Factor VIIa possesses a protease activity and belongs to the serine protease class, as do the other coagulation factors IXa, Xa, XIa, XIIa, plasma kallikrein and thrombin. Another feature common to these coagulation factors is that they are initially present in zymogenic form and protein bonds have to be cleaved for their protease activity to be displayed.

[0010] A large number of protease inhibitors which at least inhibit the active forms of these zymogens, e.g. thrombin, have been described (W. B. Lawson et al., 1982, Folia Haematol. Leipzig 109 (1982) 1, pp. 52-60). They include sulfonyl fluorides, such as phenylmethylsulfonyl fluoride (PMSF), p-aminoethylbenzenesulfonyl fluoride (AEBSF) and 4-aminophenylmethanesulfonyl fluoride (p-APMSF), organofluorophosphates, such as diisopropyl fluorophosphate (DFP), chloromethyl ketones and also peptides, such as leupeptins or proteins of the serpin family, such as C1 inhibitor, antithrombin III and aprotinin.

[0011] The protease inhibitors suffer from the disadvantage that, in the PT, they inhibit the coagulation factors IXa, Xa, XIa, XIIa, plasma kallikrein and thrombin in the sample to be determined in addition to inhibiting the factor FVII or FVIIa which has been carried over. On this basis, they would prolong coagulation time and give a false impression of a pathological value. It is to be expected, therefore, that these inhibitors would not be suitable for being used in a PT reagent. Surprisingly, however, it is possible to employ these inhibitors under specific conditions without prolonging the coagulation times of the sample; however, the FVII sensitivity of the reagent is nevertheless improved.

[0012] Another option for inhibiting the enzymic activity of a protein is that of preparing specific antibodies which inhibit the active center of the protein. It is possible to obtain a variety of antibodies against coagulation factor VII, including at least one polyclonal antibody which inhibits the enzymic activity of FVII/FVIIa. Simply adding the antibody to a PT reagent would be disadvantageous since the antibody also inhibits the FVII/FVIIa of the sample. In the present invention, it is demonstrated that there is, surprisingly, a concentration range for adding the anti-FVII antibody in which the coagulation time of a normal plasma is not prolonged but that of an FVII-deficient plasma is prolonged markedly, such that the FVII sensitivity of the reagent is substantially increased.

[0013] It has also been previously reported that serine proteases can be oxidatively inhibited (S. E. Lind et al., 1993, Blood 82, 5 15522-1531). This method has the disadvantage that the fatty acids of the phospholipids can be oxidized in addition to the disadvantage that the coagulation cascade serine proteases in the sample are inhibited (Dasgupta, A. et al., Live Science 1992, 50, 875-882). The oxidation products which are produced in this connection likewise inhibit the PT (T. W. Barrowcliffe et al., 1975, Thrombos. Diathes. haemorrh. 33, 271).

[0014] Surprisingly, however, the oxidation processes can be controlled such that it is possible to disregard the undesirable side reactions, i.e. while the coagulation time of normal plasmas is not prolonged, the FVII sensitivity of the reagent is improved. Suitable oxidizing agents are hypochlorite, hydrogen peroxide, permanganate and manganese dioxide. Many antioxidants, such as ascorbic acid, thiols, such as glutathione, acetyl cysteine, dithioerythreitol and tocopherols and tocopherol derivatives, such as di-tert-butyl-p-hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT), Trolox and propylgallate are able to exert an oxidative effect in the presence of metal ions. The metal ions which catalyze the oxidation include, in particular, iron, copper and zinc. The metal-catalyzed oxidation is particularly suitable for inhibiting residual FVII/FVIIa activities.

[0015] The process of the present invention uses simple methods for heat-treating the tissue extract selectively; without thereby restricting ourselves to one particular reaction mechanism, it might be possible to explain the efficacy of the process on the basis that the specific heat treatment removes residual FVII/FVIIa activities. The heat treatment is advantageously carried out for a short time at high temperatures.

[0016] At low temperatures of between +40 and +60 C., inactivation of the FVII activities depends on a number of factors, inter alia the dimensions and the thermal conductivity of the vessel employed and the solution used, some of which cannot be controlled. The heating time can also vary widely. The heating temperature is advantageously between +65 C. and +160 C., preferably between +80 C. and 140 C., particularly preferably between +90 and +120 C.

