The method of early identification, determining the severity of the disease and evaluation of the treatment of sepsis, as well as the means to implement this method


(57) Abstract:

The method can be used in medicine, in particular for the early detection of sepsis. Determine the content of the peptide of procalcitonin and/or formed from it incomplete peptide that is not Mature calcitonin, and determine the presence and quantity of a particular peptide is indicative of the presence of sepsis, its severity and/or success of treatment. The determination is conducted by immunometric analysis, which uses at least two monoclonal antibodies, or a combination of the first monoclonal or polyclonal antibodies with a second monoclonal antibody which possess a specificity of procalcitonin or formed of peptides. These antibodies included in the kit for detection of procalcitonin in quantities of from 0.1 to 500 ng/ml of serum sample or plasma. The method provides a more accurate diagnosis of sepsis and at an earlier stage of its development. 2 C. and 12 C.p. f-crystals, 2 tab.

The present invention relates to a method of diagnosis of sepsis, in particular for the early detection and identification of the severity of sepsis, and to carry out certain already known peptide and possibly some of its fragments are reliable biological markers of diseases of this type, found in high concentrations, and they can be relatively simply defined classical detection methods.

According to the latest ideas about this disease, the term "sepsis", as used herein, encompasses the clinical picture, in which, as a rule, are fever, leukocytosis, changes in consciousness, giperdinamicheskim circulation ("thermal shock") and hypermetabolism condition, mainly because of the infestation properly sterilized tissue by microorganisms and at the same time, a positive detection of pathogenic microorganisms in the blood, which was previously regarded as characteristic of sepsis, it becomes less important for the diagnosis of sepsis". It is shown that in clinical studies, disease prognosis in patients with sepsis, does not depend on the force of infection, particularly bacterial infection and depends on the strength of septic reactions (see G. Pilz, S. Fateh-Moghadem and K. Werdan in Krankenpflege-Journal 29 (1991), pp. 483-492 and see publication). Accordingly, along with the analysis of positive blood cultures or instead to define sepsis are determined by different laboratory parameters and hemod is lesne, if necessary, utilizing the computer, the so-called scoring systems such as APACHE (Acute discrimination end Chronic Health Evalution) II Score, described in the above publication. However, it is not yet known any individual parameter, suitable as a reliable biological marker, the definition of which is highly expressive, with a diagnosis of sepsis. All parameters used up to the present time, or have insufficient specificity, or do not allow to reliably assess the severity of sepsis, or do not allow for therapeutic examination, and, in addition, determination of, for example, factor tumor necrosis (TNF) or interleukin, such as interleukin 6 (IL-6), too difficult, expensive and/or requires a large investment of time in the analysis of bedridden patients.

Because of this, there is still a need for a reliable biological marker that can be relatively easily defined and quality and, in particular, the quantification of which is highly indicative for the diagnosis and assessment of the progression of sepsis.

The object of the present invention is a method of early detection and identification of the severity of sepsis, in which the definition is e which provides highly reliable results for diagnosis and assessment of the progression of sepsis.

To solve this problem is the method and means according to the invention. This invention is based on the discovery of the fact that the peptide procalcitonin as such and, if necessary, some high molecular weight products of its decomposition are highly suitable biological markers of sepsis, and is based on the fact that their concentrations in samples of biological fluids of patients allow us to make fairly accurate conclusions about the severity of septic diseases and, thus, they are important parameters for evaluation of disease progression and therapeutic examination of sepsis.

Thus, the possibility of the diagnosis of sepsis by determining the peptide of procalcitonin is a great practical interest, since other known possible biological markers that can be detected by sepsis, for example, some cytokines (interleukins, TNF), are unstable molecules that are normally present in very small concentrations, so that their definition is too difficult, requires time-consuming and therefore too expensive for diagnosis in bedridden patients. According to this izobreteniya of procalcitonin is extremely high in sepsis, so find a concentration in the range of ng (>1 ng, in particular, more than 10 ng to 500 ng or more per ml of serum sample or plasma), whereas in healthy people who have procalcitonin determine the best known methods, the content of procalcitonin not detected (concentration less than 0.1 ng/ml sample). At the same time with sepsis in the implementation of the method according to the present invention elevated concentrations of calcitonin is not detected that is distinct from that to the present time, as a rule, procalcitonin was seen as the precursor of calcitonin, which also results in the formation of calcitonin.

