MXPA96004002A - Inhibition of activity of proteasa de lisados decelula de la humana compl - Google Patents

Abstract

The present invention relates to a method for determining the presence of the Mx protein contained within cellular material contained in a sample, said sample comprising, which also contains protease, to cleave the cellular material, contained within said sample, by means of the use of a non-ionic detergent, to release said Mx protein; b. inactivate the protease, contained within said, shown by: 1. add one or more denaturing agents, selected from the group consisting of sodium dodecyl ste, urea and guanidine hydrochloride, to said sample containing the released intracellular protein and the protease to form a solution, and 2. heat said solution at a temperature of from about 50 ° C to about 60 ° C, and for a period of time from about 60 seconds to about 30 minutes, to inactivate said protease, c. reacting said solution with monoclonal antibody clones 1302.5.32 and 1302.34.16.2.44, and d. detecting the binding of said monoclonal antibody clones to said Mx protein, where the binding indicates the presence of Mx protein in said sample

Description

INHIBITION OF PROTEASE ACTIVITY OF COMPLETE HUMAN BLOOD CELL LYSTEES Field of the invention The present invention relates generally to the inhibition of protease activity in biological fluids and, more specifically to a method of inhibiting the proteolytic degradation of the Mx protein in used from whole human blood cells, and employ a protein test, Mx as the method that is indicative of the efficacy of interferon therapy. Background of the Invention Proteolytic degradation is a natural occurrence procedure in all biological domains. Proteolytic degradation also complicates scientific research if one wishes to examine the non-degraded level of proteins. The determination of intracellular protein or membrane proteins is particularly complicated by proteolytic degradation, since the cell lysate process also releases proteases. There is a variety of protease inhibitors for the inhibition of proteolytic degradation, and such conventional inhibitors are known to those of medium skill in the art. However, when the effectiveness of known protease inhibitors is not sufficient to stop the proteolytic degradation of a protein of interest, or the addition of those inhibitors only accelerated proteolytic degradation, an alternative way to stop this problem would have to be found. An example of the type of research that is complicated by proteolytic degradation is the precise determination of the protein in the biological fluid. Such a protein determination may also be useful to determine the clinical relevance of the protein pathway determination of the protein specifically induced for the species of interest. For example, it is important to evaluate the clinical efficacy of interferon therapy, which is expensive and of increasing popular interest in the treatment of conditions such as hemangiomas in infants, genetically predisposed multiple sclerosis, autoimmune diseases, certain types of cancer, and AIDS. The interferon circulation level test is technically difficult. However, by testing an intracellular protein called Mx protein specifically induced by interferon, the efficacy of interferon therapy can be determined. In a document entitled "A Whole Blood Immunoassay for the Interferon-Inducible Human Mx Protein" by Towbin et al. , "Journal of Interferon Research, 12, 67 (1992)", the authors describe a test procedure for the Mx protein in Used whole blood cell using an enzyme immunoassay. Although advances have been made in the investigation of interferon in general and the investigation of the Mx protein in particular, the objective remains to determine the uncommitted level of the Mx protein to minimize the proteolytic degradation of the Mx protein in the evaluation of the new application of interferon therapy. Accordingly, it is an object of the present invention to provide a method of inhibiting the proteolytic degradation of an intracellular protein, ie, Mx protein in used cells. It is still another object of the invention to provide an artificial matrix solution wherein an intracellular protein can be held stable against proteolytic degradation at a temperature at or below 4 ° C for at least three weeks. Brief Description of the Invention The foregoing and other objects and advantages of the invention are achieved by providing a method of inhibiting the proteolytic degradation of a thermally stable intracellular protein in cell-used. The method involves the formation of a solution containing one or more denaturing agents and unknown proteases that degrade the intracellular protein and heating the solution at a temperature and for a period sufficient to denature the protease. The solution can be defined as whole blood cells Used with detergent. The solution can be defined in part by using Blood Cells that simulate the synthetic matrix as well. When the solution contains the intracellular protein as used in the whole blood, the heating stage is carried out under conditions that do not destroy it. When the solution is free of intracellular protein, such as in Cells used to simulate the synthetic matrix, more severe conditions may be applied until all protease activity is destroyed. The present invention also provides an indirect method of determining interferon in the blood of the patient. The method involves heating a sample in the presence of denaturing agents, in order to denature 1 or more unknown proteases from Used of cells that degrade the intracellular protein of interest, for example, the Mx protein. The heating is applied at a level and for a period sufficient to denature the proteases, although not to denature the intracellular protein. The intracellular protein is determined later. The determination can be made by a test, in a way to detect the presence of the protein induced by the interferon, thus indirectly determining the biological effectiveness of the interferon therapy. Such a test may involve the steps of providing a binding partner to capture the intracellular protein by contacting the solid phase with the solution and coupling a second binding partner of the intracellular protein to the intracellular protein. The second binding partner carries a chemiluminium label, which can be detected by means of a luminometer. The coupling stages can be combined in any order. The present invention also provides an artificial matrix that is made to be free of protease. The intracellular Mx protein remains stable in this artificial protein