[0017] So that the liquid which is supplied is heated to the relevant heating temperature in as short a time as possible and can then be cooled down again in a similarly short period of time, the apparatus in which the heating is carried out is expediently equipped with at least one connection for heating medium and at least one connection for cooling medium. The apparatus advantageously comprises three regions or sections: a heating section, in which the reagent is heated to the requisite temperature, a holding section, in which the reagent is held at this temperature for a specified time, and a cooling section in which the reagent is cooled down once again to a low temperature.

[0018] The construction of the heat-transfer apparatus should be such that the difference between the temperature to which the solution is heated and the temperature of the heating medium is as small as possible, as a consequence of which the product is spared to the greatest possible extent on account of the resulting low excess temperature of the walls which are in contact with the product.

[0019] Preference is given to a process for continuously heating for a short time, as described in EP-A 0 571 771 for virus inactivation.

[0020] The heating and/or cooling time should in each case be less than 30 seconds, preferably, however, less than 5 seconds. The holding time can be between 0.1 and 30 seconds, preferably, however, between 0.5 and 20 seconds.

[0021] Without restricting the invention to the example of human placenta extract, the following example demonstrates the inactivation of residual FVII/FVIIa activities by heating, as employed for a Behringwerke AG tissue thromboplastin-containing Quick reagent (Thromborel S, Prod. No. OUHP). Preservatives, antioxidants, carbohydrates, proteins and amino acids, and combinations thereof, are advantageously added to the reagent as stabilizers. In the individual substance classes, the following compounds are particularly suitable:

[0022] Preservatives:

[0023] Mergal, sodium azide phenol, amphotericin, gentamicin, piperacilin and ciprofloxacin.

[0024] Antioxidants:

[0025] Di-tert-butyl-p-hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT), spermine, propylgallate, tocopherols, Trolox, dithioerythreitol and glutathione.

[0026] Carbohydrates:

[0027] Trehalose, sucrose, Tylose, mannitol, xanthan gum, phytagel, carrageenan and polyethylene glycol.

[0028] Proteins:

[0029] Lactoglobulin, lipoproteins and albumins, such as serum albumin, lactalbumin and ovalbumin.

[0030] Amino Acids:

[0031] Acetylcysteine, acetylmethionine, glycine, lysine, histidine and serine.

[0032] The following examples clarify the invention.

EXAMPLE 1

[0033] Step a)

[0034] 1.25 ml of a stabilizing solution were added to Thromborel S Bulkware and the whole was made up to 10 ml. 1 ml of Thromborel (7 C.) was in each case added to 1.5 ml reaction tubes, which were then incubated at 56 C. in a water bath for different times and subsequently cooled on ice.

[0035] Step b)

[0036] Quick's thromboplastin time (PT) was then determined using a Schnitger & Gross as follows:

[0037] Heat reagents and tubes to 37 C.;

[0038] Introduce 100 μl of control plasma N or FVII-deficient plasma into the tubes;

[0039] Incubate for 1 min in the thermoblock;

[0040] Add 200 μl of thromboplastin and at the same time start the measuring operation;

[0041] shake the tube briefly and place it in the measuring position of the Schnitger & Gross, and insert the electrode holder up to the limit stop;

[0042] Read off the coagulation time.

[0043] The read-off coagulation times are listed in Table 1:

TABLE 1
PT following thermal treatment
Standard human plasma FVII-deficient plasma
(sec) (sec)
min 1. 2. MV MV
Ref. 10.4 10.5 10.5 28.4 27.9 28.2
1 10.5 10.5 10.5 28.4 27.9 28.2
2 10.6 10.6 10.6 29.1 29.6 29.4
3 10.5 10.9 10.7 33.0 33.0 33.0
4 10.8 10.9 10.9 34.9 34.0 34.5
5 11.0 11.1 11.1 36.6 36.5 36.6
10  12.0 11.9 12.0 45.4 44.5 45.0
15  14.5 15.0 14.8 116.0 116.4 116.2
20  15.5 16.0 15.8 128.0 126.0 127.0

EXAMPLE 2

[0044] Thromborel S Bulkware was heated, using a dwell time of 2.05 sec, to temperatures of from 60 C. to 80 C. (see Tab. 2).

[0045] The coagulation time was determined as described in Example 1 step b).

