MXPA01007637A - METHOD OF IDENTIFYING N-TERMINAL proBNP - Google Patents
METHOD OF IDENTIFYING N-TERMINAL proBNPInfo
- Publication number
- MXPA01007637A MXPA01007637A MXPA/A/2001/007637A MXPA01007637A MXPA01007637A MX PA01007637 A MXPA01007637 A MX PA01007637A MX PA01007637 A MXPA01007637 A MX PA01007637A MX PA01007637 A MXPA01007637 A MX PA01007637A
- Authority
- MX
- Mexico
- Prior art keywords
- terminal probnp
- antibodies
- probnp
- terminal
- recombinant
- Prior art date
Links
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Abstract
The invention relates to a method of identifying N-terminal proBNP in a sample with at least two antibodies that detect different epitopes of the N-terminal proBNP. The method is used to differentiate or classify samples of healthy individuals and samples of patients of NYHAclasses I to I. The invention further relates to recombinant N-terminal proBNP, its use as standard in a method of identifying N-terminal proBNP, to antibodies that detect recombinant N-terminal proBNP and to their production.
Description
METHOD TO IDENTIFY N-TERMINAL proBNP
Description of the Invention The present invention considers a method for identifying the N-terminal proBNP in a sample with at least two antibodies that detect different epitopes of the N-terminal proBNP. The method is used to differentiate or classify samples from healthy individuals and samples from NYHA patients types I through VI. The invention concerns recombinant N-terminal proBNP, its use as a model in a method for identifying N-terminal proBNP, antibodies that detect the recombinant N-terminal proBNP and its production. Heart failure is a widespread phenomenon especially in the western world. According to the medical dictionary Roche (1993, Urban &Schwarzenberg), heart failure is the acute or chronic inability of the heart to generate the blood flow required for metabolism during exercise or even during rest to ensure rapid reflux (heart failure). Back and forth) . Therefore, the pumping function of the heart is weak. The causes of failure
REF: 131315 cardiac are very complex. Among others, the inflammatory and degenerative modifications of the cardiac muscle, a disorder - coronary perfusion, infarction and coronary damage are mentioned here. This leads to changes in peripheral blood flow, respiratory disorders, renal function and electrolyte metabolism (edema) and reduced performance of the skeletal muscle system. According to the New York Heart Association (NYHA), heart failure is divided into the following NYHA classes when using physical analysis after an effort: I means that it is completely free of pain after a normal physical effort, II means a slight limitation of physical strength, III means a strong limitation of physical strength, IV means that with each physical activity, the symptoms of insufficiency increase, and that during most of the time it also exists during rest. For an effective treatment with drugs against cardiac failure by means of glycosides, vasodilators, ACE inhibitors and / or β-blockers, it is first of all necessary to accurately diagnose cardiac failure and classify it, if possible, according to the degree of severity and also monitor the course of treatment. According to the latest technology, some serum markers are discussed for early diagnosis of heart failure, such as ANP (natriuretic atrial peptide hormone N-terminal) and prOaANP, CNP (C-natriuretic peptide), adrenomedullin, neuropeptide Y, endothelin and BNP (cerebral natriuretic peptide). The ANP and proANP are generally suitable markers for the diagnosis of heart failure; however, they are not very stable or only have a very short half-life in the blood, which represents an impediment to diagnostic quantifications (Clin.Sci.95 (3) (1998), 235-239; Cleland et al., Heart. 75 (1996), 410-413). A frequently cited and significant marker is BNP (cerebral natriuretic peptide). Originally, BNP is identified in the brains of pigs. It is a cardiac hormone that structurally and functionally resembles ANP (natriuretic atrial peptide) (Sudoh et al., Nature 332 (1988), 78-81). The human BNP consists of 32 amino acids and is secreted mainly in the cardiac ventricles and circulates in the blood plasma of humans. The use of BNP as a diagnostic marker is known, for example, from EP-A-0 542 255. BNP has an intramolecular disulfide bridge and is not very stable in the manner of an analyte presumably due to its physiological function. hormone which must unfold very fast. Therefore, its use as a diagnostic marker is limited (Masuta et al., Clin.Chem. Vol.44 No. 6 Supplement A (1998), 130; Tsuji et al., Clin.Chem. 40 (1994). , 672). The precursor molecule of BNP, ie proBNP consists of 108 amino acids, of which the above mentioned 32 C-terminal amino acids (77-108) called BNP, develop the true hormonal effect. The N-terminal amino acids 1-76 that are released from the precursor are called N-terminal proBNP. Apart from BNP (77-108), the N-terminal proBNP also circulates in the plasma as well as its other cleavage products (1-76) (Hunt et al., Biochem. Biophys. Res. Com. 214 (1995), 1175-1183) so that the N-terminal proBNP is also relevant as a marker of heart failure. The fact that the proBNP precursor molecule also occurs in the plasma is not completely resolved. However, it is described (Hunt et al., Peptides, Vol. 18, No. 10 (1997), 1475-1481) that a low release of proBNP (1-108) in the plasma is detectable, but because the partial unfolding is very fast at the N-terminal end, some amino acids are absent. This molecule is called high molecular weight BNP in the literature. WO 93/24531 (US 5, 786, 163) describes an immunological method for identifying N-terminal proBNP and the antibodies used therefor. To obtain these antibodies, peptides synthetically produced from the N-terminal proBNP sequence are used. The production of the antibodies by means of a peptide immunization is possible in principle but the affinity with respect to the complete molecule is generally very low in order to achieve the necessary sensitivity in a test procedure. In addition, there is a danger that when the peptides are used, the antibodies that are obtained can, for example, identify the C-terminus of the peptide and therefore, bind only to this fragment of the entire molecule. From this, it turns out that these antibodies can not bind to the entire molecule or only in very low proportion. In WO 93/24531 the polyclonal antibodies against a single peptide derived from the N-terminal proBNP are produced. It is shown that the antibodies that are produced are agglutinated to the immunization peptide (amino acids 47-64) in the competitive analysis format. However, the antibodies are not shown to be able to bind to the native N-terminal proBNP in the manner of a complete molecule in a sample. In addition, the two-layer analysis described in WO 93/24531 in a sample can not be carried out as described since there is no appropriate conventional material nor antibodies against two different epitopes. Another problem in cutting-edge technology is the sensitivity of the analysis. With the competitive analysis carried out in WO 93/24531, wherein peptide 47-64 competes in a labeled form with a sample with the unlabeled conventional peptide 47-64 to bind polyclonal antibodies of rabbit serum, only reaching a very moderate competition after 48 hours of incubation of which only a low limit of detection of approximately 250 fmol / ml can be derived. This is neither sufficient for the differentiation of healthy individuals or patients suffering from heart failure nor for a differentiated classification of patient samples in the degrees of severity of heart failure. In addition, long incubation periods of competitive analyzes are not acceptable for routine quantifications of samples in automated laboratories.