solution at a temperature of 4 ° C for at least three weeks. In accordance with one aspect of the invention, the solution includes used whole blood cell. In accordance with another aspect, the solution includes a synthetic matrix that resembles the Used whole blood cell. Other advantages, novel features and objects of the invention will become apparent from the following detailed description of the invention, in conjunction with the accompanying drawings. Brief Description of the Drawings In the drawings: Fig. 1 is a graphic illustration of the comparative degradation of Mx protein in normal human plasma, the Used whole blood, the used of packaged blood, and free bovine serum albumin. protease, in units of OD at 405 nm against the incubation length at 37 ° C in minutes; Fig. 2 is a standard curve for Mx antigen in a chemiluminium immunoassay according to the present invention, derived from solutions in which the proteolytic degradation of Mx has been inhibited according to a method of the present invention, in solutions in where the hematocrit is 15, 30, 45 70% in units of thousands of units of relative light (RLU) against the concentration Mx in units of ng / ml; Fig. 3 is a graphic illustration of the removal of proteolytic degradation of Mx protein in synthetic matrices according to the present invention, by means of heat inactivation after heating in 2M urea and 0.1% SDS, in synthetic matrices having hematocrits of 15, 30, 45 and 70%, in units of thousands of units of relative light (RLU) against time (in minutes) at 37 ° C; Fig. 4 is a graphic illustration of the removal of proteolytic degradation of Mx protein in Used whole blood according to the present invention, by heating in urea at 56 ° C for 30 minutes, in units of thousands of units of relative light (RLU) against time (in minutes) at 37 ° C; and Fig. 5 is a graphic illustration of a typical Mx protein sensitive-dose induction after administration of interferon to three human patients, determined in accordance with a test of the present invention, using 8 X 106 units / day of IFN (B / D), which shows the response in several days in units of concentration Mx (in ng / ml) in whole blood. Detailed Description of the Invention The present invention provides a method of inhibiting the proteolytic activity of a thermally stable mtcellular protein by treating cells with heat and one or more denaturing agents at a temperature and for a period sufficient to denature the protease. . Since protease activity is inhibited according to the method of the invention, a test can be reliably performed to determine the presence and / or concentration of the intracellular protein. The provision of a test sample that is stable against proteolytic degradation is advantageous when the sample must be stored for any period prior to the execution of the test. Additionally, an artificial mixture of the stable protein solution against proteolytic degradation of the protein of interest can be provided and used as a diluent for an antigen standard, controls, calibrators or the like. Inhibition of proteolytic degradation of any of a variety of thermally stable proteins is provided according to the invention. As used herein, the term "thermally stable" is intended to define the stability of the protein of interest at a temperature and for a period necessary to degrade the protease responsible for the degradation of the protein. Thermally stable proteins include, but are not limited to, some membrane proteins (e.g., carcinoembryonic antigen) and intracellular proteins, which include nuclear proteins (e.g., murine Mx protein) and cytoplasmic proteins (e.g., human Mx protein). , thermal impact proteins and cytoskeletal proteins).

In accordance with one aspect of the invention, a method of inhibiting proteolytic degradation of an intracellular protein (e.g., Mx protein) induced by interferon is provided. Such a method makes feasible the reproducible, reliable determination of the Mx protein, thus determining the clinical efficacy of interferon therapy. Approximately 30 different proteins are known to be induced by interferon. However, only 2,5-oygo- (A ') synthetase, p68 kinase and the Mx protein are known to mediate the antiviral actions of interferon, and the determination of one or more of these proteins according to the invention is by therefore highly relevant for the evaluation of interferon therapy. The determination of the Mx protein is particularly preferred for the following reasons: Mx is induced rapidly (2 hrs.) After treatment with interferon and, reaches the maximum levels in a relatively short period (approximately 36 hrs). The cellular induction of the Mx protein is not subject to feedback inhibition even at high doses of interferon therapy. Additionally, the biological half-life of the Mx protein is relatively long (T1 is 3.5-5 days). Therefore, 20% -30% of the initial Mx protein level remains even after two weeks after the end of interferon therapy. Therefore, due to its long half-life, the Mx protein is a good indicator of the effectiveness of interferon. In addition, the fact that it is easily detectable makes it an indicator of rapid, sensitive and reliable induction of interferon action on a wide dose scale of interferon. In accordance with the method of the invention, a solution containing a denaturing salt, detergents and proteases that degrade the intracellular protein was heated at a temperature and for a period sufficient to denature the protease. The solution can be formed by using the cells, for example whole human blood cells or cultured cells. The solution can also be formed by creating an artificial matrix that resembles blood. Many artificial matrices that resemble blood are suitable for use in accordance with the present invention. Preferably, an artificial matrix formulated in accordance with the present invention and composed of protease-free bovine serum albumin and crystalline bovine hemoglobin is employed. Those of ordinary skill in the art are aware of a wide variety of denaturing agents and can be used in accordance with the method of the invention, including, but not limited to, urea and guanidine hydrochloride, which are the preferred denaturing agents. The proteases to be inhibited in accordance with the present invention include virtually all those known to exist in white blood cells, including Cathepsin G, elastase, metalloproteases, etc. When the method of the invention has been carried out, that is, when a solution has been treated to denature the protease that degrades an intracellular protein, that solution can be