TAB. 2
PT following short-time heating
Standard human Standard human FVII-deficient
plasma plasma plasma
100% 25% 100%
(sec) (sec) (sec)
1. 2. MV 1. 2. MV 1. 2. MV
Ref 10.5 10.4 10.5 24 24 24.0 32.6 32.5 32.6
60 C. 10.4 10.4 10.4 23.9 24.4 24.2 37.1 37 37.1
65 C. 11 11 11.0 25 25.8 25.4 48 48.6 48.3
70 C. 11 11 11.0 25.5 26 25.8 59.1 59.6 59.4
75 C. 11.4 11.4 11.4 25.9 26.5 26.2 67.6 67.1 67.4
80 C. 11.4 11.5 11.5 26.4 26.4 26.4 68.6 69.1 68.9

EXAMPLE 3

[0046] Thromborel S Bulkware was heated, using a dwell time of 0.5-2.05 sec, to temperatures of from 75 C. to 95 C. (see Tab. 3).

[0047] The coagulation time was determined as described in Example 1 step b).

TABLE 3
PT following short-time heating
Standard human plasma FVII-deficient plasma
(sec) (sec)
1. 2. MV 1. 2. MV
75 C. 10.9 10.9 10.9 74.5 74.4 74.5
80 C. 11.1 11.1 11.1 78 78.6 78.3
85 C. 10.9 10.9 10.9 82.9 83.9 83.4
90 C. 10.4 10.9 10.7 82.4 82.5 82.5
95 C. 11 11.1 11.1 79.6 79.6 79.6
95 C. 11 10.9 11 72 70.9 71.5
(1 sec)
95 C. 11 11 11 67.4 68 67.7
(0.5 sec)
Ref 10.4 10.5 10.5 35.5 35.5 35.5

EXAMPLE 4

[0048] Thromborel S, prepared by the method described in the Examples 1a)-1b), was heated to temperatures of 65 C. and 75 C., respectively, using a dwell time of 2-16 sec (see Tab. 4). The coagulation time was determined as described in Example 1 step b).

TAB. 4
PT following short-time heating
Standard human Standard human FVII-deficient
plasma plasma plasma
100% 25% 100%
(sec) (sec) (sec)
1. 2. MV 1. 2. MV 1. 2. MV
Starting 10.5 10.4 10.5 26.9 26.5 26.7 33.6 33.5 33.6
material
2.0 sec 10.5 10.5 10.5 26 26.4 26.2 33.1 33.5 33.3
RT
15.8 sec 11.4 11.5 11.5 27.5 28.5 28.0 71.6 71.5 71.6
65 C.
7.9 sec 11 10.9 11.0 27.5 27.4 27.5 58.1 59.1 58.6
65 C.
4.0 sec 10.9 10.9 10.9 27.4 27 27.2 50 49.6 49.8
65 C.
3.0 sec 10.9 11 11.0 27 26.5 26.8 48.6 48.6 48.6
65 C.
2.0 sec 10.9 11 11.0 27.5 27.9 27.7 45.1 44.6 44.9
65 C.
3.0 sec 11 11.4 11.2 28.5 28.9 28.7 71.6 72.6 72.1
75 C.

EXAMPLE 5

[0049] Thromborel S, prepared by the method described in Examples 1a)-1b), was heated to a temperature of 90 C. using a dwell time of 3-18 sec (see Tab. 5).

[0050] The coagulation time was determined as described in Example 1 step b).

TABLE 5
PT following short-time heating
Standard human plasma FVII-deficient plasma
(sec) (sec)
1. 2. MV MV
3 sec 13.5 13.5 13.5 101.5 102 101.8
6 sec 13.5 13.4 13.5 106.5 110.5 108.5
9 sec 13.6 13.6 13.6 114.6 115.1 114.9
12 sec 13.5 13.5 13.5 124.9 125.4 125.2
15 sec 13.4 13.9 13.7 127.4 128 127.7
18 sec 13.6 13.7 13.7 131.1 132 131.6

EXAMPLE 6

[0051] Thromborel S Bulkware was heated to a temperature of 120 C. using a dwell time of from 0.5 to 1.5 sec.

[0052] The coagulation time was determined with a Behring Coagulation Timer using the PT.sec Thromborel S method.

TABLE 6
PT following short-time heating at 120 C.
TB-255
Dwell time in sec: 0.5 sec 1.0 sec 1.5 sec
Coagulation time in sec
Standard human plasma: 11.1 11.2 11.2
FVII-deficient plasma: 120.7 146.8 155

EXAMPLE 7

[0053] Thromborel S Bulkware was heated to a temperature of 85 C. using a dwell time of 3 sec. Commercially obtainable Thromborel S and heated Thromborel (TB-190) were compared with the aid of various samples.

[0054] The coagulation time was determined as described in Example 1 step b).

