DETERMINATION OF COMPONENTS ACTIVE IN PROTEOLYSIS

Abstract: 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: DETERMINATION OF COMPONENTS ACTIVE IN PROTEOLYSIS This invention relates to a freeze-dried reagent combination, preferably for use in clinical diagnosis. which enables a simple and rapid determination of com¬ ponents active in proteolysis, for example proteases and especially proenzy es, inhibitors, cofactors and • activators for proteases. The invention also concerns a new process for the preparation of such a reagent com¬ bination and use of this in the fields of coagulation and fibrinolysis . Protein-cleaving enzymes, so-called proteases, have a plurality of important functions in the body. As exemples of proteases the following can be mentioned: Trypsin and chymotrypsin which are secreted from the pancreas and participate in the digestion process; elastase, alli rein and catepsin of various types and the enzymes of the complementary system which i.a. par¬ ticipate in reactions caused by inflammatory and aller¬ gic conditions; thrombin and the factors VII a , X a , XI a and XII 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 pro- enzyme that can be converted to an active protease, and also the amounts of inhibitors and activators partici- pating in proeolytic processes are measured. Of course such measurements are of a great importance in investi¬ gations of pathological conditions. In certain treat¬ ments 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 compo¬ nents active in proteolysis, such as proteases and fac- SUBSTITUTE SHEE tors related thereto, for example proenzymes, inhibi¬ tors and activators, are based on so-called bioassays, immunologic reactions in vitro or utilization of biolo¬ gical proteins which proteins need not per se be natu- ral substrates for the relative protease. The disadvantages of the above methods are- hat they are time-consuming and laborious both in respect of performance and calibration, have a limited sensi¬ tivity and/or exhibit a lacking specificity and are also difficult to standardize. In the last few years synthetic low molecular sub¬ strates have been developed based on amino acids or short peptides and provided with a usually photometri¬ cally, 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, Martmus Nijhoff Publishers, Boston). These substrates , especially such as are provided with a photometrically measurable marker, so-called chromogenic substrates, have thus enabled the develop¬ ment of methods having an improved specificity and re- producibility. 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 chromogen¬ ic, substrates are still impaired by several shortcom¬ ings limiting the usefulness thereof. As a rule, these so-called substrate methods com¬ prise 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. A. Determination of inhibitor E e +I ^Ei-I + E Q -_ (E e >Ei) S-M—Se-i ^S + M B . Determinat i on o f co f ac t or E e + R -S E C -R + E e __ c S- le-c ■> S + M C. Determination of activator P_-__ ^ p + E <P in excess) S-M IA - s + M D. Determination of proenzyme P-^ ■ P + Ep <A in excess) Thus, the second reaction step means splitting of a substrate having a marker through the influence of a corrsponding enzyme, the amount of enzyme available for splitting consisting of the amount of active enzyme re¬ maining after or being formed through the first reac¬ tion. Thus, in all cases the amount of marker M released is proportional to the analytes I, C, A and P, respec- tively. 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-consum¬ ing 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 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 Cone 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, carry out a corre- spending number of analyses within the time of the re¬ constituted reagents being stable. So far, technique and costs have not permitted simple packages for sepa¬ rate tests as included reagents usually must be packed individually in one way or other, as in separate pack- ages or separate rooms in a packing container ("com¬ partmentalized"). At present there is only one commercially availa¬ ble package form where the reagents for a single test are physically separated in one and the same plastic container CΛ^, 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 automa¬ tically and adds samples and further reagents after preset times. SUBSTITUTE SHEET It is also possible to simplify handling of con¬ ventionally 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 paral¬ lel and in such a way that the analyte is still direct¬ ly proportional to the photometer reading. This process is either carried out in such a way that several rea¬ gents 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 biochem¬ ical reagent) in advance. Pipetting is then facilitated but a still more unstable reagent (shelf life usually only one day) is obtained. Particularly the last-men- tioned 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. Tro b. Res 1975, Vol. 6, 223-233; Kapke G.F. et al, Clin. Che . 