Determination of components active in proteolysis

The invention relates to a freeze-dried reagent combination containing all reagents required for determination of components active in a proteolysis such as proteases and especially proenzymes, cofactors, inhibitors and activators for proteases which reagent combination is enclosed in a container such as a cuvette. The invention also relates to a new process for the preparation of such a reagent combination in which process the conditions are controlled so that all reagents can exist in one single solution which is freeze-dried without the reagent being deactivated; and the use of the reagent combination in the fields of coagulation and fibrinolysis.

DESCRIPTION 
1. Technical Field 
This invention relates to a freeze-dried reagent combination, preferably 
for use in clinical diagnosis, which enables a simple and rapid 
determination of components active in proteolysis, for example proteases 
and especially proenzymes, inhibitors, cofactors and activators for 
proteases. The invention also concerns a new process for the preparation 
of such a reagent combination and use of this in the fields of coagulation 
and fibrinolysis. 
2. Background Act 
Protein-cleaving enzymes, so-called proteases, have a plurality of 
important functions in the body. As examples of proteases the following 
can be mentioned: Trypsin and chymotrypsin which are secreted from the 
pancreas and participate in the digestion process; elastase, kallikrein 
and catepsin of various types and the enzymes of the complementary system 
which i.a. participate in reactions caused by inflammatory and allergic 
conditions; thrombin and the factors VII.sub.a, X.sub.a, XI.sub.a and 
XII.sub.a consisting of proteases which participate in the chain of 
reactions leading to formation of blood coagulum, and plasmin and exogenic 
urokinase which are proteases providing the dissolution of the blood 
cagulum. 
Thus, diagnostic methods for measurement in vitro of protease activity are 
of a great importance in a plurality of clinical applications. Not only 
the amount of active protease but first of all the amount of proenzyme 
that can be converted to an active protease, and also the amounts of 
inhibitors and activators participating in proeolytic processes are 
measured. Of course such measurements are of a great importance in 
investigations of pathological conditions. In certain treatments such as 
in surgery and/or medication, in many cases a determination of the 
quantities of certain of these substances is also first carried out. 
Conventional methods for determination of components active in proteolysis, 
such as proteases and factors related thereto, for example proenzymes, 
inhibitors and activators, are based on so-called bioassays, immunologic 
reactions in vitro or utilization of biological proteins which proteins 
need not per se be natural substrates for the relative protease. 
The disadvantages of the above methods are that they are time-consuming and 
laborious both in respect of performance and calibration, have a limited 
sensitivity and/or exhibit a lacking specificity and are also difficult to 
standardize. 
In the last few years synthetic low molecular substrates have been 
developed based on amino acids or short peptides and provided with a 
usually photometrically, easily measurable marker which is easily split 
off by proteases. These substrates have to a large extent facilitated the 
methods of quantifying proteases as several of the disadvantages mentioned 
above have been eliminated by means of such substrates. (Hemker 
H.C.--Handbook of synthetic substrates, 1983, Martinus Nijhoff Publishers, 
Boston). 
These substrates, especially such as are provided with a photometrically 
measurable marker, so-called chromogenic substrates, have thus enabled the 
development of methods having an improved specificity and reproducibility. 
Moreover, by these methods activity is measured instead of amount as, on 
the other hand, is the case with for example immunologic methods and, 
thus, a linear ratio of studied parameter to measuring result is obtained 
as distinguished from log-log- and lin-log ratios mostly occurring in 
so-called bioassays. 
Despite the advantages indicated above measuring methods based on splitting 
of such, usually chromogenic, substrates are still impaired by several 
shortcomings limiting the usefulness thereof. 
As a rule, these so-called substrate methods comprise two-step reactions 
which include, on one hand, a merely biochemical reaction and, on the 
other hand, a reaction between a protease and a suitable substrate. 
Depending on which active component is to be determined the reaction 
processes can be illustrated as follows. 
##STR1## 
Thus, the second reaction step means splitting of a substrate having a 
marker through the influence of a corresponding enzyme, the amount of 
enzyme available for splitting consisting of the amount of active enzyme 
remaining after or being formed through the first reaction. 
Thus, in all cases the amount of marker M released is proportional to the 
analytes I, C, A and P, respectively. The above abbreviations have the 
following meanings, concentrations being marked by index. 
E=enzyme 
I=inhibitor 
S-M=substrate provided with marker 
M=marker 
S=substrate residue remaining after splitting off the marker 
R=reactant reacting with the enzyme through the influence of a cofactor 
C=cofactor 
A=activator 
P=proenzyme or enzyme compound which can be otherwise activated 
P=residue from activation of a proenzyme/enzyme compound 
As a rule, these two-step methods are time-consuming and laborious. As a 
plurality of pipetting steps are included and it is essential that the 
reaction times of the respective step are followed with a great 
carefullness and skill is required, moreover, when carrying out the 
analyses. Furthermore, the two-step method will limit the number of tests 
that can be carried out manually in one and the same series. 