Hunt et al., (Clinical Endocrinology 47 (1997), 287-296) also describes a competitive assay for the detection of N-terminal proBNP. For this, a complex extraction of the plasma sample is necessary before quantification; this can lead to the destruction of the analyte and error quantifications. The antiserum that is used is produced analogously to WO 93-24531 by immunization with a synthetic peptide. Hunt et al., Produce the antiserum by immunization with amino acids 1-13 of the N-terminal proBNP and the peptide of amino acids 1-21 is used as the norm. For this analysis, long incubation periods are also necessary. After a 24 hour incubation, a lower detection limit of 1.3 fmo1 / ml is reached. Therefore, there is no state-of-the-art method for detecting N-terminal proBNP that allows reliable, sensitive detection of natural N-terminal proBNP with short incubation periods. It is therefore an objective to provide a method for identifying the N-terminal proBNP in a sample avoiding as much as possible the aforementioned advantages of cutting-edge technology. In particular, high sensitivity in the analysis should be achieved to allow differentiation of patient samples from healthy individuals and patients with NYHA types I to IV. This objective is achieved with the method of identifying the N-terminal proBNP in a sample that is explained in greater detail in the claims. The method is characterized in that at least two antibodies detect different epitopes of the N-terminal proBNP that they use.
What is important in the method according to the invention is that the natural N-terminal proBNP is detected in a sample. This means that the antibodies must be able to identify and bind specifically to the intact molecule and possibly proBNP (1-108 without unfolding) and, if possible, also to the proteolytically digested partial fragments in a sample. For this method, at least two different antibodies are used that bind to different epitopes of N-terminal proBNP. These epitopes can be linear or the so-called shaped epitopes. Preferably, the epitopes are located in a manner that allows both antibodies to bind at the same time and that are not too far away from each other. Since the method according to the invention does not allow the differentiation between the N-terminal proBNP, proBNP and the originating peptides (cleavage products) NT-proBNP in the following refers to all the peptides that are identified in the analysis procedure, in particular the known N-terminal proBNP (1-76). According to the invention, the term "epitope" means the agglutination site in an immunologically binding partner such as an antigen to which an antibody agglutinates in a specific manner.An epitope is ordinarily defined by 6 to 8 amino acids. agglutination pair corresponds to the N-terminal proBNP or a partial sequence thereof The epitope to which the antibody agglutinates constitutes a partial region in the pair of algutination The epitope may be present in a linear fashion or as a conformational epitope. By means of the two antibodies with different specificities, it is possible to carry out a faster method to identify the analyte instead of the long general and competitive procedure of the cutting-edge technology.The detection method according to the invention can be carried out by means of of a homogeneous or heterogeneous analysis procedure, preferably the heterogeneous process of analysis is used and particularly preferably the two-layer process is known to the person skilled in the art. Preferably, this method of determining the N-terminal proBNP is carried out according to the following steps: a) Mix the sample with the first antibody specific for N-terminal proBNP which carries a suitable group to bind to a solid phase or mix with the first specific antibody - N-terminal proBNP that has already bound to the solid phase. b) Mix this solution with the second antibody that identifies an NT-proBNP epitope that differs from the first antibody and that carries, a marker. c) The agglutination of the immune complex to a solid phase that may already be present in step a) d) Separation of the solid phase from the liquid phase e) Detection of the marker in one or both phases. In a quantitative determination, the same quantification is carried out with a defined amount of N-terminal proBNP as the standard and after the determination of step f) of the sample is carried out, ie the comparison of the values of quantification of the standard with those of the sample, and then quantification takes place. The term "antibody" means - according to the invention, mono- or polyclonal, chimeric or humanized antibodies or other antibodies that are obtained by genetically manipulated modifications as well as all fragments known to the person skilled in the art such as F (ab ') fragments. ) 2 / Fab 'or Fab Only the immunologically specific agglutination capacity for the N-terminal proBNP must be guaranteed The first antibody specific for the N-terminal proBNP can be bound directly to the solid phase or indirectly by means of a specific system The direct agglutination of this antibody to the solid phase follows a method known to the person skilled in the art, for example in an adsorbent form.If the agglutination is indirect by means of a specific agglutination system, the first antibody is a conjugate consisting of an antibody against the N-terminal proBNP and a reaction partner for the system specific agglutination ema. A specific agglutination system means that two couples can react specifically to each other. The binder capacity can be based on an immunological reaction or on a different specific reaction. Preferably, a combination of biotin and avidin or biotin and streptavidin are used in the manner of a specific agglutination system. Other preferred combinations are biotin and antibiotin, hapten and anti-hapten, Fe fragment of an antibody and antibodies against this fragment Fe or the carbohydrate and lecithin. One of the reaction partners of the specific agglutination system is then part of the conjugate.