Peptide procalcitonin, which must be determined by the method according to the invention, and, perhaps, the products of its proteolytic cleavage, are already known, and methods for determining suitable for quantitative and qualitative immunodiagnostics analysis, also known.

Procalcitonin is a peptide consisting of 116 amino acids, and up to the present time known about him was that he appears as an intermediate product broadcast/expression of a particular gene (CALC-1), leading to the formation of a peptide of the mountains is, the AK precursor of calcitonin with this original genome (CALC - 1), excluding education procalcitonin, also regulating the formation associated with procalcitonin-gene peptide, and significantly different in length and amino acid sequence 51-116 of procalcitonin (see J. Biol.Chem., 261, 31(1986), page 14386-14391).

According to well-known information, procalcitonin is a product of proteolytic decomposition of the main protein of procalcitonin formed by a certain type of gene expression gene CALC-1, and in certain cases its appearance, as a rule, it undergoes further gradual decomposition in the process of releasing Mature calcitonin, which corresponds to the sequence of 32 amino acids (amino acids 60-81 of procalcitonin). In this process, first, along with other are formed with two larger peptide, which can be called N-procalcitonin-(1-57)-peptide and C-procalcitonin-(60 to 116)-peptide, the latter optionally cleaved before the formation of the hormone calcitonin and to peptide known as katacalcin (Biochem J. , 256, (1988), 245-250; and Cancer Research 49 (1989), 6845-6851). From the publication, placed in J. Biol. Chem., 226, 36 p. 24627-24631, it is known that in addition to prochoice kind of procalcitonin, different from the first procalcitonin last eight amino acids of the C-end. In addition, this peptide should be considered as "procalcitonin" in accordance with the present invention, since at this time the ways of immunodiagnostics analysis used in the development of the present invention, does not allow to identify the difference between the two procalcitonine and possible other closely related peptides. Therefore, "procalcitonin" in accordance with the present invention means one or many peptides, including known molecular procalcitonin, its above-described type having a different amino acid composition in the C-end, and possibly other existing varieties with comparable reactivity in the methods of election of immunodiagnostics analysis used to determine, in particular, monoclonal Immunoradiometric the analysis described below with reference to the publication in Cancer Res. 49 (1989), 6845-6851.

All these peptides comprise a peptide sequence of 57 amino acids or more, in particular 116 amino acid type full procalcitonin, and are known to follow the which correspond to amino acids 108-116 possible procalcitonin.

As for the previously made attempts to use the method of detection of calcitonin and related peptides for diagnostic purposes, it is known that calcitonin is a valuable biological marker (tumor marker) for many malignant diseases, and has developed many methods of immunodiagnostics analysis for specific definitions of calcitonin, and these methods are carried out using specific monoclonal antibodies (see, e.g., Clinica Chimica Acta, (1988), 174, pp. 35-54; Immunolo, I. 141, page 3156-3163; J. Endocr. (1988), 119. pages 351-357).

In addition, to determine calcitonin predecessors, such as procalcitonin and C-procalcitonin-(60-119)-peptide, has developed a way of immunodiagnostics definition, which is carried out in accordance with the principle Immunoradiometric analysis (IRMA) and which allows, in addition to calcitonin selectively determine procalcitonin, which uses a pair of monoclonal antibodies, one of which is specific for the outer zones of calcitonin sequence (amino acids 1-11 katacalcin or amino acids 96-107 of procalcitonin), and which is used, for example, in immobilizovannoi the AK second labeled monoclonal antibody, used for forming patterns "sandwich" IRMA, is specific to a site corresponding to amino acids 11-17 calcitonin (amino acids 70 - 76 procalcitonin). Thus, in this immunological analysis reveals only those peptides that incorporate calcitonine areas, as well as amino acids 1-11 katacalcin plot, and, therefore, are either full procalcitonin or peptide with the specified educated on it by sections, such as C-procalcitonin-(60-116)-peptide (see Cancer Res., 49 (1989), page 6845-6851). Of the many available monoclonal antibodies are selected in a known manner those who have Association constants in the range K =0,9-3,01010M-1. This known method can be used to direct the implementation of the method according to the present invention or using the same monoclonal antibodies, which can be obtained as described in J. lmmunol., so 141, No. 9 (1988), page 3156-3163 in which to determine the increase of procalcitonin as the most clear and easy way to assess for clinical purposes must be used in pairs of antibodies with similar high affinity as described above.