Besides, the usefulness of the methods is impaired by comparatively short 
shelf life (one month or less) that, as a rule, reconstituted biochemical 
reagents have. Commercial reagents available at present for these analyses 
are usually packed so that there is enough for 20-200 analyses. 
Consequently the user should need, for economical reasons, to carry out a 
corresponding number of analyses within the time of the reconstituted 
reagents being stable. So far, technique and costs have not permitted 
simple packages for separate tests as included reagents usually must be 
packed individually in one way or other, as in separate packages or 
separate rooms in a packing container ("compartmentalized"). 
At present there is only one commercially available package form where the 
reagents for a single test are physically separated in one and the same 
plastic container (ACA.RTM., DuPont, USA). The container having a 
technically complicated design is merely useful in an instrument specially 
made for this purpose, that breaks the partitions between the reagent 
compartments automatically and adds samples and further reagents after 
preset times. 
It is also possible to simplify handling of conventionally packed reagents 
to a certain extent by adapting reaction conditions and reactants so that 
the two-step process is converted to a one-step process. The two reactions 
are then allowed to proceed in parallel and in such a way that the analyte 
is still directly proportional to the photometer reading. This process is 
either carried out in such a way that several reagents are added 
immediately after one another--which does not reduce the number of 
pipetting steps--or that certain reagents are mixed (e.g. substrate and 
biochemical reagent) in advance. Pipetting is then facilitated but a still 
more unstable reagent (shelf life usually only one day) is obtained. 
Particularly the last-mentioned process has been used in automatic methods 
(see below). 
One of the advantages of the substrate methods is their applicability to 
automatic analyzers for clinical use facilitating a change into a one-step 
method in the measuring process (Bergstrom K. and Lahnborg, G. Tromb. Res 
1975, Vol. 6, 223-233; Kapke G. F. et al, Clin. Chem. 1982, Vol. 28, 
1521-1524). In this way many of the disadvantages of the substrate methods 
are eliminated. However, an investment in automatic analyzers requires 
long test series and concentration on central units to become profitable, 
which means, however, that the period from sampling until the analysis 
results are obtained may become unacceptably long. 
A comparatively complicated process of achieving one-step methods is 
described in EP-A2-0 168 738, pipetting steps and exact timing demands 
being avoided. However, the process described therein is substantially 
limited to the technically complicated method of applying to a fixed 
carrier matrix the necessary biochemical reagents and substrates either in 
separate processes using different solvents preventing reaction or by 
compartmentalizing. Moreover, an apparatus specially constructed for the 
purpose is required for reading the resulting color. 
SUMMARY OF THE INVENTION 
Thus, it is the object of this invention to provide a reagent combination 
by means of which determinations of proteolytically active components can 
be easily carried out in a single step using the substrate method, yet the 
disadvantages indicated above being eliminated. 
This object is achieved according to the invention by means of a 
freeze-dried reagent combination. 
Thus, the invention is related to a freeze-dried reagent combination 
enclosed in a container and intended for a direct or indirect 
determination of a component active in proteolysis through cleaving of a 
substrate included in the reagent combination and capable of being cleaved 
by a protease to produce a detectable response, characterized in that the 
reagent combination comprises all the reagents required for the 
determination each in a substantially unreacted form and optionally one or 
more additives known per se and is prepared by freeze-drying in a way 
known per se of a solution containing all the constituents included in the 
freeze-dried reagent combination each in a substantially unreacted form, 
said freeze-dried reagent combination having substantially its original 
activity at reconstitution e.g. in a buffer solution. 
Best and Various Modes for Carrying Out Invention 
The reagent combination is preferably enclosed in the container in an 
amount sufficient for carrying out one separate single-step substrate 
method determination of proteolytically active components, for example 
proenzymes, protease activators, cofactors or protease inhibitors or their 
activators. 
Accordingly the present reagent combination contains a substrate that can 
be cleaved by a protease to produce a detectable response. Depending on 
which proteolytically active component is to be determined the reagent 
combination also contains other reagents, usually selected from the 
following components: Proenzyme, protease activator, protease inhibitor, 
an activator for the inhibitor and a cofactor for proteolysis. The reagent 
combination preferably also contains additives, for example stabilizing 
additives as is explained more in detail below.

Useful substrates are all substrates that can be split by proteases to 
produce a detectable response. Particularly suitable substrates are such 
as give a photometrically measurable response, so-called chromogenic 
substrates. The above-mentioned synthetic substrates are then preferably 
used. Such substrates are commercially available for example from 
KabiVitrum AB Diagnostika, Molndal, Sweden, for example under the 
commercial designations S-2251 (H-D-Val-Leu-Lys-pNA.2HCl), S-2337 
(N.alpha.-benzoyl-Ile-Glu(.gamma.-piperidide)-Gly-Arg-pNA.HCl), S-2366 
(&lt;Glu-Pro-Arg-pNA.HCl), S-2390 (H-D-Val-Phe-Lys-pNA.2HCl) and S-2732 
(N.alpha.-succinoyl-Ile-Glu-(.gamma.-piperidide)-Gly-Arg-pNA.HCl). In 
addition to these substrates which are described in the following 
illustrative examples further examples of substrates are reported in Table 
I below which are useful according to the invention. However, the said 
substrates are only illustrative but not limitative of the present 
invention. 