The other reaction pair of the first agglutination partner in the specific agglutination system is a layer of the solid phase. Streptavidin or avidin is preferably used. The agglutination of the other reaction partner of the specific agglutination system to an insoluble carrier material can be carried out according to methods known to the person skilled in the art. Covalent agglutination as well as adsorbent is suitable here. In the solid phase, test tubes or microtiter plates made of polystyrene or similar plastics are suitable and coated on the inner surface with a reaction partner of the specific agglutination system. Other substances which are suitable and particularly preferred are particulate substances such as latex particles, magnetic particles, molecular screening materials, glass corpuscles, plastic tubes and others. Porous, and stratiform carriers such as paper or nitrocellulose can also be used as carriers. The magnetic evaluations coated with the corresponding agglutination partner of the specific agglutination system described above are used in a particularly preferred manner. After the termination of the analysis reaction, these microparticles can be separated from the liquid phase for the detection reaction process, for example by filtration, centrifugation or in the case of the magnetic particles by means of a magnet. The second specific antibody identifies a different epitope of the N-terminal proBNP compared to that of the first antibody. The distance of the two epitopes in the molecule must be large enough so that a simultaneous agglutination of the antibodies to the N-terminal proBNP is possible without reservations; if not, no two-layer complex can be structured.
The detection of the specific agglutination reactions between the antibodies against the N-terminal proBNP and the N-terminal proBNP can be carried out in different ways. Generally, the second antibody is labeled. The usual markers are the chromogens, fluorophores, which are substances suitable for chemo- or electrochemiluminescence, radioisotopes, haptens, enzymatic markers or substances capable of structuring a specific agglutination pair such as biotin / streptavidin. The immune complex is then detected by means of a signal emitted by the marker. The second antibody can for example be labeled with the hapten digoxigenin. This hapten is once again agglutinated by another specific antibody for digoxigenin. This specific antibody for digoxigenin is found in itself, marked by an enzyme, such as, for example, peroxidase. Then the final detection is carried out by means of a change in color or extinction that occurs when a particular substrate is added to the peroxidase. All biological liquids known to the person skilled in the art can be used as samples for the procedure of the method for identifying the N-terminal proBNP. Preferred samples are body fluids such as whole blood, blood serum, blood plasma, urine or saliva. The use of blood serum and plasma is particularly preferred. Apart from the so-called wet analyzes with the analysis reagents in a liquid phase, all the usual formats for a dry analysis suitable for the detection of antigens, haptens, peptides, proteins, antibodies, etc. can also be used. These dry analyzes or test strips described for example in EP-A-0 186 799 combine all the analysis components in a single carrier, except the sample to be analyzed. The detection reaction begins when the test strip comes into contact with the liquid sample. The method according to the invention is characterized in that the lower limit of detection for the N-terminal proBNP is less than 1 fmol / ml (corresponds to 1 pmol / 1). The high sensitivity of <1 fmol / ml according to the invention is achieved without long periods of incubation. The total period of a microtiter analysis is less than 2 hours, preferably about 15 minutes with the most sensitive detection methods such as electrochemiluminescence. An upper limit with respect to the concentration to be detected practically does not exist for this detection method. The upper technological limit is usually given by the quantification method that is used. The method mainly also detects very high concentrations of N-terminal proBNP. Another advantage of the method according to the invention is a good differentiation of the samples of patients with and without cardiac failure or by means of the quantification values obtained. The detection method is so sensitive that even a differentiation of individuals without a coronary disease and patients suffering from mild heart failure or only a mild onset of NYHA types I and II. This early establishment of the onset of heart failure can influence the decision to begin early treatment with drugs and thus clearly prolong the patient's survival rate. Another subject of the invention is the N-terminal proBNP which is produced recombinantly. The N-terminal proBNP is the N-terminal part which consists of amino acids 1-76 and which is released from the proBNP precursor molecule consisting of 108 amino acids. The N-terminal proBNP also covers parts of this that can occur in the blood due to the cleavage reactions of this molecule. No non-recombinant N-terminal proBNP is known up to now in cutting-edge technology since its production is not easily possible due to the short amino acid sequence. The chemical synthesis of a peptide of more than 30 amino acids is due to the error sequences that occur and production markedly decreased by synthetic cycle, there is no alternative compared to the recombinant production of a host organism. For a diagnostic detection method, however, a control model or material is always necessary to determine the analyte quantitatively on the one hand and to verify the functional capacity of the analysis on the other. If quantification is desired, a quantitative calibration should be carried out when using conventional series. However, this calibration is only useful in the case of the material that is used as the norm and that shows the same or a similar behavior in the immunological analysis with respect to the analyte. It is important that the standard has a sufficient structural and in particular immunological similarity to the analyte so that the agglutination of the standard to the detection antibody resembles that of the natural molecule in the sample. This conventional material for a N-terminal proBNP detection method is not provided by the cutting-edge technology. Only short synthetic peptides are described. According to the invention it is now possible for the first time to produce a DNA sequence coding for the N-terminal proBNP with the aid of a genetic synthesis and achieve a recombinant expression of the N-terminal proBNP in E. coli. Example 1 explains the only steps to follow_. Another subject of the invention is therefore the use of the recombinant N-terminal proBNP as a model in a method for identifying the N-terminal proBNP in a sample by means of at least two antibodies that recognize different epitopes of proBNP N -terminal.