1982, Vol. 28, 1521-1524) . In this way many of the disadvantages of the substrate methods are elimi¬ nated. 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 sub tantially limited to the technically complicated method of apply¬ ing to a fixed carrier matrix the necessary biochemical reagents and substrates either in separate processes using different solvents preventing reaction or by com¬ partmentalizing. Moreover, an apparatus specially con- structed for the purpose is required for reading the resulting color. Thus, t is the object of this invention to pro¬ vide a reagent combination by means of which determina- 5 tions of proteolytically active components can be easi¬ ly 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 10 by means of a freeze-dried reagent combination as de¬ fined in claim 1. Thus, the invention is related to a freeze-dried reagent combination enclosed in a container and intend¬ ed for a direct or indirect determination of a co po- 15 nent active in proteolysis through cleaving of a sub¬ strate 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 deter ina- 20 tion each m a substantially unreacted form and option¬ ally one or more additives knovm 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 un- 25. " reacted form, said freeze-dried reagent combination having substantially its original activity at reconsti- fcution e g in a buffer solution. The reagent combination is preferably enclosed in the container in an amount sufficient for carrying out 30 one separate single-step substrate method determination of proteolytically active components, for example pro- enzymes, protease activators, cofactors or protease in¬ hibitors or their activators. Accordingly the present reagent combination con- 35 " tains a substrate that can be cleaved by a protease to produce a detectable response. Depending on which pro- SUBSTITUTE SHEET teolytically active component is to be determined the reagent combination also contains other reagents, usu¬ ally 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 exemple 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 chromo- gemc substrates. The above-mentioned synthetic sub¬ strates are then preferably used. Such substrates are commercially available for example from abiVitrum AB Diagnostika, Molndal, Sweden, for example under the commercial " designations S-2251 (H-D-Val-Leu-Lys- -pNA-2HCl), S-2337 (NCC-benzoy1- Ile-Glu(^-piperldide )- -Gly-Arg-pNA-HCl) , S-2366 ( <Glu-Pro-Arg-pNA* HC1 ) , S-2390 (H-D-Val-Phe-Lys-pNA*2HCl) and S-2732 CNOC-succi- noyl-Ile-Glu-(^ / '-piperιdide)-Gly-Arg-pNA*HCl) . In addi¬ tion to these substrates which are described in the following illustrative examples further examples of substrates are reported in Table I below which are use- ful according to the invention. However, the said sub¬ strates are only illustrative but not limitative of the present invention. Table I Analyte Enzyme Desiqnatjon Structure 1. antifactor X F Xa S-2767 XJ Boc-D-Arg-Gly-Arg-pNA S-2775 x) Succinoyl-D-Arg-Gly-Arg-pNA CBS 31.39xx) CM 3 S0 2 -D-Leu-Gly-Arg-pNA 2. Anti LJ-ftrαmbin x) thrombin S-2266»' tl-D-Val-Leu-Arg-pNA 3. ant i lasmin plasmin S-2403 ■x' ) <Glu-Phe-Lys-pNA Chromozym- I** 5 Tosyl-Gly-Pi'o-Lys-pNA .. 5. 6. x) Mønufuturer KabiVitrum AB xx) Manufacturer Pentapharm AG, Basel, Sch eiz Abbrevia ions.: 80 C = t-butyloκycarbαnyl CIIA = cyclohexy lalanin CIIG = cyclohexylglycin Tosyl = ρ-toluer.e,.v. sul Fpnyl The invention is generally applicable to determi¬ nation of proteolytically active components according to the basic principles for the substrate de ermina ion 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 essen¬ tially 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 fibrino- lysis except the combination of reagents required for determination of 0C«. -antitrypsin and F VIII. However, it is possible that such conditions possibly will be established also for these combinations after further expe imentation . 