TABLE I 
__________________________________________________________________________ 
Analyte Enzyme 
Designation 
Structure 
__________________________________________________________________________ 
1. antifactor X.sub.a 
F Xa S-2767.sup.x) 
Boc--D--Arg--Gly--Arg-pNA 
S-2775.sup.x) 
Succinoyl-D--Arg--Gly--Arg-pNA 
CBS 31.39.sup.xx) 
CH.sub.3 SO.sub.2 --D--Leu--Gly--Arg-pNA 
2. Antithrombin 
thrombin 
S-2266.sup.x) 
H--D--Val--Leu--Arg-pNA 
3. antiplasmin 
plasmin 
S-2403.sup.x) 
&lt;Glu--Phe--Lys-pNA 
Chromozym-'L.sup.xx) 
Tosyl-Gly--Pro--Lys-pNA 
4. heparin 
FXa see antifactor Xa! 
5. Fibrin monomer 
plasmin 
S-2466.sup.x) 
H--D--Glyn--Phe--Lys-pNA 
PL-1.sup.xx) 
H--D--Nle--CHA--Lys-pNA 
6. t-PA plasmin 
S-2390.sup.x) 
H--D--Val--Phe--Lys-pNA 
Chromozym-P'L.sup.xx) 
Tosyl-Gly--Pro--Lys-pNA 
7. FX FXa S-2732.sup.x) 
Succinoyl-Ile--Glu-(.gamma.-piperidide)-Gly--Arg 
-pNA 
S-2765.sup.x) 
Benzyloxycarbonyl-D--Arg--Gly--Arg-pNA 
Spectrozyme Xa.sup.xx) 
Methyloxycarbonyl-D--CHG--Gly--Arg-pNA 
8. Plasminogen 
plasmin 
S-2406.sup.x) 
&lt;Glu--Leu--Lys-pNA 
Chromozym-PL.sup.xx) 
Tosyl-Gly--Pro--Lys-pNA 
9. Protein C 
Protein C.sub.a 
S-2288.sup.x) 
H--D--Ile--Pro--Arg-pNA 
S-2401.sup.x) 
&lt;Glu--Thr--Arg-pNA 
__________________________________________________________________________ 
.sup.x) Manufacturer KabiVitrum AB 
.sup.xx) Manufacturer Pentapharm AG, Basel, Schweiz 
Abbreviations: 
BO C = tbutyloxycarbonyl 
CHA = cyclohexylalanin 
CHG = cyclohexylglycin 
Tosyl = ptoluene sulfonyl 
The invention is generally applicable to determination of proteolytically 
active components according to the basic principles for the substrate 
determination method provided such freeze-drying conditions can be 
established for a solution of the reagents under which conditions these 
components do not lose their activity, for example react with each other, 
but recover essentially their original activity after reconstitution. 
So far it has been possible to establish suitable conditions for all 
reagent combinations of interest, especially in the fields of coagulation 
and fibrinolysis except the combination of reagents required for 
determination of .alpha..sub.1 -antitrypsin and F VIII. However, it is 
possible that such conditions possibly will be established also for these 
combinations after further experimentation. 
The invention is well suited for determination of the proteolytically 
active components mentioned above for the substrate determination method. 
Thus, suitable reagent combinations are, when the proteolytically active 
component consists of a) an inhibitor, b) a cofactor, c) an activator or 
d) a proenzyme, a chromogenic substrate which can be split by protease in 
combination with a) a protease, b) a protease and a reactant which can 
react with the protease under the influence of the cofactor to be 
determined, c) a proenzyme or another activable enzyme compound and, 
respectively, d) an activator for the proenzyme. Embodiments of the 
invention preferred at present appear from the illustrative examples. 
The present invention also relates to a process for preparation of the 
freeze-dried reagent combination. 
More specifically, the invention relates to a process for preparation of 
the freeze-dried reagent combination, which process is characterized in 
that a solution containing all the reagents required for the determination 
and possibly one or more additives known per se is produced in a solvent 
such as water under conditions that are controlled in such a way that the 
reagents remain substantially unreactive in the resulting solution and in 
a subsequent freeze-drying of the solution; and that the solution is 
freeze-dried in the container in a way known per se, the freeze-dried 
reagent combination being obtained wherein the reagents are included in a 
substantially unreacted form and wherein the reagents have substantially 
original activity after reconstitution of the reagent combination, e.g. in 
a buffer solution. 