For reasons of immunization, only synthetic peptides, and short N-terminal proBNP derivatives have been used in the latest technology. The disadvantage of immunizations with peptides is that most of the time only antibodies of very low affinity are obtained or the antibodies that are obtained only react with linear epitopes and the unfolded antigen can not naturally be bound in the sample (see example 3). ). Therefore it is important to use for the production of antibodies, an immunogen with a sufficient similarity to the analyte to be detected. Only in this way can it be ensured that the antibody binds with a high affinity to the natural analyte in the sample. One subject of the invention is therefore also the use of the recombinant N-terminal proBNP as an immunogen for the production of antibodies against the N-terminal proBNP. Another subject of the invention are antibodies against the recombinant N-terminal proBNP. The definition of the term antibody corresponds to the definition given in the paragraphs that concern the analysis procedure. Preferably, the antibodies according to the invention specifically identify the epitopes in the N-terminal part in the N-terminal proBNP of 76 amino acids long, preferably in the amino acid region from 10 to 66, particularly preferred in the 10 to 50 region. or 10 to 38. A useful localization of the epitopes identified by the antibodies is achieved even when the N-terminal proBNP which is at its printeolitically digested ends in the sample contains those epitopes. The stability of the analyte in the sample is thus of a more or less secondary importance. Epitopes in the preferred regions of the N-terminal proBNP can occur in linear form or as shaped epitopes. A preferred subject of the invention therefore are the monoclonal antibodies that are produced by the MAB M 10.1.1 and MAB M 13.4.14 cell lines, which are deposited and received on January 26, 1999 with the DSMZ (German collection of microorganisms and cell cultures) GmbH, Braunschweig, Germany. The antibodies that are produced by these two cell lines are IgG type antibodies. The cell lines M 10.1.11 and M 13.4.14 are also subject of the invention. Another subject of the invention are the antibodies which are like those of those cell lines M. 10.1.11 and M 13.4.14 which are produced in an equivalent manner and are suitable for binding specifically to the N-terminal proBNP. The expression "antibodies produced in an equivalent manner" means that antibodies are obtained by immunization with recombinant N-terminal proBNP A subject of the invention are also methods for the production of antibodies that bind specifically to N-terminal proBNP. production of polyclonal antibodies which is preferably carried out according to the following steps: immunization of an organism such as, for example, sheep with recombinantly produced N-terminal proBNP, isolation of the antibodies, detection of the most reactive epitopes and purification of the antibodies by immunoabsorption in the appropriate peptides This method is described in example 2.
The production of monoclonal antibodies is preferably carried out according to the following steps: immunization of a suitable organism for example mice with recombinant N-terminal proBNP produced and selection of the clones with respect to their reactivity of the antibodies with the natural N-terminal proBNP in different patient serum pools. This method is described in example 3. The invention is explained in greater detail in the following examples:
Example 1
Method for the production of recombinant N-terminal proBNP (1-76)
1. Cloning of recombinant N-terminal proBNP
The nucleotide sequence of the N-terminal proBNP (amino acid sequence 1-76) is produced by means of genetic synthesis. To obtain an optimal expression of the gene in E. coli, the DNA sequence fits the codons that are most frequently used in E. coli. The sequences of the oligonucleotides that are used for the production of the gene are the following: The production of the gene is carried out with these primers when using PCR (polymerase chain reaction). The amplified gene is cloned into a suitable vector as' e.g. vector pUC19 and then sequenced. For the cloning of the gene in the expression vector pQE8, the gene is cut from the vector pUC19 by means of the restriction cutting sequences Bam Hi and Hind III and then ligated into the vector pQE8 which allows an expression of the proteins with the marker of Histidine-N-terminal and transformed into E. coli M15 [pREP4].