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 chromo- genic substrate which can be split by protease in com¬ bination with a) a protease, b) a protease and a reac- tant which can react with the protease under the influ¬ ence, 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 illus- trative examples. The present invention also relates to a process for preparation of the freeze-dried reagent combination which process is defined in claim 5. More specifically, the invention relates to a pro- cess for preparation of the freeze-dried reagent combi¬ nation, which process is characterized in that a solu- UI IS 1JT ~ ~ r i ' E£ tion containing all the reagents required for the de¬ termination 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 result¬ ing solution and in a subsequent f eeze-drying of the solution; and that the solution is freeze-dried in the container in a way knovm per se, the freeze-dried rea¬ gent combination being obtained wherein the reagents are included in a substantially unreacted form and wherein the reagents have substantially original acti¬ vity after reconstitution of the reagent combination, e g n a buffer solution. It is essential according to the invention that all the reagents included in the freeze-dried combina¬ tion are dissolved in the same solution at least m the very freeze—drying process. The process will be especi¬ ally simple to carry out if a stock solution is first prepared from all reagents concerned under conditions controlled according to the invention, said stock solu¬ tion also containing possible additives being thereaf¬ ter 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 d etyl sul oxide. According to a suitable embodiment of the inven¬ tion 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 solu tions of reagents are adjusted, for example by provid¬ ing 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 prepa¬ ration 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 solu- tion. 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 rea¬ gent 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 fur¬ ther 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 incuba- tion and reaction periods are eliminated. Moreover, lower demands are made on the technical skill of the <§} • _ £_ _!} ά J a .___/ _ ■ -w --= ■ >- Λ»—-a 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 pro- cesses 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 inven¬ tion provides the possibility of obtaining a quick re¬ sponse in acute situations as the determination can be carried out locally ("bedside") and as no preparative work such as preparation of reagent solutions, estab¬ lishment of standard curve or running of controls is necessar . Moreover, the use of the product according to the invention often means a considerable saving of resour¬ ces 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 analy- sis,, 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 de¬ pending on which proteolytically active component one wishes to determine. Thus, the preparation of this pro¬ duct must take place under conditions that are con- trolled with respect to which reagents are to be inclu¬ ded 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 proenzy es. 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- -defmed amount of an enzyme inhibited by the formation of l:l-complex with the inhibitor is added to the samp¬ le and that the excess of enzyme is determined by addi- tion 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 extreme¬ ly 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 al¬ most complete activity after reconstitution of the freeze-dried combination, for example by addition of a suitable buffer. In determination of protease inhibitors the pro¬ cess of the invention is based on the fact that such a pH-range has been found within which the enzyme activi¬ ty 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 SUBSTITUTE OK? 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 reeze-drying or are present after freeze-drying in such low amounts that the sample can be prepared before the analysis in currently used buf¬ fers 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 for¬ mic 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-dry- mg and during a following storage of the reagent mix¬ ture and in order to promote a rapid dissolution of the reagent mixture when adding the sample and minimizing reagent adorption at the reagent container additives are suitably used, such as inorganic salts, inactive proeins, e.g. albumin, sugar, e.g. mannitol and/or sur¬ factants such as polyethylene glycol (PEG, Carbowa_*2> 8000 available from Union Carbide, USA) and Tritor® 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 analy¬ sis, 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 impor- S_r*'JH_ SE_S* = tance 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 condi¬ tions, 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 m , which should preferably be 2-8xl0-^ mol/1. Commercially available synthetic substrates are usually utilized, for example those indicated in table I and such as are described in the illustrative examp¬ les. Suitable substrate concentrations are 1-20x10"^ mol/1. The concentration should be so high that there is a linear