[0055] Samples:

PT100: Standard human plasma Batch designation: 502556
(98% of st.)
Ctrl.P: Control plasma P Batch designation: 512628
MACI: Marcumar plasma I. Batch designation: 951201
MACII: Marcumar plasma II. Batch designation: 951202
PATH-I: Pathoplasma I. Batch designation: 502883
PATH-II: Pathoplasma II. Batch designation: 502973
FVII: Factor VII-deficient plasma Batch designation: 500756

[0056] 1 IU/ml Hep: Standard human plasma supplemented with 1 IU of heparin/ml.

[0057] MAC-02 and MAC-03: Plasma pools from orally anticoagulated patients

[0058] 50% FVII and 2% EVII: dilutions of FVII-deficient plasma in standard human plasma.

[0059] PT16.75: Standard human plasma diluted to 16.75% in physiological NaCl solution.

[0060] % of standard: % of the standard (estimate using the following relationship):

C x−(PT 16.75% −PT 100%)/(4.97*PT x−4*PT 100% +PT 16.75%)

[0061] ISI: international sensitivity index:

[0062] PR: PT(Marcumar plasma)/PT((standard human plasma)

[0063] ISIk: ISI of the calibrated reagent;

[0064] ISIx: ISI of the uncalibrated reagent

ISI k =ISI x*log(PR x)/log(PR k)

[0065]

TAB. 7
PT following short-time heating
Thromborel S TB-190
% of st. PR INR % of st. PR ISI
PT100 11.15 10.05
PT16.75 41.9 46.2
1 IU/ml Hep. 12.25 84.9% 1.10 1.11 10.1 99.3% 1.00
MACI 34.95 20.6% 3.13 3.43 31.2 25.6% 3.10 1.09
MACII 66 10.1% 5.92 6.82 67.8 11.2% 6.75 1.01
MAC-02 42.2 16.6% 3.78 4.21 39.9 19.6% 3.97 1.04
MAC-03 60.9 11.1% 5.46 6.26 58.4 13.1% 5.81 1.04
PATH-I 29.25 25.5% 2.62 2.83 25.2 32.4% 2.51
PATH-II 51.45 13.3% 4.61 5.21 44.75 17.3% 4.45
Crtl.P 21.2 38.1% 1.90 2.00 20.8 40.4% 2.07
50% FVII 12.65 80.5% 1.13 1.15 11.45 83.9% 1.14
2% FVII 31.1 23.7% 2.79 3.03 39.45 19.8% 3.93
FVII 47.75 14.5% 4.28 4.81 138.5 5.4% 13.78
/ISI = 1.08

EXAMPLE 8

[0066] a)

[0067] Thromborel S Bulkware is short-time heated (temperature 95 C., dwell time 0.5 sec) and aliquots were then treated with different protease inhibitors (Table 8). The stabilizers were added after the samples had been incubated overnight. The reference does not contain any inhibitor.

[0068] b)

[0069] Quick's thromboplastin time (PT) is then determined using a Schnitger & Gross as follows:

[0070] Heat reagents and tubes to 37 C.;

[0071] Introduce 100 μl of plasma into the tubes;

[0072] Incubate for 1 min in the thermoblock;

[0073] Add 200 μl of thromboplastin and at the same time start the measuring operation;

[0074] shake the tube briefly, place it in the measuring position of the Schnitger & Gross, and insert the electrode holder up to the limit stop;

[0075] Read off the coagulation time.

[0076] The read-off coagulation times are listed in Table 8:

[0077] Standard human plasma (Behring Diagnostics) having a coagulation activity of approx. 100%, a 1:4 dilution of the standard human plasma (StHPl) in physiological sodium chloride solution (25%) and an FVII-deficient plasma (FVII-MP, Behring Diagnostics) were employed as samples. The spread was calculated from the ratio of the coagulation times for the dilution of the StHPl and the StHPl, and the FVII sensitivity was calculated from the ratio of the coagulation times for FVII-MP and STHPL.