overridden with the intracellular protein without risk of proteolytic degradation. Such a solution may serve as a diluent for an antigen standard in a test, and may contain used whole blood cells, or a synthetic matrix that resembles the Used of whole blood cell. In accordance with a preferred embodiment of the present invention, such a solution remains stable at a temperature of 4 ° C for at least 3 weeks. According to another embodiment, the solution can serve as a standard, or it can comprise a sample in a test, for example a sample of whole human blood. When the solution contains whole blood cells or cultured cells, a Used agent is advantageously included in the solution before heating in the presence of the denaturing agents. Thus, the cells can be used and the protease denatured in a single step. A variety of Used agents are suitable for use in accordance with the present invention, including, but not limited to non-ionic detergents such as monodisperse and polydisperse polyoxyethylenes, homogeneous and heterogeneous. Preferred used agents include "Tergitol N P-40" (available from Union Carbide) or Triton X-100 (available from Rohm and Haas), which must be added in an amount such that its concentration, when the sample is heated in the presence of other denaturing agents (such as urea and hydrochloride and guanidine) should be sufficient to denature the protease. Although it is not important if the cells are used before or at the same time as the protein is solubilized, it is important that the non-ionic detergent used to remove the cells be included in the denaturing medium, since the non-ionic detergent assists the process of denaturation. In the case when the Used is not required (for example, when a synthetic matrix is used), the non-ionic detergent must still be added together with 1 or more other denaturing agents (eg, urea or guanidine hydrochloride) and the detergent anionic (for example SDS) to ensure that denaturation occurs. Depending on the cell involved, other agents of Use may be appropriate. For example, in the case of red blood cells, water is sufficient to remove the cell. However, when the cell is used using an agent in addition to a non-ionic detergent, the non-ionic detergent must still be used to denature the protease. Although SDS combined with heat has been used in the past to mask the charge of the native configuration of the proteins, it therefore frequently results in its denaturation (see Laemmli, Nature 227: 680! 970)), the use of the SDS, the denaturant (for example urea) and heating in a controlled manner in the present invention results only in the destruction of the proteases, without denaturing the protein of interest. The solution is heated in the presence of a denaturing agent at a temperature and for a period sufficient to denature the protease. The temperature and time of heating should be selected to sufficiently denature the protease, and when the solution contains the intracellular protein, the temperature and time should be selected so as not to denature the intracellular protein. A temperature of not less than 50 ° C must be selected and the solution must be heated for at least 60 seconds. If the intracellular protein is present in the solution, the solution should be heated to a temperature of about 50 ° C to about 60 ° C for a period of 15-30 minutes. If the solution contains only the artificial matrix (that is, it contains protease contamination although it does not yet contain the protein of interest), more severe conditions may be employed before the protein of interest is added to the matrix. For example, such a solution can be heated to a temperature from about 50 ° C to about 100 ° C for a period of about 1 minute to 1 hour or more, preferably at a temperature of about 56 ° C for about 1 hour (see Manwaring , WH (1906) on the destruction of complement by heat, TR Chicago Path Soc. 6: 425). When a solution contains the intracellular protein, the solution conditions must be maintained within a scale compatible with the survival of that protein. Specifically, the pH of the solution should be maintained within a range of 7.0-8.0, and the ionic strength of the solution should be maintained at a level of no more than about 4M. As noted above, a solution according to the present invention that contains a thermally stable intracellular protein (e.g., protein Mx) and that is free of a protease that degrades the Mx protein facilitates a reliable and reproducible test to determine the intracellular Mx protein. As used herein, the term "determines" is intended to define the detection of the intracellular protein at the limit of the test, or the determination of the concentration in the solution of the intracellular protein. Many types of tests are known in the art that can be modified for use in a determination in accordance with the present invention. General test types include, for example, direct, indirect, competitive and heterogeneous or homogeneous intercalation type tests such as those described in U.S. Patent No. 5,252,459, issued October 12, 1993 to Tarcha et al. And incorporated herein by reference. When the whole human blood is to be tested for the Mx protein according to a particularly preferred embodiment of the present invention, an assay method can be carried out as follows: A solution containing the proteins derived from the cells is formed of whole blood by, as described above, the combination of the Used agent, 1 or more denaturing agents (preferably urea) plus sodium dodecylsulfate (SDS), the detergent selected to denature the protease and solubilize the Mx protein. In addition, it is important that the protease be sufficiently diluted so that the denaturing agents are effective. The solution is then heated to a temperature from about 50 ° C to about 60 ° C for a period of about 15-30 minutes, and the Mx protein is then determined. Variations of the present invention are possible for a range of proteins insofar as the temperature at which proteases are destroyed is determined and found to be lower than that temperature at which the analyte is denatured. In addition, the appropriate used agent, generally a non-ionic detergent, and a solubilizing / denaturing agent, generally an ionic surfactant, such as anionic surfactant sodium dodecyl sulfate, and denaturation salts (eg, urea or guanidine). If the sample obtained is already used, there is no need to include the non-ionic (used) agent in the system. Further variations of the present invention are possible. For example, the sample suspected of containing interferon may be a sample of whole