relationship between the enzyme concentra- tion and the marker concentration. In general a prefer¬ red substrate concentration is 5x10 " "^ mol/1. However, it should be pointed out that an optimal substrate concentration and the affinity properties of the substrate are influenced by additives in the reac- tion 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 anti- thrornbin which can be determined via a reaction with thrombin or factor Xa in the presence of heparin, anti- plasmin, which can be determined via plasmin, and kal- likrem inhibitors, and according to the invention freeze-dried reagent combinations for carrying out these determinations can be prepared. I I . Process for the preparation of a product for determination of cofactors Cofactors comprise many different types of sub¬ stances actuating enzyme reactions. Here a factor is exemplified in the first place which actuates the inhi¬ bition of certain serine proteases, primarily FX a and thrombin, with the inhibitor antithrombin, viz. hepa- rin. The method for determination of heparin and simi¬ lar substances is similar to the inhibitor method de¬ scribed 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 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 coagu¬ lation enzyme and being a cofactor in the activation of the proenzyme plasminogen via the enzyme t-PA (plasmi- nogen activator) which then serves as an activator. This enzyme i.e. the activator, is freeze-dried togeth¬ er with plasminogen, i.e. its substrate, and a chro o- genic substrate suitable for the activation product, viz. the enzyme plasmin. The process for the prepara¬ tion of the reagent combination corresponds to the pro¬ cess for preparation of a reagent combination for the inhibitor determination described above with the diffe¬ rence 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. SUBS _TτI!TTϋ3JlT£ SHEET III. Process for the preparation of a reagent combination for determination of activators The method for de ermination 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 stabi¬ lity and solubility in analogy with what has been de¬ scribed above. Moreover, certain determination methods can require the presence of reaction promoting rea- gents. 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 co - bination for determination of proenzvmes Proenzymes such as Protein C, plasminogen and fac¬ tor X are v/ell 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 embo¬ diment of the invention comprises reeze-drying of a substrate together with an activator that activates the major portion of proenzyme aoccurring 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 sub¬ strate . <__ l__ J_) _d ύ J Δ • * _=, • - .-= According to the invention reeze-drying is car¬ ried out in conventional manner, e.g. at 0,08-0.15 mbar by freezing at a temperature of -45° to -40°C, prefer¬ ably -42°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 12-24°C, preferably 22°C. The invention also relates to use of a freeze- -dried reagent combination for quantitative, semi-quan¬ titative or qualitative determination of coagulation factors and fibrinolysis factors in a sample, especial¬ ly 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 explain¬ ed in greater detail by means of the following examp¬ les, 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 n by the aid of suitable freeze-dried reagent combina¬ tions 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 de¬ scribed. a) Acetate buffer, 1000 ml, is prepared by dilut¬ ing 500 ml of a mixture of 0.2 M acetic acid and 0.2 M sodium acetate in water to 1000 ml by addition of dis¬ tilled water. The desired pH-value of 4.2 is obtained with 368 ml 0.2 M acetic acid and 132 ml 0.2 M sodium acetate. b) in order to obtain an improved stability and increased solubility of the reagent combination albumin and annitol are added to the buffer solution a) in the amounts indicated below. c) Preparation for freeze drying: The substrate S-2732, 650 mg/1 (0.8 mmol/1) and factor Xa, 1000 nkat (1000 nkat/1) are added to the buffer so¬ lution a), to which BSA, 5 g/1 (0.5 %) and mannitol, 10 g/1 (1 %) have been added. d) Preparation of a freeze-dried reagent combina¬ tion intended for determination of antifactor Xa : Each 200 μl 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°C for one hour and dried for 15 h at +22°C and a pressure of 0.08-0.15 mbar . e) Use of cuvettes d) for determination of anti¬ factor Xa : 300 μl of plasma (diluted 1:500) in 0.05 mol/1 Tris, pH 8.4, 1=0.2 containing EDTA (7.5 mmol/1), heparin (3 IU/ml3 and PEG (1 %) are added to the cuvette d) con¬ taining the freeze-dried reagent combination consisting of S-2732 (0.13 mg) FXa (0.2 nkat) and BSA and manni¬ tol . After addition the pH of the test solution