TABLE 8
Patho FVII- Spread: FVII
100% II MP Patho sens.:
Protease Concen- Coagulation times II/ FVII-MP/
inhibitors tration in sec 100% 100%
pa-PMSF 0.01 mM 10.6 22.3 144 2.1 13.6
pa-PMSF 0.025 mM 10.6 21.8 147 2.1 13.8
pa-PMSF 0.05 mM 10.6 22.2 161 2.1 15.2
pa-PMSF 0.1 mM 10.7 21.7 172 2.0 16.1
pa-PMSF 0.5 mM 10.8 23.1 298 2.1 27.6
PMSF 1.5 mM 10.7 21.9 259 2.0 24.2
Leupeptin 0.5 mg/l 10.6 22.5 121 2.1 11.4
Pepstatin 0.7 mg/l 10.6 21.6 115 2.0 10.9
Antagosan 100 KIU/ml 10.6 22.2 118 2.1 11.1
Reference 10.7 21.4 135 2.0 12.6

EXAMPLE 9

[0078] Stabilizers were added to Thromborel S Bulkware according to Example 8a), which bulkware had been short-time heated at a temperature of 85 C. and using a dwell time of 3 sec, and different concentrations of the protease inhibitor diisopropyl fluorophosphate (DFP) were also added to some aliquots; the reference does not contain any inhibitor.

[0079] The coagulation time was determined using a Behring Coagulation Timer (PT.sec Thromborel S). Standard human plasma, the control plasma Patho II (Behringwerke AG) and an FVII-deficient plasma were employed as samples.

[0080] The spread was calculated from the ratio of the coagulation times of the Patho II and the StHPl, and the FVII sensitivity was calculated from the ratio of the coagulation times for FVII-MP and STHPL.

TABLE 9
Patho Spread: FVII
100% II FVII-MP Patho sens.:
Protease Concen- Coagulation times II/ FVII-MP/
inhibitors tration in sec 100% 100%
DFP 1 mM 10.0 41.9 389 4.2 38.9
DFP 0.33 mM 9.9 40.2 331 4.1 33.5
DFP 0.1 mM 9.9 40.7 213 4.1 21.6

EXAMPLE 10

[0081] Preparation of an Anti-FVII Antibody

[0082] The FVII immunizing antigen was isolated from human plasma. After having been adsorbed on aluminum hydroxide, the bound proteins were eluted with 0.3 M sodium phosphate, pH 7.4. Following a subsequent precipitation with 16% ethanol, the supernatant was precipitated once again with 25% ethanol and the sediment was purified further using AE cellulose. The adsorbed proteins were eluted with a solution which contains 3% sodium citrate, 0.15 M sodium chloride and 0.09% EDTA, pH 8.0. Following further purification by means of PVC electrophoresis and column chromatography using Sephadex-G 100, the pure FVII was then used for the immunization.

[0083] Sheep were immunized by standard methods and the anti-FVII antibody was isolated from the serum.

[0084] Thromborel S Bulkware according to Example 8a) was short-time heated to a temperature of 85 C. using a dwell time of 3 sec and additionally treated with different concentrations of the above-described polyclonal antibodies.

[0085] The coagulation time was determined using a Behring Coagulation Timer (PT.sec Thromborel S). Standard human plasma, the control plasma Patho II and an FVII-deficient plasma were employed as samples. The spread was calculated from the ratio of the coagulation times of the Patho II and the StHPl, and the FVII sensitivity was calculated from the ratio of the coagulation times for FVII-MP and STHPL.

TABLE 10
FVII
Concen- 100% Patho II FVII-MP Spread: sens.:
tration Coagulation times Patho II/ FVII-MP/
Antibody μg/ml in sec 100% 100%
AB 6.25 10.1 42.3 400 4.2 39.8
AB 0.625 10.0 41.2 301 4.1 30.2
AB 0.0625 9.9 40.4 163 4.1 16.5
Reference 0 9.9 39.2 136 4.0 13.8

EXAMPLE 11

[0086] Thromborel S Bulkware according to Example 8a) was short-time heated to a temperature of 85 C. using a dwell time of 3 sec and additionally treated with ascorbic acid, ascorbic acid and iron, and also ascorbic acid and copper. The stabilizers were added after an incubation of from 1 to 6 hours.

[0087] The coagulation time was determined as described in Example 8b).

[0088] Standard human plasma, the control plasma Patho II and an FVII-deficient plasma were used as samples. The reagents were stored at 4 C. and 37 C. and the coagulation time was repeated after 7 and 14 days. The spread was calculated from the ratio of the coagulation times of the Patho II and the StHPl, and the FVII sensitivity was calculated from the ratio of the coagulation times for FVII-MP and the STHPL.