It is essential according to the invention that all the reagents included 
in the freeze-dried combination are dissolved in the same solution at 
least in the very freeze-drying process. The process will be especially 
simple to carry out if a stock solution is first prepared from all 
reagents concerned under conditions controlled according to the invention, 
said stock solution also containing possible additives being thereafter 
distributed on containers and freeze-dried in these containers. However, 
it is of course also possible to prepare several stock solutions 
containing one or some of the reagents concerned and possible additives 
which solutions are thereafter brought together and freeze-dried 
simultaneously as one single solution in the respective container. Water 
is a suitable solvent even if additives of other solvents may be used, for 
example acetic acid, methanol and dimethyl sulfoxide. 
According to a suitable embodiment of the invention such an amount of the 
solution (or solutions) is introduced into each container as is intended 
for one single test. A cuvette or a well in a microtiter plate or the like 
is then preferably used as container to which a sample can be added after 
which the container is directly transferred to a standard equipment for 
clinical analyses, for example for photometry. 
According to the invention it is essential that the conditions in the 
preparation of the solution of all the reagents or in the combination of 
several solutions of reagents are adjusted, for example by providing the 
solvent with suitable additives of stabilizing soluble components and/or 
that the solvent is adjusted to such a pH-range, preferably by means of 
buffer salts, so that a mixture of such components usually reactive with 
each other can be dissolved in the preparation of the freeze-dried reagent 
combination without undesired but per se expected reactions taking place 
in the preparation. How the conditions are to be adjusted is dependent on 
the reagents included in the reagent combination and will be explained 
more in detail below. 
The establishment of such controlled conditions has enabled a simultaneous 
freeze-drying according to the invention of all the reagents in one single 
solution. The mixture of reagents in the final form of the product is 
present in a freeze-dried state without any special compartmentalizing of 
the components included in the mixture which mixture has a good stability, 
an immediate dissolution of all the components included in the reagent 
mixture being achieved upon addition of a sample dissolved in a buffer for 
the freeze-dried reagent combination. 
Thus, through the invention a product is provided which enables the 
utilization of all the advantages of the substrate determination methods 
and by which further advantages are achieved. For example, the need of 
several pipetting steps is eliminated as only pipetting for handling of 
the sample is required. Likewise the demands on accuracy as regards 
keeping correct incubation and reaction periods are eliminated. Moreover, 
lower demands are made on the technical skill of the operator and no 
specially constructed instrument for using the reagent combination is 
required for carrying out the analysis. 
Another advantage of the products according to the invention is a 
considerably longer shelf life than for the corresponding reconstituted 
reagents and that they can be standardized by the manufacturer which 
provides a considerably simplified process for calculating the test result 
in comparison with previously known processes of the same type which 
usually require the establishment of a standard curve. 
The present freeze-dried reagent combination is thus extremely well suited 
for routine use utilizing current instruments for carrying out single 
tests in small and/or big clinics when these have no automated analyzer in 
operation and/or no qualified personnel in service. Moreover, the product 
according to the invention provides the possibility of obtaining a quick 
response in acute situations as the determination can be carried out 
locally ("bedside") and as no preparative work such as preparation of 
reagent solutions, establishment of standard curve or running of controls 
is necessary. 
Moreover, the use of the product according to the invention often means a 
considerable saving of resources and material in comparison with current 
products available on the market, such as "kits", which are not suited for 
determination of a few samples or in so far as they are designed for 
performance of a single analysis, said performance requiring special 
equipment. 
As pointed out above the reagent combination of the invention contains a 
substrate capable of being cleaved by proteases and in addition other 
reagents depending on which proteolytically active component one wishes to 
determine. Thus, the preparation of this product must take place under 
conditions that are controlled with respect to which reagents are to be 
included in the reagent combination, i.e. depending on which active 
component is to be determined. The process is explained in the following 
more in detail with respect to embodiments thereof that are important at 
present, viz. determination of inhibitors, cofactors, activators and 
proenzymes. 
I. Process for the Preparation of a Product for Determination of Inhibitors 
Determination of the amount of protease inhibitor in a sample is based on 
the fact that a known and well-defined amount of an enzyme inhibited by 
the formation of 1:1-complex with the inhibitor is added to the sample and 
that the excess of enzyme is determined by addition of a known and 
well-defined amount of a suitable substrate. 
According to the invention it has surprisingly been found that it is 
possible under conditions suited for production on a large scale to 
prepare a solution containing the enzyme as well as the substrate under 
conditions controlled in such a way that neither the enzyme nor the 
substrate is destroyed in the solution or in the process of freeze-drying. 
It is then extremely surprising that the enzyme does not react with the 
substrate in the preparation of the reagent combination although the 
enzyme and the substrate will achieve almost complete activity after 
reconstitution of the freeze-dried combination, for example by addition of 
a suitable buffer. 
In determination of protease inhibitors the process of the invention is 
based on the fact that such a pH-range has been found within which the 
enzyme activity in respect of the substrate is negligible, however no 
denaturation of the enzyme or any hydrolysis of the substrate taking 
place. Such a range is preferably pH 3-5 and most preferably pH is 4.2. 
The optimum value within the range varies somewhat depending on which 
enzyme and which substrate are to be utilized in the process. 