is 8.2. The reaction is allowed to take place at room tempera¬ ture (25°C) or at 37°C and is interrupted after 8 min through addition of 300 μl 5 % AcOH. The absorbance of the solution at 405 ran is there- after 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. la have already been prepared at the manufacturer of the reagent combination. This curves show the relationship between dose and response and are the basis of factors ■■ _ r 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. la. The correlation to a generally used and commer¬ cially available "kit" for determination of antithrom¬ bin (CoatesQ-9 Antithrombin, KabiVitru AB, Sweden) is shown in Fig. lb. Exemple 2 In this example a process for the preparation of a reagent combination intended for determination of anti¬ thrombin 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 manni¬ tol is prepared m a cuvette in analogy with example 1. b) Use of the reagent combination -in determination of antithrombin. 300 μl plasma (diluted 1:80) in the same buffer as in- dicated in example le) is added to the cuvette from a), the contents of which being freeeze-dried . The reaction is allowed to take place at 37°C and interrupted after 8 mm through addition of 300 μl' of 5 % acetic acid. Measuring is carried out in analogy with example le), 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 anti- piasmin 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 μl plasma (diluted 1:30) in 0.05 mol/1 Tris, pH = 8.3 comprising 0.15 mol/1 methylamine is added to the cuvette from a), the contents of which being freeze- -dried. The reaction takes place at 37°C and is inter- rupted after 8 min by addition of 300 μl of ' 5 % AcOH. Measurement is carried out in analogy with exemple le) . The standard curve shown in Fig. 3 is obtained. The above examples show reagent combinations in¬ tended 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 combi¬ nation for determina ion of heparin and its use are de- scribed in this example. a) A cuvette compring a reagent combination for determination of heparin and. containing S-2732 (0.2 g) , FXa (0.5 nkat) and BSA and mannitol is prepared in analogy with example 1. b) Use of the reagent combination in determina ion of heparin. 300 μl plasma (diluted 1:15) in 0.05 mol/1 Tris, pH = 8.4, I = 0.2 containing EDTA (7.5 mmol/1) 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 addi¬ tion of 300 μl of 5 % acetic acid. It is measured in analogy with example le) . 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 dis- solved in 7.0 ml 0.03 mol/1 sodium acetat buffer, pH = 4.9 containing 0.01 % Tween*^ 80 and mixed with 2.5 mg __ £_ £ ϊϊm-m 8 human Glu-plasminogen dissolved in 0.8 ml sterile water. 100 mg BSA dissolved in 0.5 ml water and 3.S μg t-PA dissolved in 0,8 ml sodium acetate buffer are ad¬ ded to the resulting solution after which additional buffer is added so that a total volume of 20 ml is achieved. Portions of 200 μl of the final solution are distributed on microcuvettes and freeze-dried according to example id) . b) Use of the reagent combination in determination of fibrin monomer. 300 μl plasma (diluted 1:41) in 0.063 mol/1 Tris, pH = 8.5 containing 0.01 % Tweeri® 80 is added to the cuvette rom a) , the contents of which being freeze- -dried and consisting of S-2390 (0.12 mg) , plasminogen (25 μg) and t-PA (0.036 μg) as well as mannitol, Tween 80 and BSA. The reaction takes place at room tempera¬ ture and is interrupted after 20 min by addition of 300 μl of 20 % acetic acid. It is measured in analogy with example le) . The standard curve shown in Fig. 5 is ob- tained. An embodiment intended for determination of an ac¬ tivator for an enzyme reaction is illustrated in the following example. Example 6 A process for the preparation of a reagent combi¬ nation for determination of t-PA and its use are de¬ scribed 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 con- taming 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 fibrm- (ogen) (3.75 mg) and mannitol (15 mg) . Portions of 200 μl of the final solution are distributed on microcuvet- tes and freeze-dried according to example Id) . > J3§> ihw.