So, for example, the is obtained by using CT-TT (Calcitonin-Titanosilicate) as the immunogen according to previously described procedures immunization (Motte and others, J. lmmunol., 138 (1987), 3332) to generate antibodies having the binding characteristics similar to the characteristics of the binding CT21. Antibodies having the same characteristics as X, can be obtained by immunization of mice, Bizzy with cell lines with distinct antireproductive properties (Biozzi, etc., J. Exp. Med., 132 (1970), 152) with KS-TT (Katacalcin-Technoclassica), resulting according to the described procedure are four injections, each consisting of 15 g of peptides inserted in different ways: subcutaneously in full adiuvante's adjuvant (FCA); subcutaneously in incomplete adjuvant's adjuvant (FIA); intraperitoneally in FIA and intravenous 0.15 mol/l sodium chloride. Interval immunization is from 13 to 30 weeks. Three days after the last intravenous injection of the conjugate mice kill, remove the spleen and splenocytes merge with cell line murine myeloma NSI using 40% polyethylene glycol (Bellet, etc., J. Clin. Endocrinol. 10 Metab., 56 (1983), 530). Surfaced of hybridoma may be selected for the production of specific antibodies by enzyme immunosorption assay (ELISA). After cloning by limiting dilution of hybridoma cells intraperitoneally implanted hairless mice B is purified from ascitic fluids by precipitation using 50% ammonium sulfate at a temperature of 4oC and chromatography protein A, as described in the publication Manil and others (J. lmmunol. Methods 90 (1986), 25). Conjugates of the hapten-carrier CT-TT or KC-TT can be obtained by separate binding CT or KC with TT by using glutaraldehyde as a coupling reagent (Audibert and others, Proc. Natl. Acad Sci., USA, 79 (1982), 5042).

For definitions described in the following examples, we used a pair of monoclonal antibodies, which included the above antibody mAbKC01 and antibody mAbCT21, with the antibody mAbCT21 with respect to its binding with a molecule of procalcitonin, as well as with respect to its affinity, very close to the antibody mAbCT08 described in the above publication (the constant of Association Kasn= 3,01010M-1). Hybridoma producing monoclonal antibodies KC01 and CT21, were deposited in the DSM - Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Nascheroderweg IB, 38124 Braunschweig, Germany) according to the decision of the Budapest Treaty, under the formal rooms DSMACC2124 and DSMACC2125, respectively; the date of the Deposit of 20 April 1993.

The described method in accordance with the publication in Canser Res., 49 (1989), page 6845-6851, allows to obtain a concentration of procalcitonin or C-procalcitonin-(60-119)-peptide, or both in the samples, or if the. what it concerns the methods that can be used to determine procalcitonin according to the invention, the details are set out in the above publication, and in some of the publications that are included in the present description as reference materials for additions.

As described in the above publication, an attempt was made to determine procalcitonin in order to check the suitability of the marker of the tumor. It was found that patients with tumors observed pattern of parallel changes in the concentrations of procalcitonin and calcitonin, on what basis it was concluded that both of them are formed from neoplastic C cells in the thyroid gland. In the above publication also States that the concentration of procalcitonin increase in patients who are not suffering from malignant diseases, but with serious viral infections. In these cases, was not observed simultaneous increase of the concentrations of calcitonin. These patients had sepsis and their disease had no relationship to sepsis.

After according to the present invention was first established that there is a direct relationship between Ko the order of increasing concentration of procalcitonin without a simultaneous increase in the concentration of calcitonin in sepsis, as well as increasing the concentration of obviously lesser extent, allowing for the most part directly to distinguish these cases from sepsis in patients suffering from serious viral diseases. As one patient with sepsis who were thyroidectomy, you can find nevertheless an increase in the concentration of procalcitonin to concentration, which is significant in sepsis, it is obvious that in sepsis, procalcitonin is not formed in the thyroid gland, which is responsible for this other body. If, in connection with the increased concentration of procalcitonin with viral hepatitis to assume that other specified organ is the liver, the increase in the concentration of procalcitonin can be explained in the first case, the direct impact of viral diseases on hepatocytes and in another case an indirect, but more effective exposure to endotoxins produced by bacteria responsible for sepsis, the same hepatocytes. However, it should be borne in mind that this explanation is a working hypothesis, but not by theory, it is experimentally proven.