2. Expression of N-terminal proBNP in E. coli
For the expression of the gene in E. coli, a culture that is left overnight in a recombinant E. coli clone is transfected 1/60 into a Luria culture broth (with
100 μg / ml ampicillin and 50 μg / ml kanamycin) and an OD 550 of 1 is induced with IPTG
(isopropylthiogalactoside, 1 mM final concentration). After induction, the cultures are incubated further for 4 hours at 37 ° C. The cultures are then centrifuged and the cell pellet is harvested in 50 mM of a Na-phosphate buffer, pH 8.0; 300 mM NaCl. After decomposition of the cell suspension by means of ultrasound, the suspension is centrifuged and the supernatant is applied on a Ni-NTA (nitrile-triacetate) column. After a washing step with 50 mM of a Na-phosphate buffer, pH 8.0; 300 mM NaCl; 20 mM imidazole, the N-terminal proBNP labeled with histidine is eluted with 50 mM Na-phosphate buffer, pH 8.0; 300 mM NaCl; 300 mM imidazole. The eluted fractions are collected and dialyzed against 50 M Tris pH 8.0. To separate the impurities, the product that was subjected to dialysis is applied to a Q-sepharose column. The mass of the purified N-terminal proBNP is determined by means of MALDI-TOF.
Example 2
Production of polyclonal antibodies against N-terminal proBNP
1. Immunization
The sheep are immunized with recombinant N-terminal proBNP (1-76) in a complete Freund's adjuvant. The dose is 0.1 mg per animal. Immunizations are repeated at 4-week intervals for a period of 10 months. 6 weeks after the first immunization and then once a month the serum samples are obtained and analyzed for their sensitivity and titration.
2. Purification of polyclonal antibodies from sheep serum
Starting with the unprocessed serum of a sheep immunized with recombinant N-terminal proBNP, the lipid components are removed by delipidation with aerosil (1.5%). After this the immunoglobulins are separated with ammonium sulfate (2M). The dissolved precipitation is subjected to dialysis against 15 mM KP04, 50 mM NaCl pH 7.0 and subjected to chromatography by means of sepharose DE7? E. The IgG fraction, P.AB < rec. NT-pro-BNP > S-IgG (DE) are found in the eluate.
3. Sequential affinity chromatography for the production of polyclonal antibodies specific for NT-pro-BNP
For the purification of polyclonal antibodies specific for NT-proBNP which are directed against amino acids 1-21, PAB < rec. NT-pro-BNP > M-IgG (IS, 1-21) The C-terminal biotinylated peptide HPLGSPGSASDLETSGLQEQR-Bi (1-21 Bi, SEQ ID NO 7) is used. The affinity matrix is produced by charging 10 ml of methacrylate polymer particles coated with streptavidin (Boehringer Mannheim, Reference 1529188) with 1 mg of peptide (1-21-Bi). With 10 ml of the affinity matrix, one column is filled and equilibrated with 50 mM KP04, 150 mM NaCl pH 7.5 (PBS). For the first step of affinity sequential chromatography 850 mg PAB < NT-pro-BNP > S-IgG (DE) are agglutinated in the column. The eluate is preserved for a second step (see below). The column is washed with PBS and 20 mM KP04, 500 mM NaCl, 0.1% Triton X-100, 0.5% Na-deoxycholic acid pH 7.5. The IgG binds specifically to the affinity matrix and is eluted with ImmunoPure © Gentle Ag / Elution buffer (Pierce, Product No. 21013). The affinity matrix is regenerated with 1M propionic acid and stored in PBS / NaN3. In the same manner described above the peptide Bi-ELQVEQTSL (Bi-30-38 SEQ ID NO 8) is used for the production of an affinity matrix for the purification of immunoglobulins specific for NT-proBNP- which are directed against amino acids 30 to 38. PAB < rec. NT-pro-BNP > M-IgG (IS, 30-38) are harvested from the eluate for the first affinity purification.
4. Biotinylation of PAB < NT-pro-BNP > S-IgG (IS, 1-21)
Purified affinity antibodies are dialyzed against a biotinylation buffer (100 mM KPO4, 70 mM NaCl pH 8.0) and thereafter the solution is adjusted to a protein concentration of 1 mg / ml. The D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester is dissolved in DMSO and added to the antibody solution in a molar ratio of 1: 7.5. The reaction is terminated by adding L-lysine and the excess of the labeling reagent is removed by dialysis.
. Digoxygenylation of PAB < NT-pro-BNP > S-IgG (IS, 30-38)
The purified affinity antibodies are dialyzed against the digoxygenylation buffer (100 mM KP04, 70 mM NaCl pH 7.6) and then the solution is adjusted to a protein concentration of 1 mg / ml. The digoxigenin-3-CME-N-hydroxysuccinimide ester is dissolved in DMSO and added to the antibody solution in a molar ratio of 1: 5. The reaction is terminated by adding L-lysine and the excess of the labeling reagent is removed by dialysis.
Example 3
Production and detection of monoclonal antibodies against N-terminal proBNP (1-76)
1. Obtaining monoclonal antibodies against N-terminal proBNP (1-76)
Balb / c mice, 8-12 weeks of age, are subjected to an intraperitoneal immunization with 100 μg of N-terminal proBNP recombinant antigen, with a complete Freund's adjuvant. After a period of 6 weeks, three more immunizations are carried out at 4 week intervals. One week after the last immunization, a blood sample is taken and the antibody titer is determined in the serum of the test animals. From the spleen of positively reacting mice, B-lymphocytes are obtained and subjected to fusion with a permanent myeloma cell line. The fusion is carried out according to the method that is well known from Kóhler and Millstein (Nature 256, 1975, p.