-≤δ*1A-4-3 b) Use of the reagent combination in determination of t-PA. 300 μl of pretreated plasma (diluted 1:125) in 0.05 mol/1 Tris, pH = 8.3 containing 0.01 % Tweer® 80 is added to the cuvette from a), the contents of which be¬ ing freeze-dried and consisting of S-2251 (120 μg) , plasminogen (0.25 CU) and CNBr-digested fibrin(ogen) (50 μg) as well as mannitol. The reaction is allowed to take place at 37°C and interrupted after 2 h and 45 min through addition of 300 μl 20 % AcOH. It is measured in analogy with example le) and the standard curve shown in Fig. 6 is obtained. Embodiments intended for determination of proen- zymes are illustrated in the following example. Example 7 A process for the preparation of a reagent combi¬ nation 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 CaCl2 (0.1 mol/1) is added to the resulting solution so that a total volume of 20 ml is obtained. Portions of 200 μl of the final solution are distributed on microcuvettes and freeze-dried according to example Id) . b) Use of the reagent combination in determination of factor X. 600 μl of plasma (diluted 1:60) in 0.05 mol/1 Tris, pH = 7.8 containing Polybrene© ( 20 ιng/1, Aldrich, USA) are added to the cuvette from a), the contents of which be¬ ing freeze-dried and consisting of S-2337 (0.24 mg) and RVV (9 μg) as well as mannitol, CaC1.2 and NaCl. The re- action takes place at 37°C and is interrupted after 3 mm. through addition of 200 μl of 20 % AcOH. It is measured in analogy with example le) and the standard curve shown in Fig. 7 is obtained. Examp1e 8 In this example a process for the preparation of a reagent combination for determination of plasminogen and its u ' se are described. a) S-2251 (30 mg) is dissolved in 16 ml of distil¬ led water and mixed with 4 ml of a solution containing streptokinase (80 000 IU) and 12 mg of albumin (20 %) . Portions of 200 μl of the resulting final solution are distributed on microcuvettes and freeze—dried according to example Id) . b) Use of the reagent combination in determination of plasminogen. 300 μl of plasma (diluted 1:20) in 0.05 mol/1 Tris, pH - 7.4 and I = 0.05 are added to the cuvette from a), the contents of which being freeze-dried and -consisting of S-2251 (0.3 mg) and Strepokmase (800 IU) as well as albumin. The reaction is allowed to take place at 37°C and is interrupted after 3 min by addition of 300 μl 5 % AcOH. It is measured in analogy with example le) and the standard curve shown in Fig. 8 is obtained. Example 9 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^ C-solution (10 U/ml) , Pentapharm, Switzerland) is added. Portions of 200 μl of the resulting solution are distributed on microcuvettes and freeze-dried according to example Id) . b ) Use of the reagent combination in determination of protein C. 300 μl plasma (diluted 1:11) m 0.025 mol/1 Tris, pH = 8.4 containing 0.1 % PEG are added to the cuvette from SUBSTITUTE SHEET a), the contents of which being freeze-dried and con¬ sisting of S-2366 (0.12 mg) and Prota<£Bt (0.045 U) . The reaction is allowed to take place at 37°C and is inter¬ rupted after 7 min through addition of 300 μl 20 % AcOH. It is measured in analogy with example le) 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 T Trriiss tris(hydroxymethyl )-a inomethane EDTA ethylenediamine tetraacetic acid PEG polyethylene glycol AcOH acetic acid Tweeri®30 polyoxyethylene sorbitan onooleate (Atlas Chemical Industries, USA) Glu glutamic acid t-PA plasminogen activator S-2251 H-D-Val-Leu-Lys-pNA* 2HC1 S-2337 Nα-benzoy l - I l e-Glu ( ^pipe idide ) -Gly-Arg- -pNA - HC l S-2366 <Glu-Pro-Arg-pNA-HCl S-2390 H-D-Val-Phe-Lys-pNA- 2HC1 S2732 Nα-succinoy1- I le-Glu( -Gly-Arg- pNA-HCl - -ppNNAA p-nitroanilide A 405 absorbance at 405 n 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 < Hep ri ) IU unit related to international standard (Streptokinase) CU (plas- casein unit rninogen) U 1 U = the amount of ProtaoS activating (Prota S* 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 A405 for samp¬ les 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 corre- sponding illustrative example.

Claims: Claims 1. Freeze-dried reagent combination enlosed a container and intended for determination of a component active in proteolysis directly or indirectly by cleav- ing of a substrate included in the reagent combination and capable of being cleaved by a protease to produce a detectable response, c h a r a c t e r i z e d in that the reagent combination comprises in a substanti¬ ally unreacted form all reagents required for the de- termination and optionally one or more additives known per se; and is prepared by freeze-drying in a way known per se a solution containing all the constituents in¬ cluded in the freeze-dried reagent combination in a substantially unreacted form, said freeze-dried combi- nation having substantially its original activity at reconstitution as in a buffer solution. ' 2. Reagent .combination as claimed in claim 1, c h a r a c t e r i z e d in that an amount of the reagent combination intended for one single determina- tion is enclosed in a container having an undivided in¬ terior volume, which container preferably can be used directly in a measuring apparatus intended for the re¬ sponse detection. 