blood, packed blood cells, cultured tissue cells, a solution containing whole blood cells, synthetic matrices to which the Mx protein is added. to simulate the Used of whole blood or similar. Other variations will become apparent to those with regular experience in the art. The following examples are intended to illustrate the benefits of the present invention, although they do not exemplify the full scope of the invention. For example, although a specific denaturing agent, solubilization detergent and Used agent are exemplified, a variety of such agents may be employed. While the determination of the Mx protein and the corresponding preparation of control and standard solutions containing the Mx protein are exemplified, it is understood that a variety of thermally stable proteins are included, including, but not limited to, the scope of the present invention. a, those induced by interferon or other cytokines or other biological response modifiers. It is understood that these and other modifications and their equivalents are within the scope of the present invention. Examples Materials and Methods Protease inhibitors phenylmethylsulfonylfluuride (PMSF), aprotonin, antipain, chymostatin, leupeptin, pepstatin A, tosyl-lysine chloromethyl ketone (TLCK), tosyl-phenylalanine chloromethyl ketone (TPCK), epsilon-amino-caproic acid (EACA), elastin, and E-64 were purchased from "Sigma Chemical Co." (St. Louis, MO). A non-ionic detergent, N P-40 (used to solubilize leukocytes), 2-mercaptoethanol (2-ME), protease-free bovine serum albumin (BSA-PF), BSA (BSA-RIA) grade radioimmunoassay (RIA) ) and crystalline bovine hemoglobin (bHB) were also purchased from "Sigma Chemical Co". Sodium dodecylsulfate (SDS) was purchased from "BioRad Laboratories" (Hercules, CA). Some proteases, that is, elastases (porcine pancreas and human leukocytes), Cathepsin G (human leukocytes) were also "Sigma" chemicals. Crystalline trypsin was obtained from "Worthington Biochemicals" (Freehold, NJ) and, PEFAbloc ™, an analogue of phenylsulfonyl fluoride, was obtained from "Pentapharm AG" (Basel, Switzerland). All other chemicals were reactive grade chemicals from "Mallinkrodt" (Paris, KY). "DEAE Sephadex a25" and 12.5% "Phast gel" were obtained from "Pharmacia Biotech Inc." (Piscataway, NJ). The goat serum from "Ventrex Laboratory" (Portland, ME) was heat inactivated and filtered through a "Millipore" filter of 0.2 um before use. The "Immobilized Protein -A Affinity Pak ™" was purchased from "Pierce Chemicals Co." (Rockford, I L) and was used as described by the manufacturer. Example 1 Investigation of Proteolytic Degradation of Protein Mx An investigation of the degradation of the Mx protein in normal human plasma, Used of whole blood, Used of packed blood cell (in the absence of plasma,; BCL), in synthetic matrices that resemble the ones used in whole blood, and in protease-free controls. The protein in cultured cell lines (ie, WISH, CHO, 3T3) were induced with interferon (B / D) (Ciba-Geigy, Basel, Switzerland), and the cells were used and stored frozen at -80 ° C until that were used. An ELISA test showed that the endogenous Mx protein present in the frozen cell samples showed a much lower amount of immunoreactive Mx protein after freeze-thaw than an original fresh sample. In contrast, the recombinant Mx protein produced in E. coli, purified to homogeneity and stored at -80 ° C until used, retained 100% of its initial immunoreactivity under repeated freezing and thawing. A known amount of purified rMx protein was fixed within two different BSA preparations, ie, BSA-PF (protease-free) and BSA-RIA, as well as within fresh whole blood drawn from a normal healthy volunteer. Both the Whole Blood Purpose and the two different BSA preparations contained a Used agent, specifically 2% (v / v) detergent NP-40 as described by Towbin, et al. , previously referred.

Those preparations were further diluted with a medium containing a denaturing agent, specifically 2M urea, and a solubilization detergent, specifically 0.1% SDS and a pH regulating salt, ie, 50mM Tris-HCl (pH 8.0). A final protein concentration was adjusted to 1%. The Mx protein was fixed within a synthetic matrix composed of BSA-PF and crystalline bHB. These samples were incubated at 37 ° C for up to 120 minutes. The aliquots of the samples were periodically removed and the amount of Mx protein remaining in the solution was correlated with a signal drop (RLU's) during the time as determined in a chemiluminium immunoassay. The Mx protein bound within BSA-PF experienced minimal degradation during this incubation period. In contrast, the BSA-RIA rapidly degraded the Mx protein within the first 30 minutes and the Mx protein in the Whole-blood whole was degraded continuously through an incubation period of 2 hours. The Mx protein fixed within a synthetic matrix composed of BSA-PF and crystalline bHB was also degraded.

Since the Mx protein was minimally degraded in BSA-PF, it was assumed that the crystalline bH B must have been the source of this protease activity. In fact, synthetic matrices with increasing concentrations of hemoglobin exhibited a higher degree of proteolytic degradation of Mx. The Mx protein was degraded more rapidly in Used blood cell packed (in the absence of plasma), than in the Used whole blood. Fig.1 graphically illustrates the results of this test, showing the significant decrease of the Mx protein in normal human plasma (NHP), the used ones of whole blood (WBL) and in the used ones of packed blood cell (BCL), compared to a control (1% BSA). It was obvious from this investigation that the Mx protein is subjected to proteolytic degradation in a variety of biological fluids, mainly the ones used in whole blood. These results indicated a definite need to eliminate protease activity in the synthetic as well as the used matrices of whole blood before the amount of Mx protein can be reliably and reproducibly determined in clinical samples. Example 2 Preparation of Whole Blood Lysates Whole blood lysates were prepared from normal healthy volunteers by adding 2% (v / v, final concentration) of NP-40 detergent to freshly extracted blood, collected in tubes containing EDATA or heparin and that served as untreated controls. Clinical samples from Interferon (B / D) clinical trials were also prepared in the same manner as the normal whole blood samples and were kept frozen at -80 ° C until used. Example 3: Formulation of synthetic matrices for Mx protein Series of synthetic matrices that simulate whole blood lysates of individuals with multiple hematocrits were formed.