495-497). The primary cultures of hybrid cells that are constructed here are cloned in a usual manner, for example by using the commercially available cell sorter or by "limiting dilution." Only those cloned cultures are processed where - in a suitable assay procedure - they react positively with the recombinant N-terminal proBNP and identify the natural N-terminal proBNP in the patient's serum (see subsection 2.) By this way, various hybridoma cell lines producing the monoclonal antibodies according to the invention are collected. ascites, 5 x 106 hybridoma cells are injected intraperitoneally into Balb / c mice that have been treated 1-2 times with 0.5 ml Pristan before this.After a period of 2-3 weeks, the ascites fluid can be obtained from the abdominal region of the mice, from which the antibodies can be isolated in the usual way. Monoclonal antibodies are specifically directed against human N-terminal proBNP. In the subsequent they are called MAB M 10.1.11 or MAB M 13.4.14. Both monoclonal antibodies belong to subclass IgGl, kappa. By means of this method, both clones of the hybridoma cell lines M 10.1.11 and M 13.4.14, which are deposited with the DSMZ as mentioned above, can be isolated.
2. Detection analysis for antibodies against proBNP and recombinant NT-proBNP peptides
To identify the presence and specificity of antibodies against NT-proBNP in the serum of immunized mice, the culture supernatant of the hybrid cells or the ascites fluid, the clones are evaluated according to the following principles of analysis:
a) Reactivity with recombinant N-terminal proBNP
Microtiter plates (Nunc, Maxisorb) are agglutinated with 2.5 μg / ml of recombinant NT-proBNP as an antigen in a charge buffer
(Boehringer, 0.2 M carbonate / sodium bicarbonate, pH 9.3-9.5, Catalog No. 726 559) 100 μl / well, for one hour at room temperature under stirring. The afterload is carried out in PBS buffer (phosphate buffered saline solution), Oxid, Code-BR 14A) and 1% Byco C, for 30 minutes. Subsequently, the washing is carried out in a wash buffer (0.9 sodium chloride solution, 0.05% Tween 20). The incubation of the antibody sample is carried out with 100 μ / well for one hour at room temperature under stirring. Another washing step with a washing solution takes place twice later. After this, another incubation is carried out with the PAB antibody detection conjugate <; M-Fcy > S-Fab-peroxidase (Boehringer Mannheim, catalog No. 1500 686), 100 mU / ml, 100 μl / well, for one hour at room temperature under stirring. After another washing step with a washing buffer, the peroxidase activity is established in the usual way (for example with ABTS®, for 30 minutes at room temperature, the extinction difference is read in U at 405 nm by means of of an ELISA reader.
b) Reactivity with N-terminal proBNP peptides
In this case the streptavidin-loaded microtiter plates are bound with NT-proBNP-biotin peptide conjugates of sequences 1-10, 8-18, 1-21, 16-30, 30-38, 39-50, 50 -63 or 64-76 as an antigen, 250 ng / ml of PBS buffer (phosphate buffered saline, Oxid, Code-BR 14a) with 0.5% Byco C, 100 μl / well for one hour at room temperature under stirring. Subsequently, the washing is carried out with a washing buffer (0.9 sodium chloride solution, 0.05% Tween 20). The incubation of the antibody sample and the detection reaction are carried out as described in step a). Due to the reactivity with certain NT-proBNP peptides, the position of the epitope can be determined.
c) Reactivity with natural N-terminal proBNP in the patient sample Microtiter plates (Nunc, Maxisorb) are agglutinated with 5 μg / ml PAB < of human proBNP > S-IgG (IS, (1-21) or (30-38) S-IgG in a charge buffer (Boehringer, 0.2 M carbonate / sodium bicarbonate, pH 9.3-9.5, Catalog No. 728 559), 100 μl / well, for one hour at room temperature under agitation The afterload is carried out in the buffer PBS (phosphate buffered saline, Oxid, Code-BR 14a) and 1% Byco C, for 30 minutes. Washing is carried out with a wash buffer (0.9 sodium chloride solution, 0.05% Tween 20) .Incubation with the natural antigen in the patient's plasma, diluted in PBS buffer, is carried out with 100 μl / well for one hour at room temperature under stirring After another washing step, the incubation of the antibody sample is carried out with 100 μl / well for one hour at room temperature while stirring. times with a wash solution and another incubation more with the antibody conjugate of detecc PAB ion < M. Fcy > S-Fab-peroxidase (Boehringer Mannheim GmbH, catalog No. 1500 686), 100 mU / ml, 100 μl / well, for one hour at room temperature under stirring. After another washing step with the washing buffer, the peroxidase activity is established in the usual way (for example with .ABTS ®, for 30 minutes at room temperature, the difference of extinction is read in mU at 405 nm by medium of an ELISA reader).