3. Reagent combination as claimed in claim 1 or 2 intended for determination of a component active in proteolysis and consisting of a) an inhibitor, b) a cofactor, c) an activator or d) a proenzyme, c h a ¬ a c t e r i z e d in that in addition to the sub¬ strate, capable of being cleaved by proteases and pre- ferably being a chro ogenic substrate, and possible ad¬ ditives it contains a) a protease, b) a protease and a reactant capable of reacting with the protease under the influence of the cofactor to be determined, c) a proenzyme or an enzyme compound capable of being acti- vated or d) an activator for the proenzyme. ^Uϋ I j _ . i ____ • •*_ * ■a ' --_-._*. I 4. Reagent combination as claimed in claim 3, c h a r a c t e r i z e d in that it is intended for determination of 1) antifactor X a , 2) heparin, 3) fib¬ rin monomer or 4) protein C and comprises 1) factor X a , Nα-succinoyl-Ile-Glu(^-piperidide) -Gly-Arg-pNA-sub- strate; bovine serum albumin and mannitol; 2) factor X a , HCC-succinoyl-Ile-Glu(.ϊ'-ρiperidide)-Gly-Arg-plA-sub- strate, bovine serum albumin and mannitol, 3) plasmino¬ gen, H-D-Val-Phe-Lys-pNA-substrate, t-PA, mannitol, surfactant and bovine serum albumin or 4) protein C-ac- tivator and <Glu-Pro-Arg-pNA-substrate. 5. Process for the preparation of a freeze-dried reagent combination according to claim 1 and intended for determination of components active in proteolysis directly or indirectly through cleaving of a substrate included m the reagent combination and capable of be¬ ing cleaved by a protease to produce a detectable re¬ sponse, c h a r a c t e r i z e d m that a solution containing all the reagents required for the determina- tion and optionally one or more additives known per se is produced in a container in a solvent such as water under conditions controlled in such a way that the rea¬ gents remain substantially nonreactive in the resulting solution and in a following freeze-drying of the solu- tion; and that the solution is freeze-dried in the con¬ tainer in a way known per se, the freeze-dried reagent combination being obtained wherein the reagents are in¬ cluded m a substantially unreacted form and wherein the reagents have substantially original activity after reconstitut on of the reagent combination such as in a buffer solution. 6. Process as claimed in claim 5, c h a r a c ¬ t e r i z e d in that the container contains such a quantity of solution in the freeze drying as is suit- able for one single determination, said container hav¬ ing an undivided interior volume. SUBSTITUTE SKEE" 7. Process as claimed in claim 5 or 6, c h ¬ r a c t e r i z e d in that the conditions are con¬ trolled, preferably by means of a buffer, to a pH-value of 3-8.5 when a reagent combination intended for deter- mination of cofactors or activators is prepared, and a pH-value of 3-5 when a reagent combination intended for determination of inhibitors or proenzymes is prepared. 8. Process as claimed in claim 5, 6 or 7, c h a ¬ r a c t e r i z e d in that the freeze-drying is car¬ ried out at 0.08-0.15 mbar through freezing at a tempe¬ rature of -45° to -40°C, preferably -42°C for a time of 1-5 h, preferably i h, and a subsequent drying for a time of 12-20 h, preferably 15 h, at a temperature of 12-24°C, preferably 22°C. 9. Process as claimed in any of the preceding claims, c h a r a c t e r i z e d in that the solu¬ tion contains additives, such as inactive protein, for example albumin; sugar, for example mannitol; and/or surfactant, for example polyethylene glycol . 10. Process as claimed in any of the preceding claims, c h a r a c t e r i z e d in that a reagent combination is prepared intended for determination of a component active in proteolysis and consisting of a) an inhibitor, b) a cofactor, c) an activator or d) a pro- enzyme and that the solution of the reagent combina¬ tion, which is freeze-dried, contains in addition to the substrate capable of being cleaved by protease, and possible additives a) a protease, b ) a protease and a reactant which can react with the protease under the influence of the cofactor to b determined, c) a pro¬ enzyme or an enzyme compound capable of being activated or d) an activator for the proenzyme. 11. Use of a freeze-dried reagent combination as claimed in claim 1 for quantitative, semi-quantitative or qualitative determination of coagulation factors and fibrinolysis factors in a sample, especially a biologi- cal one, such as whole blood, blood plasma, blood se¬ rum, cerebrospinal liquid, lung liquid or urine in a one-step process through addition of the sample to the freeze-dried reagent combination enclosed in a contain¬ er, p referably a cuvette, and reading of the resulting response in a way known per se. f S "^T

Inventors: AURELL LEIF, FRIBERGER PETTER, AURELL, LEIF, FRIBERGER, PETTER
Applicants: KABIVITRUM AB
Application year: 1988
Main classification: C12Q1
IPC codes: C12Q1, G01N33