Furthermore, it is advantageous that such a buffer is utilized for 
adjustment of optimal pH, the salts of which leave at freeze-drying or are 
present after freeze-drying in such low amounts that the sample can be 
prepared before the analysis in currently used buffers having a pH of 
6.5-9.5 so that optimal reaction conditions, i.e. pH 6.5-9.5, can be 
obtained for the substrate-enzyme reaction as well as for the 
enzyme-inhibitor reaction. Suitable buffers for the process of preparing 
reagents are weak organic acids such as formic acid or acetic acid mixed 
with the respective salt. Also other acids having a pKa-value in the same 
range such as citric acid, ascorbic acid etc. can be used but as a rule 
lower concentrations are required with the result that a considerably less 
stable pH is attained. 
To avoid degradation processes during freeze-drying and during a following 
storage of the reagent mixture and in order to promote a rapid dissolution 
of the reagent mixture when adding the sample and minimizing reagent 
adsorption at the reagent container additives are suitably used, such as 
inorganic salts, inactive proteins, e.g. albumin, sugar, e.g. mannitol 
and/or surfactants such as polyethylene glycol (PEG, Carbowax.RTM. 8000 
available from Union Carbide, USA) and Triton.RTM. X-100 (Rohm & Haas, 
USA). 
As shown in the following examples the excellent results obtained when 
using the reagent combination according to the invention are due to the 
fact that it is possible to find reaction conditions for the analysis, for 
example pH, ionic strength and buffer salts, which are appropriate for 
both the reactions, i.e. in the determination of inhibitor for the 
substrate-enzyme reaction as well as for the inhibitor-enzyme reaction. 
The choice of substrate is also of a great importance when utilizing the 
present invention. Besides the fact that the substrate, as pointed out 
above, must be cleavable by protease to give a detectable response it is 
also essential that such a substrate is selected for a certain reaction as 
reacts effectively enough with the enzyme in order that reasonable 
measuring conditions, preferably reaction times shorter than 10 min, will 
be achieved but which is still not a substrate effective enough to prevent 
the enzyme molecule from reacting with the inhibitor. This condition can 
partly be expressed with the bonding ability of the substrate to the 
enzyme, K.sub.m, which should preferably be 2-8.times.10.sup.-4 mol/l. 
Commercially available synthetic substrates are usually utilized, for 
example those indicated in table I and such as are described in the 
illustrative examples. Suitable substrate concentrations are 
1-20.times.10.sup.-4 mol/l. The concentration should be so high that there 
is a linear relationship between the enzyme concentration and the marker 
concentration. In general a preferred substrate concentration is 
5.times.10.sup.-4 mol/l. 
However, it should be pointed out that an optimal substrate concentration 
and the affinity properties of the substrate are influenced by additives 
in the reaction mixture, and therefore strict general selection criteria 
cannot be defined. 
Of course the choice of enzyme is dependent on which inhibitor is to be 
determined. Such enzymes are commercially available for a plurality of 
inhibitors of clinical interest, for example for several important 
protease inhibitors occurring in plasma such as antithrombin which can be 
determined via a reaction with thrombin or factor Xa in the presence of 
heparin, antiplasmin, which can be determined via plasmin, and kallikrein 
inhibitors, and according to the invention freeze-dried reagent 
combinations for carrying out these determinations can be prepared. 
II. Process for the Preparation of a Product for Determination of Cofactors 
Cofactors comprise many different types of substances actuating enzyme 
reactions. Here a factor is exemplified in the first place which actuates 
the inhibition of certain serine proteases, primarily FX.sub.a and 
thrombin, with the inhibitor antithrombin, viz. heparin. The method for 
determination of heparin and similar substances is similar to the 
inhibitor method described above except that the inhibitor, in this case 
antithrombin, must be added in excess. 
Due to this the enzyme amount must be considerably increased in order that 
correct reaction conditions might be obtained. Otherwise the process is 
based on the same principles as the inhibitor method above exemplified for 
antithrombin and factor X.sub.a. Thus, the same conditions apply as in the 
process for preparation of a reagent combination for inhibitor 
determination according to the process I. 
Another cofactor that can be determined is fibrin monomer. This is a plasma 
protein converted by a coagulation enzyme and being a cofactor in the 
activation of the proenzyme plasminogen via the enzyme t-PA (plasminogen 
activator) which then serves as an activator. This enzyme i.e. the 
activator, is freeze-dried together with plasminogen, i.e. its substrate, 
and a chromogenic substrate suitable for the activation product, viz. the 
enzyme plasmin. The process for the preparation of the reagent combination 
corresponds to the process for preparation of a reagent combination for 
the inhibitor determination described above with the difference that the 
pH-range that can be used for the freeze-drying also comprises neutral pH 
and, thus, pH is suitably 3-8.5, and preferably 5-7. 