These synthetic matrices were composed of BSA-PF and bHB in PBS as follows: Hematocrit 1 5% 30 0% 45.0% 70.0% bH B 5g% 1 0.0 g% 1 5.0g% 23.0g% BSA 7g% 5.5g% 4.5g % 2.5g% The purpose of these synthetic matrices was to investigate the potential effects of the variable hemoglobin content on the signal reading of an Mx test and to define the most suitable hemoglobin content to formulate a synthetic matrix. Example 4 Development of the test for Mx 1 protein determination. Monoclonal antibodies to Mx protein Two separate monoclonal antibodies, one directed to the C-terminal (clone 1302.5.32) and the other to the N-terminal portion (clone 1302.34.16.2.44) of the Mx protein were used as capture and detector antibodies in an intercalated type immunoassay. The cell lines that produced those monoclonal antibodies were identified as Hybridoma Mx 1302.5.32 and Hybridoma Mx 1302.34.16.2.44. Those cell lines were deposited at the American Type Culture Collection (ATCC) Patent Depository (12301 Parkiawn Dr., Rockville, Maryland 20852, E. U.) and the * ATCC numbers given were ATCC HB-1 1836 (for the Mx hybridoma) 1302.5.32) and ATCC HB-1 1837 (for Hybridoma Mx 1302.34.16.2.44). The deposit was made according to the Budapest Treaty. Those antibodies were purified from fluids from mouse acites using medium of protein-A Sepharose and tested to be > 95% pure by densitometric screening of the SDS_PAGE gel stained with Coomassie Blue (20). The monoclonal antibody of clone 1302.5.32, directed to the C-terminus of the Mx protein, was conjugated to paramagnetic particles (PMP) using the glutaldheido activation method of Whitehead et al. as described in U.S. Patent 4,554,088. The PMP-conjugated antibody was suspended at 10 mg / ml in PMP wash buffer, which contained 0.25% BSA (protease-free), 0.7% bovine gamma globulin (BGG, Pentex, Miles Scientific, Naperville, IL), and 0.1% sodium azide in saline regulated at its pH with phosphate (PBS) and used as a solid phase capture antibody. The monoclonal antibody of clone 1302.34.16.2.44, directed to the N-terminus of the Mx protein, was labeled with acridinium ester using the dimethylacridinium ester activated with N-hydroxysuccinimide (DMAE- N HS, Ciba-Corning Diagnostics Corp., Walpole , MA) at a molar ratio of DMAE: antibody = 20: 1 at room temperature for 30 minutes with constant agitation. The free DMAE and the DMAE-labeled antibody were separated by chromatography on a DEAE_Sephadex A25 column in PBS. Fractions of 1 ml were collected and the labeled antibody fractions were monitored using an MLA-I or I I luminometer (Ciba-Corning Diagnostics Corp. Oberlin, OH). The fractions containing the DMAE-labeled antibody were combined, diluted to a final concentration of 1012 units of relative luminicence (RLU / ml in PBS containing 1% BSA-PF, 2% NP-40 and 0.1% azide). Sodium Both antibody preparations were stored at 4 ° C until they were used 2. Development of a chemiluminium test Purified recombinant Mx protein (rMx) derived from the inclusion bodies of E. coli (see Horisberger, et al. "CDNA Cloning and Assignment to Chromosome 21 from IFI-78-k Gene, The Human Equivalent of Murine Mx Gene", "Somatic Cell &Molecular Genetics, 14, 123 (1988)) was used as the antigen standard. Protein was determined by both a BioRad test using a BSA standard and quantitative Western blotting of a 2-D gel as described by Towbin et al., referred to above .. The amount of Mx protein was confirmed in a modified version of an EL test. ISA originally published by Towbin et al. for whole blood lysates. The modified version used a larger sample volume (sample of 50ul against 20ul) and larger amounts of primary antibody (50ul against 40ul) and secondary 100ul against 50ul). The purified rMx protein served as the antigen standard in the modified ELISA test. For the chemiluminium immunoassay of the Mx protein, all samples or synthetic matrices containing a known amount of Mx protein were incubated in 12 x 75 mm plastic tubes simultaneously with DMAE-labeled detector antibody and PMP-conjugated capture antibody. The incubation period varied from 30 minutes to 120 minutes at 37 ° C in a water bath. At the end of each incubation period, the bound-solid phase immune complex was separated with a magnetic separating grid, "Magic Rack ™ (Ciba-Corning Diagnostics Corp., E. Walpole, MA) for three minutes at room temperature. unbound or antibody was discarded by decanting The separated pellets were resuspended in 1 ml of deionized water using a "Multi-Tube Vortexer" (Model 4010, Corning, NY) and the PMP pellets were separated, and the material was removed These pellets were washed once more with 1 ml of deionized water and finally suspended again in 0.1 ml of deionized water before counting in an MLA-I or II luminometer For automated tests, the ACS was used : 180 ™ (Ciba-Corning Diagnostics Corp. Oberlin, OH) and the data were analyzed using the mathematical algorithms generated by a statistical program 3. Preparation of the detector antibody with DMAE label. Following the DMAE labeling procedure described in the Materials and Methods section, the DMAE-labeled antibody was obtained with a specific activity of 7 x 101 1 units of relative luminicence (RLUs) per mg of detector antibody (clone 1302.34.16.2.44 ), with the luminicepcia determined by means of a luminometer and the concentration of protein determined by a BioRad protein test. The detector antibody was purified with protein-A-Sepharose medium as suggested by the manufacturer. The DMAE-labeled antibody reacted with the solid phase-bound Mx protein in a dose-dependent manner, similar to that of the biotinylated antibody used in the ELISA test. This result indicates that the DMAE labeling did not destroy the immunoreactivity of the detector antibody for Mx antigen. 4. Preparation of PMP-bound antibody The conjugate capture antibody (clone 1302.5.32) was conjugated to PMP at a coupling efficiency of 74%, which results in 150 mg of bound antibody per gm of PMP. The PMP-conjugated antibody reacted in a separate dose form with a separate epitope of rMx, which is not occupied by the DMAE-labeled antibody. 