3. Results: Monoclonal and polyclonal antibody reaction pattern against proBNP N-ter inal
a) MAB reactivity (c = 5 μg / ml) of the immunization with the N-terminal proBNP peptides:
Table 1
The monoclonal antibodies that are obtained from immunizations with different peptides react strongly with the corresponding peptides. The reactivity with the recombinant N-terminal proBNP can only be recognized with two monoclonal antibodies while no reaction occurs with the natural N-terminal proBNP in a group of patients (see table 1).
b) Reactivity of monoclonal antibodies (MAB) from immunization with recombinant N-terminal proBNP:
Table 2
The monoclonal antibodies obtained from immunization with recombinant N-terminal proBNP only partially react with the peptides, but very strongly with the recombinant N-terminal proBNP or with the native N-terminal proBNP in a pool of patients. The non-reaction of the only monoclonal antibodies with the peptides indicates the identification of the so-called shaped epitopes (see table 2).
c) Reactivity of P.AB from immunization with N-terminal proBNP:
Table 3:
The PAB that is obtained shows the strongest reaction with the peptides 1-21 and 30-38. For this reason these epitopes are selected and the PAB is positively immunosorbed with the help of these peptides. The PAB immunoabsorbed with peptide 1-21 shows the strongest reaction with the 8-20 region and a clearly reduced reaction with the N-terminal sequence 1-10. The PABs immunoabsorbed in this way react very strongly with the recombinant N-terminal proBNP and in the format between two layers of PAB / PAB with the natural sample (see table 3).
Example 4
Highly sensitive immunoassay for the determination of NT-proBNP
By using the antibodies that are produced in Example 2 and 3, a highly sensitive immuno-lactation can be structured. In general, all test formats apply two antibodies with different recognition for epitopes and are suitable. As an example, the so-called ELISA between two layers is described. As a solid phase, a microtiter plate (MTP) coated with streptavidin is used. 10 μl of an untreated sample or calibrator is agitated with the pipette together with 100 μl of buffer containing both epitope-specific antibodies in the MTP wells and then incubated for one hour at room temperature. As an antibody, 1 μg / ml of PAB < rec.NT-proBNP > S-IgG (IS. 1-21) biotinylated and 0.5 μl digoxigenylated PAB < rec.NT-proBNP > S-IgG (IS, 30-38) is used. After this, the solution is extracted and washed three times with 350 μl of wash buffer. After this, 100 μl of the conjugate solution is added via a pipette and incubated again for one hour at room temperature. In the manner of a conjugate, an anti-digoxin-antibody-POD conjugate with a concentration of 100 mlU / ml is used. The conjugate solution is then extracted and washed three times with 350 μl of wash buffer. At the end of this, an ABTS® substrate solution is poured into the wells and quantified for 30 minutes at room temperature. After reaching the substrate reaction at 30 minutes, the microtitre plate is quantified directly in an MTP reader at a wavelength of 405 nm - and against the reference wavelength of 495 nm. To determine the sensitivity, a calibration curve is established and the precision of the zero standard (n = 21) is determined. As a calibrator, human plasma is used with EDTA which is then structured with the recombinant N-terminal proBNP at the concentration that is required. Bovine plasma is used as the zero standard. The results are shown in Table 4.
Table 4:
Extinction (mean) Standard deviation (n = 21)
Calibrator at 0 131 mU 5.7 mU fmol / ml Calibrator b: 5.04 268 mU fmol / ml Calibrator c: 19.9 746 mU fmol / ml Calibrator d: 50.5 1500 mU fmol / ml Calibrator e: 100.9 2401 mU fmol / ml
Based on the gradient of the calibration curve of 22.5 mU x ml / fmol and an SD of 5.7 mU, the following lower detection limit is given according to the Kaiser formula:
LDL = 3 SD standard zero / Ce gradient = 3 x 5.7 / 22.5 = 0.76 fmol / ml.
Example 5
Determination of the stability of the N-terminal proBNP sample
With the help of a two-layer ELISA described in Example 4, the analyte stability of the N-terminal proBNP is quantified. For this, blood of four NYHA type II-III patients is drawn into collecting tubes containing EDTA and preserved at room temperature for 3 days. Each day a sample is taken and the concentration of the N-terminal proBNP is quantified. The reference sample as well as the samples for the determination of stability in plasma with EDTA, are directly cooled to 4 ° C-8 ° C and centrifuged for a period of 15 minutes. The plasmas with EDTA, are preserved at 4 ° C and at room temperature and then quantify at different time intervals within a duration of fatigue of 24 hours. The results are shown in Table 5.
Table 5:
These data prove that the N-terminal proBNP is completely stable within the time limits analyzed and can therefore be used as a routine parameter. This result is inconsistent with the literature (Hunt et al., Clinical Endocrinology, 47, 287 (1997)) and confirms what is believed that by selecting and designing this analysis format with 2 specific antibodies, the epitopes of these antibodies do not they are at the outer end of the analyte, and the stability of the analyte can be influenced.
Example 6
Determination of the diagnostic sensitivity of the -valuation of N-terminal proBNP For the determination of the diagnostic sensitivity of the analysis described in example 4, it is used again. For this, 114 healthy individuals and 235 patients of the NYHA classification between groups I and IV are quantified. Normally, it is particularly critical to differentiate healthy individuals from NYHA type I patients. With this highly sensitive titration, an average value of 6. 6 fmol / ml NR-proBNP with a standard deviation of 7.3 fmol / ml is quantified at 110 healthy blood donors. The lowest level quantified is 0.2 fmol / ml. This clearly shows that a sensitivity of < 1.0 fmol / ml is necessary to accurately detect the reference region. With this distribution, the upper region of the normal value (97.5% percent) that is determined is 26.6 fmol / ml. Assuming a reference region of 0-26.6 fmol / ml, only 16 patients of the 233 NYHA patients type I-IV show a value in the conventional region. This corresponds to the clinical sensitivity of 93.3%. If only patients with NYHA type I are considered as 30 of 37 patients that are detected as positive which corresponds to a sensitivity of 81.1%. This result confirms that by highly sensitive assessment for the N-terminal proBNP, a clear differentiation between patients suffering from NYHA type I with heart failure and a normal healthy collective is possible. With the latest technology assessments (Dagubatti et al., Cardiovascular Research 36 (1997), 246) available to date, this could not be achieved. It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the model for the manufacture of the objects or products to which it refers.