TABLE 11
Concentrations in mM
Sample: SHPL
Ascorbic acid: 0 1 1 1
Fe(II)SO4: 0 0 0.01 0
CuCl: 0 0 0 0.01
Stabilization: after 6 h after 6 h after 1 h after 6 h
Sample: 100%
18 h/4 C. 12.9 13.7 15 13.7
7 days: 4 C. 12.9 14 15.9 13.8
7 days: 37 C. 13.4 14.7 16.4 14.6
14 days: 4 C. 12.9 14.1 16 13.9
14 days: 37 C. 14.8 15.3 17.1 15.5
Sample: FVII
18 h/4 C. 126.4 157.7 139.8 243.3
7 days: 4 C. 118.8 154.3 153.2 240.8
7 days: 37 C. 114.7 181.1 167.5 261.7
14 days: 4 C. 118.2 161.6 157.9 245.8
14 days: 37 C. 120.2 195.8 179 258.1
Sample: Patho II
18 h/4 C. 47.6 51.4 62.7 48.5
7 days: 4 C. 46.6 51.3 64.2 48.2
7 days: 37 C. 48.6 51.5 60.3 48.8
14 days: 4 C. 46 51.1 64.2 48.1
14 days: 37 C. 57 53.1 62 53.6
FVII sens.: FVII/100%
+18 h/4 C. 9.8 11.5 9.3 17.8
7 days: 4 C. 9.2 11.0 9.6 17.4
7 days: 37 C. 8.6 12.3 10.2 17.9
14 days: 4 C. 9.2 11.5 9.9 17.7
14 days: 37 C. 8.1 12.8 10.5 16.7
Spread: Patho II/100%
18 h/4 C. 3.7 3.8 4.2 3.5
7 days: 4 C. 3.6 3.7 4.0 3.5
7 days: 37 C. 3.6 3.5 3.7 3.3

EXAMPLE 12

[0089] Thromborel S Bulkware according to Example 8a) was short-time heated to a temperature of 120 C. using a dwell time of 0.75 sec and adjusted to pH 7.5 with 0.16 mM sodium carbonate; different concentrations of H2O2 were then added and the mixtures were incubated at 37 C. for 90 min. Stabilizers and 6 mM acetylcysteine were added to the solutions. The pH value was adjusted to 6.5 and the coagulation time was determined according to Example 8b).

[0090] Standard human plasma, the control plasma and an FVII-deficient deficient plasma were used as samples. The FVII sensitivity was calculated from the ratio of the coagulation times for FVII-MP and STHPL.

TABLE 12
Coagulation time in sec FVII-sens.:
Concentration of H2O2 100% FVII-MP FVII-MP/100%
0.3%  15 156 10.4
0.03%  11.5 143 12.4
0.003% 11.4 139 12.2
 0.0003% 11.7 133 11.4
 0.00003% 11.9 132 11.1
11.9 131 11.0

EXAMPLE 13

[0091] Thromborel S Bulkware according to Example 8a) was short-time heated at a temperature of 120 C. using a dwell time of 0.75 sec and then adjusted to pH 7.5 with 0.16 mM sodium carbonate; different concentrations of chloramine T were subsequently added and the mixtures were incubated at 37 C. for 90 min. Stabilizers and 6 mM acetylcysteine were added to the solution. The pH value was adjusted to 6.5 and the coagulation time was determined using a Behring Coagulation Timer (PT.sec Thromborel S).

[0092] Standard human plasma, the control plasma Patho II and an FVII-deficient plasma were used as samples. The spread was calculated from the ratio of the coagulation times of the Patho II and the StHPl, and the FVII sensitivity was calculated from the ratio of the coagulation times for the FVII-MP and the STHPL.

TABLE 13
Spread: FVII-sens.:
Concentration Coagulation time in sec Patho II/ FVII-MP/
of chloramine T 100% Patho II FV-MP 100% 100%
0.1 mM 13 38.2 177.4 2.9 13.6
0.01 mM 11.7 34.7 141.6 3.0 12.1
0.001 mM 11.5 34.2 140.1 3.0 12.2
11.6 34.2 142 2.9 12.2

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7148067Aug 31, 2004Dec 12, 2006The Board Of Trustees Of The University Of IllinoisTissue factor Factor VII and a phosphatide in dried form with calcium, sodium and dipeptides
US7682808Feb 23, 2006Mar 23, 2010The Board Of Trustees Of The University Of IllinoisCoagulation and fibrinolytic cascades modulator
WO2006031387A1 *Aug 23, 2005Mar 23, 2006S The Board Of Trustees Of TheThromboplastin reagents
Classifications
U.S. Classification435/13, 530/384, 435/184, 435/212
International ClassificationG01N33/573, C07K14/745, G01N33/86, C12Q1/56
Cooperative ClassificationG01N2333/96447, G01N33/86, C12Q1/56
European ClassificationG01N33/86, C12Q1/56
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