III. Process for the Preparation of a Reagent Combination for Determination 
of Activators 
The method for determination of activator is built on the principle of 
freeze-drying a suitable proenzyme together with a substrate suitable for 
the activated proenzyme in the presence of additives promoting stability 
and solubility in analogy with what has been described above. Moreover, 
certain determination methods can require the presence of reaction 
promoting reagents. Thus, it is possible according to the invention to 
freeze-dry simultaneously all the reagents that are required for 
determination of for example t-PA, viz. plasminogen, plasmin substrate and 
stimulator of fibrin or polylysin type, and still maintain their activity, 
the conditions previously described being used which in analogy with the 
process II above also comprise neutral pH. Thus, a suitable pH-range is 
3-8.5 and preferably 6-7. 
IV. Process for the Preparation of a Reagent Combination for Determination 
of Proenzymes 
Proenzymes such as Protein C, plasminogen and factor X are well suited for 
determination by means of the substrate technique in the one-step 
embodiment provided the activator utilized as reagent in the presence of 
substrate for the corresponding activated proenzyme is fast-acting. Thus, 
the process according to this embodiment of the invention comprises 
freeze-drying of a substrate together with an activator that activates the 
major portion of proenzyme a occurring i the sample within some minute. 
The resulting reagent combination is intended for determination of 
proenzyme, the sample being diluted so much that inhibitors of the 
activated proenzyme occurring by nature in the sample are present in such 
a low concentration that these do not disturb the reaction between 
activated proenzyme and its substrate. 
According to the invention freeze-drying is carried out in conventional 
manner, e.g. at 0.08-0.15 mbar by freezing at a temperature of -45.degree. 
to -40.degree. C., preferably -42.degree. C. for a time of 1-5 h, 
preferably 1 h, and a following drying for a time of 12-20 h, preferably 
15 h, at a temperature of 12.degree.-24.degree. C., preferably 22.degree. 
C. 
The invention also relates to use of a freeze-dried reagent combination for 
quantitative, semi-quantitative or qualitative determination of 
coagulation factors and fibrinolysis factors in a sample, especially a 
biological one, such as whole blood, blood plasma, blood serum, 
cerebrospinal liquid, lung liquid or urine, in a one-step process by 
addition of the sample to the freeze-dried reagent combination enclosed in 
a container, preferably a cuvette, and reading in a way known per se of a 
response received. 
The process according to the invention is explained in greater detail by 
means of the following examples, which are not limiting per se, with 
reference to the enclosed drawings, wherein FIGS. 1-9 show standard curves 
for a number of different components active in proteolysis and established 
at a wavelength of 405 nm by the aid of suitable freeze-dried reagent 
combinations according to the invention. 
EXAMPLE 1 
In this example a process for the preparation of a freeze-dried reagent 
combination for determination of a protease inhibitor, antifactor Xa and 
its use are described. 
a) Acetate buffer, 1000 ml, is prepared by diluting 500 ml of a mixture of 
0.2M acetic acid and 0.2M sodium acetate in water to 1000 ml by addition 
of distilled water. The desired pH-value of 4.2 is obtained with 368 ml 
0.2M acetic acid and 132 ml 0.2M sodium acetate. 
b) in order to obtain an improved stability and increased solubility of the 
reagent combination albumin and mannitol are added to the buffer solution 
a) in the amounts indicated below. 
c) Preparation for freeze drying: 
The substrate S-2732, 650 mg/l (0.8 mmol/l) and factor Xa, 1000 nkat (1000 
nkat/l) are added to the buffer solution a), to which BSA, 5 g/l (0.5%) 
and mannitol, 10 g/l (1%) have been added. 
d) Preparation of a freeze-dried reagent combination intended for 
determination of antifactor Xa: Each 200 .mu.l of the solution c) is 
transferred to the respective microcuvette of plastic (Kartell Art. No. 
1938). The cuvettes are placed in a freeze-drier at -42.degree. C. for one 
hour and dried for 15 h at +22.degree. C. and a pressure of 0.08-0.15 
mbar. 
e) Use of cuvettes d) for determination of antifactor Xa: 300 .mu.l of 
plasma (diluted 1:500) in 0.05 mol/l Tris, pH 8.4, I=0.2 containing EDTA 
(7.5 mmol/l), heparin (3 IU/ml] and PEG (1%) are added to the cuvette d) 
containing the freeze-dried reagent combination consisting of S-2732 (0.13 
mg) FXa (0.2 nkat) and BSA and mannitol. 
After addition the pH of the test solution is 8.2. The reaction is allowed 
to take place at room temperature (25.degree. C.) or at 37.degree. C. and 
is interrupted after 8 min through addition of 300 .mu.l 5% AcOH. 
The absorbance of the solution at 405 nm is thereafter read in a photometer 
and the result is compared with samples containing antifactor Xa in known 
amounts, by means of which the standard curves shown i FIG. 1a have 
already been prepared at the manufacturer of the reagent combination. This 
curves shows the relationship between dose and response and are the basis 
of factors calculated by the manufacturer and utilized by the user for 
calculation of the analyte in question. 