5. Development of the chemiluminium test for the MxA protein. Effect of the biochemical nature and total protein concentration of the matrices on the exit of the chemiluminium signal Since the whole blood lysates consist mainly of hemoglobin and serum albumin, it was examined the effect of these two proteins on the chemiluminium signal output. The effect of the total protein concentration of the synthetic matrix on the exit of chemiluminium signal was also examined. Hemoglobin at the same concentration of 1 g% (w / v) exhibited only 1/10 of the chemiluminium signal output compared to that of 1% BSA. On the other hand, 2 g% of the total protein (dilution of 1: 10 of goat serum + 15 g% of human hemoglobin) generated only% of the signal output generated with 1% of total protein. Therefore, the biochemical nature of the protein, (ie the hemoglobin against BSA) as well as the total protein concentration both influence the signal output of the chemiluminium test. Fig. 2 graphically illustrates that, on the dilution of matrices up to 20 times with a pH regulator (eg, 50mM Tris-HCl (pH 8.0)) containing 2M urea + 0.1% SDS, the four matrices representing the hematocrit among 15 % and 70% generated similar signal output. The protease inactivation procedure works best at this dilution. Therefore this dilution procedure was incorporated as part of the standard sample preparation. B. Effect of detergent concentration on non-specific binding level and signal output Despite 2% (v / v) of detergent NP-40 present in whole blood lysates or synthetic matrices, the final detergent concentration in the test mixture was low, as the solutions were diluted 20 times with a medium containing 2M urea (denaturing agent and 0.1% SDS (solubilizing detergent) .Therefore, several samples were tested. NP-40 concentrations (ie 0.5, 1 and 2%) in the medium containing the detector antibody to determine whether the detergent concentration (0.2%) would be sufficient to block the non-specific binding of the DMAE-labeled antibody to the phase solid (PMP) It was found that the concentration of NP-40 at 2% (v / v) in the medium containing the antibody labeled with acrinide ester gave the best signal-to-noise ratio below this level of detergent ( 0.525% NP-40 in the test mixture), the level of non-specific binding was high, particularly in the lower scale of the Mx protein concentrations (below about 4 ng / ml), whereas the output signal was lower if the concentration detergent was above this scale. It was also found that including 50 mM 2-ME in the test mixture raised the level of non-specific binding without increasing the solubility of the Mx protein. Therefore, the 2-ME included in the original pH buffer to increase the solubility of the Mx proteins was omitted in the denaturing medium, consequently in the test mixture. C. Effect of incubation time on the sensitivity level of the test. Since level of Mx protein in normal healthy volunteers was at the limit of detection of the current test, it was examined whether the sensitivity limit of the test could be extended by a longer incubation period. As the incubation length was increased from 30 minutes to 2 hours, the absolute signal output was higher. However, it did not extend the limit of sensitivity or improve the accuracy of the test at the lower end of the antigen concentrations. Therefore, an incubation length of 30 minutes was selected. 6. Dilution and recovery of the Mx protein In order to determine that the Mx protein test produced a linear-sensitive dose curve over a wide range of Mx protein concentration, the Mx protein was serially diluted in the same test medium and it was tested at seven different concentrations of Mx. The average recovery of the Mx protein tested in three ACS instruments: 180 separate gave an average recovery of 95.9%, with a limit of sensitivity of 1 ng / ml, which indicates that the performance of the test is independent of the concentration. Example 5 Inhibition of Proteolytic Degradation of a Thermally Stable Protein A known amount of recombinant Mx protein was fixed within whole blood lysates or synthetic matrices and incubated at 37 ° C. The aliquots of the samples were periodically extracted and kept on ice until they were tested for the residual Mx protein. The removal of protease activity in whole blood lysates and synthetic matrices was examined after the first dilution of cell lysates or synthetic matrices with various volume ratios of a solution containing a denaturing agent and a detergent. solubilization, specifically 2M urea + 0.1 (w / v)% SDS in 50mM of a pH buffer solution of Tris-HCl (pH 8.0). The diluted mixtures were subjected to heat treatment at 56 ° C for 30 minutes or at 90 ° C for 60 seconds in a water bath. The effectiveness of heat treatment in the destruction of protease activity of synthetic matrices or whole blood lysates after fixation with a known amount of Mx protein within the heat-treated medium and observing changes in the level of Mx protein on incubation at 37 ° C for up to 120 minutes. A regulated solution was prepared in its pH containing 2M urea + 0.1% SDS + 50mM Tris-HCl (pH 8.0), followed by heat treatment to investigate the effect on protease activity. Synthetic matrices or whole blood lysates were diluted up to 20 times (v / v) with the above solution and subjected to heat treatment at 90 ° C for 60 seconds or at 56 ° C for 30 minutes. Following the heat treatment, the solutions were incubated at 37 ° C for 60 minutes and the residual protease activity was determined with aliquots of the samples taken during this incubation period. As shown in Fig.3, the Mx protein fixed within the four different synthetic matrices, which had been inactivated with heat according to the present invention, remained stable for at least 1 hour at 37 ° C, indicating that virtually the protease activity of