The occurrence of elevated concentrations of procalcitonin with a serious viral diseases is the effect uvelicheny only to a relatively small extent, until such values that can be detected also in the case of serious viral diseases, such viral diseases must be excluded before the diagnosis of sepsis.

In addition, in patients with chronic renal failure and, consequently, with impaired excretion of the peptide should be raised concentrations of peptides type of procalcitonin, but this increase is not clinically is the same as in the case of an increase in patients without this disease. However, the doctor, whose clinical diagnosis, can easily take into account these circumstances.

The present invention is not limited to the use of the above specific method of analysis for the detection of procalcitonin, but encompasses other methods of detection that are already known, which include methods using other monoclonal or polyclonal antibodies, for example, the methods implemented by using specificity to N-procalcitonin-(1-57)-peptide and, in particular, amino acids 51-57. Thus, for General use polyclonal antibodies to the zones of N-procalcitonin-(1-57)-peptide instead mAbKC01 30 along with labeled monoclonal and is that in both cases reached similar concentrations of procalcitonin, and, based on the fact that if you are using these polyclonal antibodies, detected zones are present within the same molecule only if not subjected to procalcitonin peptide, this confirms the conclusion that in sepsis concentration is not subjected to procalcitonin really improved and formed its partial peptides in most cases play a secondary role.

The method according to the invention in principle can be carried out by determining procalcitonin other way than immunodiagnostics, for example, by liquid chromatography high pressure (ghvd), if these methods provide sufficient sensitivity and if there is or can be achieved specificity of these methods.

Moreover, although the definition of procalcitonin according to the invention is at present mainly in samples of serum or plasma, the method according to the invention in principle encompasses the definition of procalcitonin in other biological fluids such as whole blood and urine, if it turns out that in these liquids concentration of procalcitonin can be measured reproduce 108, 6 (1990), pp. 1097-1101, reported that in patients suffering from sepsis, plasma concentration of the peptide CGRP, which is associated with calcitonin, slightly raised in the range of picograms (PG) (14,9+3,2 PG/ml compared with 2,0+0,3 PG/ml in control patients). These findings do not allow to conclude on the concentrations of other related peptides, and significantly lower absolute concentrations and significantly lower relative increase in sepsis compared with normal concentration relative to the concentration in the method according to the invention confirm that the definition of CGRP as a marker of sepsis may not be used.

In addition, the publication of the Lancet, 1, (1983), page 294, it is reported that in the case of a major meningococcaemia children observed concentrations of calcitonin increased two to three times, but in the next publication in Rediatr. Res. , 18. (1984), page 811 was fixed that the designated substance, in all probability, is not subjected to the effects of calcitonin, but there was no indication that this substance is actually defined. The observed increase of up to approximately three times the value from the normal rate in these cases was compared with uvelichit about these publications are not relevant to the present invention.

The method according to the invention is described below in more detail with reference to clinical data, which is the proof of the appropriateness of the information provided.

All definitions of procalcitonin was performed according to the method of determining procalcitonin described in Cancer Res., 49, (1989), page 6845-6851, using monoclonal antibodies KC01 (DSM ACC2124) and CT21 (DSM ACC2125) (see above).

These examples illustrate the present invention but they should not be construed as limiting the invention.

Example 1. The determination of concentrations of procalcitonin in hospitalized children with various diseases.

Determined concentration of procalcitonin in different groups of hospitalized children with various diseases.

The results are shown in table. 1 (see tab. 1 and 2 at the end of the description).

The data obtained show that in patients with sepsis is detected up to 180 ng/ml pCT (concentration of procalcitonin), while in patients with "normal" viral diseases pCT concentration is increased to 2 mg/ml, for cases of extremely serious viral diseases concentrations of pCT in patients with sepsis, which was simultaneously watching the progress of the illness according to the evaluation of APACHE II Score, disease severity.

In 20 patients who had undergone treatment by intravenous infusion of Pseudomonas-IgG for treatment of sepsis after cardiac operations, observed the concentration of pCT in five days, and at the same time observed the patient's condition according to APACHE II Score. The results are shown in table. 2.