The reaction was found to have a low temperature dependence which is also 
apparent from FIG. 1a. 
The correlation to a generally used and commercially available "kit" for 
determination of antithrombin (Coates.RTM. Antithrombin, KabiVitrum AB, 
Sweden) is shown in FIG. 1b. 
EXAMPLE 2 
In this example a process for the preparation of a reagent combination 
intended for determination of antithrombin and its use are described. 
a) A freeze-dried reagent combination intended for determination of 
antithrombin and containing S-2366 (0.1 mg), thrombin (0.7 nkat) as well 
as BSA and mannitol is prepared in a cuvette in analogy with example 1. 
b) Use of the reagent combination in determination of antithrombin. 300 
.mu.l plasma (diluted 1:80) in the same buffer as indicated in example 1e) 
is added to the cuvette from a), the contents of which being freeze-dried. 
The reaction is allowed to take place at 37.degree. C. and interrupted 
after 8 min through addition of 300 .mu.l of 5% acetic acid. Measuring is 
carried out in analogy with example 1e), the standard curve shown in FIG. 
2 being obtained. 
EXAMPLE 3 
In this example a process for the preparation of a reagent combination 
intended for determination of antiplasmin and its use are described. 
a) A cuvette containing a freeze-dried reagent combination intended for 
determination of antiplasmin and containing S-2251 (0.3 mg), plasmin (0.3 
nkat) and BSA and mannitol is prepared in analogy with example 1. 
b) Use of the reagent combination in determination of antiplasmin. 300 
.mu.l plasma (diluted 1:30) in 0.05 mol/l Tris, pH=8.3 comprising 0.15 
mol/l methylamine is added to the cuvette from a), the contents of which 
being freeze-dried. The reaction takes place at 37.degree. C. and is 
interrupted after 8 min by addition of 300 .mu.l of 5% AcOH. Measurement 
is carried out in analogy with example 1e). The standard curve shown in 
FIG. 3 is obtained. 
The above examples show reagent combinations intended for determination of 
protease inhibitors. The two immediately following examples illustrate 
reagent combinations intended for determination of cofactors. 
EXAMPLE 4 
A process for the preparation of a reagent combination for determination of 
heparin and its use are described in this example. 
a) A cuvette comprising a reagent combination for determination of heparin 
and containing S-2732 (0.2 mg), FXa (0.5 nkat) and BSA and mannitol is 
prepared in analogy with example 1. 
b) Use of the reagent combination in determination of heparin. 300 .mu.l 
plasma (diluted 1:15) in 0.05 mol/l Tris, pH=8.4, I=0.2 containing EDTA 
(7.5 mmol/l) and PEG (1%) is added to the cuvette from a), the contents of 
which being freeze-dried. The reaction is carried out at room temperature 
and interrupted after 6 min through addition of 300 .mu.l of 5% acetic 
acid. It is measured in analogy with example 1e). The standard curve shown 
in FIG. 4 is obtained. 
EXAMPLE 5 
In this example a process for the preparation of a reagent combination for 
determination of fibrin monomer and its use are described. 
a) S-2390 (12 mg) and mannitol (19 mg) are dissolved in 7.0 ml 0.03 mol/l 
sodium acetate buffer, pH=4.9 containing 0.01% Tween.sup.R 80 and mixed 
with 2.5 mg human Glu-plasminogen dissolved in 0.8 ml sterile water. 100 
mg BSA dissolved in 0.5 ml water and 3.6 .mu.g t-PA dissolved in 0.8 ml 
sodium acetate buffer are added to the resulting solution after which 
additional buffer is added so that a total volume of 20 ml is achieved. 
Portions of 200 .mu.l of the final solution are distributed on 
microcuvettes and freeze-dried according to example 1d). 
b) Use of the reagent combination in determination of fibrin monomer. 
300 .mu.l plasma (diluted 1:41) in 0.063 mol/l Tris, pH=8.5 containing 
0.01% Tween.RTM. 80 is added to the cuvette from a), the contents of which 
being freeze-dried and consisting of S-2390 (0.12 mg), plasminogen (25 
.mu.g) and t-PA (0.036 .mu.g) as well as mannitol, Tween.RTM. 80 and BSA. 
The reaction takes place at room temperature and is interrupted after 20 
min by addition of 300 .mu.l of 20% acetic acid. It is measured in analogy 
with example 1e). The standard curve shown in FIG. 5 is obtained. 
An embodiment intended for determination of an activator for an enzyme 
reaction is illustrated in the following example. 
EXAMPLE 6 
A process for the preparation of a reagent combination for determination of 
t-PA and its use are described in this example. 
a) S-2251 (9 mg) dissolved in 13.5 ml distilled water is mixed with 0.75 ml 
of an aqueous solution containing plasminogen (18.75 CU) and mannitol (15 
mg). The resulting solution is cooled and mixed with 0.75 ml of an aqueous 
solution containing CNBr-digested fibrin(ogen) (3.75 mg) and mannitol (15 
mg). Portions of 200 .mu.l of the final solution are distributed on 
microcuvettes and freeze-dried according to example 1d). 
b) Use of the reagent combination in determination of t-PA. 