the cell lysates had been abolished. Essentially identical results were obtained by heating at 90 ° C for 60 seconds. As shown in Fig. 4, the Mx protein fixed within six different individual whole blood lysates remained also stable for at least one hour at 37 ° C followed by the heat inactivation procedure according to the present invention. The inhibition method of the present invention was also tested with several commercially available proteases, ie, Cathepsin G and elastase from human leukocytes and trypsin and type IV elastase from the porcine pancreas. It was determined that the method of the present invention is also effective in removing the enzymatic activity of the purified proteases to an equimolar ratio of enzyme (E) to substrate (S). In order to determine the stability of the Mx protein contained in the frozen whole blood lysates, a known amount of purified rMx protein was fixed in four different whole blood lysates derived from normal healthy volunteers and stored at 4 ° C, - 20 ° C and -80 ° C. The endogenous levels of the Mx protein in those whole blood lysates had previously been determined and found to be negligible. An aliquot of the stored sample was extracted every week and diluted immediately with a volume of 20x of 2M urea + 0.1% SDS in 50mM TrisHCI pH buffer (pH 8.0) and heated at 56 ° C for 30 minutes in order to to minimize the subsequent degradation of the Mx protein during the test procedure. The Mx protein maintained in the whole blood lysates experienced an appreciable degree of autolysis even at -80 ° C and the most pronounced destruction at 4 ° C (25% destruction in 1 week). In contrast, the Mx protein maintained in heat-treated lysates according to the present invention remained stable at both -80 ° C and 4 ° C for at least three weeks, further demonstrating the effectiveness of this simple procedure in stopping Proteolytic degradation of the Mx protein in cell lysates. The whole blood lysates were prepared with freshly harvested blood to normal healthy volunteers and served as normal untreated controls. Ninety-eight samples of frozen whole blood lysates derived from a clinical interferon (B / D) test for the Mx protein were treated using the manual test. A total of 26 patients with various types of malignancies had been treated with doses of interferon (B / D) from 2 x 106 up to 64 x 106 units / day for days 1, 2, 3 and 7, or 8 or 9. treated groups of three patients with each dose of interferon (B / D) at 2.4 8, 16, 32, 64 and 25 million units per day. Fig. 5 shows a typical response to dose-dependent Mx induction for the three patients who were treated with IFN (B / D) at 8 x 106 units / day. The data was collected according to the method of the invention.Example 6 Comparison between manual versus automated test The maximum incubation time in the automated test on the ACS: 180 is only 7.5 minutes, while the duration of the incubation in the manual test can be adjusted. To compare the performances of the manual versus the automated test, it was chosen to incubate the test mixture for 30 minutes at 37 ° C in the manual test. The results showed that the values of the manual test are scarcely higher than those of the automated test, considering the longer incubation time of the manual test. The two tests exhibited an excellent linear correlation (R = 0.987). Example 7 Comparative Example: Conventional Protease Inhibitors At least 14 different protease inhibitors listed in the Materials and Methods Section including an elastase inhibitor, elastin and cysteine protease inhibitor, E-64, failed to block the Protease activity either from whole blood lysates or synthetic matrices. In fact, the inclusion of these protease inhibitors in the used whole blood exacerbated the proteolytic degradation of the Mx protein. The use of a high concentration of chaotropic salts (ie 3M sodium thiocyanate or, potassium chloride), denaturation salts such as 8M urea or 6M guanidine-HCl or, exposure to extreme pHs (pH2 or 1 1) failed all in the arrest of the proteolytic degradation of the Mx protein. Those skilled in the art will readily appreciate that all of the parameters listed herein are intended to be exemplary and the actual parameters will depend on the specific application for which the sealing and grinding arrangements are being used. It is therefore understood that the above embodiments were presented only by way of example and that, within the scope of the appended claims and equivalents thereof, the invention may be implemented in a manner different from that specifically described.

Claims (25)

1. CLAIMS 1. A method of proteolytic inhibition of a sample of thermally stable intracellular protein contained within a cell, comprising: the cell to release the protease and said intracellular protein, if said protein and protease are contained within the cell, and denature said protease through a. adding one or more denaturing agents to said sample containing the released intracellular protein and the protease to form a solution; and b. heating said solution to a temperature and for a period sufficient to denature said protease but not to denature the intracellular protein.
2. 2. The method of claim 1, wherein said cell is Used by the use of a non-ionic detergent.
3. 3. The method of claim 2, wherein the non-ionic detergent is octyl-phenoxy-polyethoxy-ethanol.
4. 4. The method of claim 1, wherein the denaturing agents are sodium dodecyl sulfate and one or both selected from the group consisting of urea and guanidine hydrochloride.
5. The method as claimed in claim 1, wherein said heating step comprises heating said solution to a temperature from about 50 ° C to about 60 ° C for a period from about 60 seconds to about 30 minutes.
6. The method as claimed in claim 1, wherein the source of said protease and the intracellular protein is selected from the group consisting of whole blood, whole blood cells, packed cells, cultured cells and a synthetic matrix that resembles the whole blood.