From the data table. 2 shows that in the case of reactions to successfully carry out the treatment of sepsis (sensitivity to impact) pCT concentration decrease with improvement in clinical status, whereas these concentrations pCT remain almost invariably high in case of lack of response (no sensitivity to the effects) of treatment. From this table it is also clear that in the case insensitivity treatment of the initial concentration of pCT significantly higher than in the case of sensitivity to treatment, and, thus, the severity of the disease in the first case more. In comparison with the data obtained by the evaluation method of the APACHE II Score data of the pCT values show significant differences sepsis on the severity of the disease, as confirmed by the mortality..

In addition, this is with reliable information about the successful course of treatment, so if you want, then perhaps an early conclusion about the change in the course of treatment selected, such as selecting a different antibiotic drug.

Similar results can be obtained in patients with burns who develop sepsis because of skin transplants, which were subjected to treatment of various antibiotic drugs. Successful treatment is always accompanied by a significant decrease in the concentration of the pCT, and in one case at an early stage to adjust the insensitivity to the first treatment the first antibiotic drug, which can be defined as the concentration of pCT remain constant, by replacing the antibiotic drug with a response to the second antibiotic drug, the suitability of which is based on the fact that the concentration of pCT instantly reduced.

1. The method of determining the state of casinogo disease, characterized in that in the sample of biological fluid of the patient determine the content of the peptide of procalcitonin and/or formed from it incomplete peptide that is not Mature calcitonin, and determine the presence and quantity of a particular peptide is until the decomposing those to determine procalcitonin and/or N-procalcitonin-(1-57)-peptide and/or C-procalcitonin-(60-116)-peptides.

3. The method according to p. 1 or 2, characterized in that in the exercise of immunodiagnostics way to find the appropriate peptide or peptides through the use of monoclonal antibodies or a combination of the first monoclonal or polyclonal antibodies with a second monoclonal antibody which as such or in combination with each other have the specificity of procalcitonin or generated from it, peptides, and which can make the differentiation between them and Mature calcitonin and peptide CGRP.

4. The method according to p. 3, characterized in that procalcitonin determined by immunometric analysis, which uses at least two monoclonal antibodies, the first monoclonal antibody for the extraction of procalcitonin of the analyzed sample, and the second to indicate, moreover, these antibodies have specificity with respect to different parts of the amino acid sequence of procalcitonin, so the possible differentiation between procalcitonin and products of proteolytic cleavage.

5. The method according to p. 4, titulo, which has specificity towards another adjacent portions of amino acid sequence of procalcitonin compared with the first monoclonal antibody.

6. The method according to any of paragraphs. 3-5, characterized in that procalcitonin find by immunometric analysis, which selectively detects the complete procalcitonin and/or C-procalcitonin-(60-116)-peptide.

7. The method according to any of paragraphs. 3-5, characterized in that procalcitonin find by immunometric analysis, which selectively detects the complete procalcitonin and/or N-procalcitonin-(1-57)-peptide.

8. The method according to p. 7, characterized in that the content of procalcitonin more than 1 ng/ml of sample are indicative of the possible presence of sepsis, and the fact that the content of procalcitonin in the range from 10 ng/ml to 500 ng/ml or more are associated with increasing severity of sepsis and worsened prognosis.

9. The method according to p. 8, characterized in that at the same time in a parallel analysis to discover the content of calcitonin and the fact that no evidence of elevated concentrations of calcitonin helps to make a diagnosis of sepsis.

10. The method according to p. 8 or 9, characterized toget to be excluded the existence of a serious viral diseases, as the cause of increased concentration of procalcitonin.

11. The method according to any of paragraphs. 1-10, wherein the sample is serum or plasma is used as a sample of biological fluid.

12. The method according to any of paragraphs. 1-11, characterized in that the monoclonal antibody is a monoclonal antibody X (DSM ASS) or antibody SC (DSM ACC 2125).

13. Toolkit for implementing the method according to any one of paragraphs. 1-12, characterized in that for the detection of procalcitonin in the amount of from 0.1 to 500 ng/ml of serum sample or plasma these tools include: one (or many) of immobilized (immobilized) the first monoclonal or polyclonal antibody (or a monoclonal or polyclonal antibodies) on the carrier for binding of procalcitonin in the sample; additional monoclonal antibody carrying a marker and linking procalcitonin in the field, which is different from the connecting region of the first antibody (or the first antibody); and General auxiliary funds.

14. The Toolkit under item 13, in which one or more monoclonal antibody is an antibody X (DSM ASS) or antibody SC (DSM ASS), respectively.


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