300 .mu.l of pretreated plasma (diluted 1:125) in 0.05 mol/l Tris, pH=8.3 
containing 0.01% Tween.RTM. 80 is added to the cuvette from a), the 
contents of which being freeze-dried and consisting of S-2251 (120 .mu.g), 
plasminogen (0.25 CU) and CNBr-digested fibrin(ogen) (50 .mu.g) as well as 
mannitol. The reaction is allowed to take place at 37.degree. C. and 
interrupted after 2 h and 45 min through addition of 300 .mu.l 20% AcOH. 
It is measured in analogy with example 1e) and the standard curve shown in 
FIG. 6 is obtained. 
Embodiments intended for determination of proenzymes are illustrated in the 
following example. 
EXAMPLE 7 
A process for the preparation of a reagent combination for determination of 
factor X and its use are described in this example. 
a) S-2337 (24 mg) and mannitol (120 mg) dissolved in 6.5 ml of distilled 
water are mixed with 3.5 ml of an aqueous solution containing Russel's 
Viper Venom (RVV, Miami Serpentarium Labs, USA) (0.9 mg) and NaCl (60 mg). 
A solution of CaCl.sub.2 (0.1 mol/l) is added to the resulting solution so 
that a total volume of 20 ml is obtained. Portions of 200 .mu.l of the 
final solution are distributed on microcuvettes and freeze-dried according 
to example 1d). 
b) Use of the reagent combination in determination of factor X. 
600 .mu.l of plasma (diluted 1:60) in 0.05 mol/l Tris, pH=7.8 containing 
Polybrene.RTM. (20 mg/l, Aldrich, USA) are added to the cuvette from a), 
the contents of which being freeze-dried and consisting of S-2337 (0.24 
mg) and RVV (9 .mu.g) as well as mannitol, CaCl.sub.2 and NaCl. The 
reaction takes place at 37.degree. C. and is interrupted after 3 min. 
through addition of 200 .mu.l of 20% AcOH. It is measured in analogy with 
example 1e) and the standard curve shown in FIG. 7 is obtained. 
EXAMPLE 8 
In this example a process for the preparation of a reagent combination for 
determination of protein C and its use are described. 
a) S-2366 (12 mg) is dissolved in 19.55 ml of distilled water and 0.45 ml 
of Protac.RTM. C-solution (10 U/ml), Pentapharm, Switzerland) is added. 
Portions of 200 .mu.l of the resulting solution are distributed on 
microcuvettes and freeze-dried according to example 1d). 
b) Use of the reagent combination in determination of protein C. 
300 .mu.l plasma (diluted 1:11) in 0.025 mol/l Tris, pH=8.4 containing 0.1% 
PEG are added to the cuvette from a), the contents of which being 
freeze-dried and consisting of S-2366 (0.12 mg) and Protac.RTM. C (0.045 
U). The reaction is allowed to take place at 37.degree. C. and is 
interrupted after 7 min through addition of 300 .mu.l 20% AcOH. It is 
measured in analogy with example 1e) and the standard curve shown in FIG. 
9 is obtained. 
In these examples abbreviations have been used having the following 
meanings 
______________________________________ 
BSA bovine serum albumin 
Tris tris(hydroxymethyl)-aminomethane 
EDTA ethylenediamine tetraacetic acid 
PEG Polyethylene glycol 
AcOH acetic acid 
Tween .RTM. 80 
polyoxyethylene sorbitan monooleate (Atlas 
Chemical Industries, U.S.A.) 
Glu glutamic acid 
t-PA plasminogen activator 
S-2251 H--D--Val--Leu--Lys-pNA.2HCl 
S-2337 N.alpha.-benzoyl-Ile--Glu(.gamma.-piperidide)-Gly--Arg- 
pNA.HCl 
S-2366 &lt;Glu--Pro--Arg-pNA.HCl 
S-2390 H--D--Val--Phe--Lys-pNA.2HCl 
S2732 N.alpha.-succinoyl-Ile--Glu(.gamma.-piperidide)-Gly--Arg- 
pNA.HCl 
pNA p-nitroanilide 
.sup.A 405 
absorbance at 405 nm 
nkat nanokatal (1 katal = the amount of enzyme 
activity that splits 1 mol substrate per 
sec. under specified conditions) 
1 U unit related to international standard 
(Heparin) 
1 U unit related to international standard 
(Streptokinase) 
CU (plas- casein unit 
minogen) 
U 1 U = the amount of Protac .RTM. activating 
(Protac .RTM.) 
protein C included in 1 ml of normal human 
citrate plasma. 
______________________________________ 
The standard curves shown on the drawings have been obtained, preferably at 
the manufacturer of the reagent combinations, by measurement of A.sub.405 
for samples having varying known amounts of the component to be determined 
by means of the respective standard curve in analogy with the measurements 
carried out in the corresponding illustrative example.