7. The method of claim 1, wherein the intracellular protein is the Mx protein.
8. 8. A method of inhibiting the protease contained within a sample consisting of cellular contents comprising: adding one or more denaturing agents to said sample to release the intracellular protein and the protease and heating said sample to a temperature and during a sufficient period to denature said protease but not to denature the intracellular protein.
9. The method of claim 8, wherein the sample comprises a synthetic matrix into which the protein is added after the protease is inactivated.
10. The method of claim 8, wherein said heating step comprises heating said solution to a temperature of from about 50 ° C to about 1 00 ° C for a period from about 1 minute to about 1 hour or more.
11. The method of claim 10, wherein the heating takes place at about 56 ° C for about 1 hour.
12. 12. The method of claim 1, which also comprises determining said intracellular protein in the solution.
13. The method as claimed in claim 12, said step of determining comprising: providing a first antibody of said intracellular protein coupled to a solid phase insoluble in said solution; allowing said antibody to capture the intracellular protein by contacting said solid phase with the solution; reacting a second antibody of the intracellular protein for said intracellular protein, the second antibody carrying a label; and determining said label, wherein said enabling and coupling steps can be performed in any order.
14. 14. The method as claimed in claim 13, wherein said first and second binding partners are independently selected from the group consisting of monoclonal antibody clones with access ATCC numbers HB-1 1836 and HB-1 1837.
15. 15. The method as claimed in claim 13, wherein the label comprises an acridinium ester.
16. 16. The method as claimed in claim 15, wherein said label comprises dimethylacridinium ester activated with N-hydroxysuccinimide.
17. 17. A method of determining interferon in a sample, comprising: forming a sample containing a thermally stable intracellular Mx protein induced by interferon, an unknown protease that degrades said intracellular Mx protein, a denaturing agent and a detergent selected to solubilize said intracellular Mx protein; heating said sample to a temperature and for a period sufficient to denature said protease but not to denature the intracellular protein; and determining the intracellular protein in said solution.
18. 18. The method as claimed in claim 17, wherein the heating step comprises heating said sample to a temperature of from about 50 ° C to about 60 ° C for a period from about 15 to 30 minutes.
19. 19. A solution on which a clinical test is conducted, comprising an inactivated protease matrix that stimulates a Used cell and remains free of protease activity for at least 3 weeks at 4 ° C, wherein said protease was inactivated by (1) releasing any protease contained within said solution by the use of denaturing agents and (2) heating to denature any released protease.
20. A method of inhibiting proteolytic degradation of an Mx protein contained within a cell, comprising: Using the cell to release the protease and said intracellular protein, if said protein and protease are contained within the cell, and denaturing said protease through a. adding one or more denaturing agents to said sample containing the released intracellular protein and the protease; and b. heating said solution to a temperature and for a period sufficient to denature said protease but not to denature the intracellular protein.
21. 21. A method of claim 20 in which a. said cell is Used by the use of a non-ionic detergent, b. the denaturing agents are selected from the group consisting of sodium dodecyl sulfate, urea and guanidine hydrochloride, and said heating step comprises heating said solution at a temperature from about 50 ° C to about 60 ° C for a period of about from 60 seconds to about 30 minutes.
22. 22. A method of claim 22 wherein the non-ionic detergent is "Tergitol N P-40" and the denaturing agents are urea and sodium dodecylisulfate.
23. 23. A method of determining the amount of Mx protein contained within a cell comprising: a. Inhibit the proteolytic degradation of the Mx protein contained within the cell by 1. if the protein and protease are contained within said cell, use the cell to release the intracellular protein and the protease, and 2. denature said protease by a. add one or more denaturing agents to the sample containing the released intracellular protein and the protease and b. heating said solution to a temperature and for a period sufficient to denature the protease but not to denature the intracellular protein.
24. 24. A method of claim 23 wherein a. said cell is Used by the use of a non-ionic detergent, b. said denaturing agents are selected from the group consisting of sodium dodecyl sulfate, urea and guanidine hydrochloride, and c. said heating step comprises heating said solution to a temperature from about 50 ° C to about 60 ° C for a period from about 60 seconds to about 30 minutes.
25. 25. A method of claim 23 wherein said method of determining the amount of Mx protein comprises the use of monoclonal antibody clones with accessing ATCC numbers HB-1 1836 and HB-1 1837. SUMMARY OF THE INVENTION A method of inhibiting the proteolytic degradation of a thermally stable intracellular protein is described. The method involves adding one or more denaturing agents to a sample containing the protease and the protein of interest and heating the resulting solution at a temperature and for a period sufficient to denature the protease. The method optionally includes a step to use the cell if the protein of interest is contained in a cell in order to release said protein. Additionally, a method of determining Mx protein induced by interferon in a blood sample is described. The method involves adding a sado agent to a blood sample, a denaturing agent and a detergent selected to solubilize the Mx protein. The sample containing the Mx protein is then heated to a temperature from about 50 ° C to about 60 ° C for a period from about 1 hour to about 30 minutes, and the Mx protein is then determined in the solution. A solution is also described that includes a synthetic matrix that resembles the whole blood cell lysates, or the actual whole blood cell lysates, to which a known concentration of a thermally stable intracellular protein is added to prepare a material of control. In addition, a protease-free solution that degrades the intracellular protein is described, said solution remaining stable at 4 ° C for at least three weeks.