- LIST OF SEQUENCES < 110 > Diagnostics Roche GmbH < 120 > Method to identify proBt.'P ..- terminal < 130 > 51810A O-SZ < 140 > < 141 > < 160 > 8 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 17 < 212 > DNA < 213 > E. coli < 400 > 1 ccggatccca cccg'ctg 17
< 210 > 2 < 211 > 79 < 212 > ? DN < 213 > E. coli • < 400 > 2 cgggatccca cccgctgggt tccccgggtt ccgcttccga cctggaaacc tccggtctgc 60 aggaacagcg taaccacct 79
< 210 > 3 < 211 > 70 < 212 > DNA < 213 > E. coli < 400 > 3 cggttccagg gaggtctgtt caacctgcag ttcggacagt ttaccctgca ggtggttacg 60 ctgttcctgc 70
< 210 > 4 < 211 > 71 < 212 > DNA < 213 > E. coli < 400 > 4 cagacctccc tggaaccgct gcaggaatcc ccgcgtccga ccggtgtttg gaaatcccgt 60 gaagttgcta c 71
< 210 > 5 < 211 > 87 < 212 > DNA < 213 > E. coli < 400 > 5 cccaagctta acgcggagca cgcagggtgt acagaaccat tttacggtga ccacggatac 60 cttcggtagc aacttcacgg gatttcc 87
< 210 > 6 < 211 > 19 < 212 > DNA < 213 > E. coli '. "...
< 400 > 6 cccaagctta acgcggagc 19
< 210 > 7 < 21 l > 21 < 212 > PRT < 213 > E. coli < 400 > 7 His Pro Leu Gly Se r Pjro Gly S * r Wing Being Asp Leu Glu Tnr Ser Gl and 1 5 10 15 Leu din Olu ßln Arg 20 ''
< 210 > 8 < 211 > 9 < 212 > PRT < 213 > E. coli < 400 > 8 Glu Leu Gln Val Glu Gln Thr Ser Leu 1 5
Claims (19)
1. A method for identifying N-terminal proBNP in a sample, characterized in that at least two antibodies that detect different N-terminal proBNP epitopes are used.
2. The method of compliance in claim 1, characterized in that the antibodies can agglutinate simultaneously to the N-terminal proBNP.
3. The method of compliance in claims 1 or 2, characterized in that the method is carried out heterogeneously.
4. The method of compliance in claim 3, characterized in that the method is carried out in the form of two layers.
5. The method according to any one of the preceding claims, characterized in that the lower limit of detection for the N-terminal proBNP is below 1 fmol / ml.
6. The method according to any of the preceding claims, characterized in that by means of the values obtained, a differentiation of the samples taken from healthy patients and patients with heart failure or NYHA-types I to IV can be done .
7. The method according to claim 6, characterized in that by means of the values obtained, a differentiation of the samples taken from healthy patients and patients with NYHA-type I can be made.
8. The use of a method according to any of the aforementioned claims, characterized in that it is for the differentiation between samples that are made from healthy patients and patients with heart failure or NYHA-types I to IV.
9. The recombinant N-terminal proBNP.
10. The use of the recombinant N-terminal proBNP characterized in that it is a model in a method for identifying the N-terminal proBNP according to claims 1 to 7.
11. The use of recombinant N-terminal proBNP characterized in that it is for the production of antibodies against N-terminal proBNP.
12. Antibodies against the recombinant N-terminal proBNP.
13. The antibodies as claimed in claim 12, characterized in that they bind specifically in the range of amino acids 10 to 66 of the N-terminal proBNP.
14. The antibodies as claimed in claim 12 or 13, characterized in that they are obtained by immunization of a suitable organism with the recombinantly produced N-terminal proBNP.
15. The antibodies as claimed in claims 12 to 14, characterized in that they are obtained from the cell lines M 10.1.11 (DSM ACC 2386) or M 13.4.14. (DSM ACC 2387) deposited with the DSMZ on 226.01.1999.
16. The antibodies as claimed in claim 15, characterized in that they are produced in an equivalent manner with the N-terminal proBNP in comparison to those that are produced from the cell lines M 10.1.11 (DSM ACC 2386) or M 13.4. 14 (DSM ACC 2387).
17. The cell lines M 10.1.11 (DSM ACC 2386) or M 13.4.14 (DSM ACC 2387) deposited with the DSMZ on 26.01.1999.
18. A method for the production of polyclonal antibodies as claimed in claims 12 to 14 or 16, characterized in that it contains the steps of immunization of a suitable organism with the recombinantly produced N-terminal proBNP, the isolation of antibodies, the detection of the majority of the reactive epitopes and the purification of the antibodies by immunoabsorption with the appropriate peptides.
19. The method for the production of monoclonal antibodies, as claimed in claims 12 to 16, characterized in that it contains the steps of immunization of a suitable organism with the N-terminal proBNP that is produced recombinantly and the selection of the clones with respect to the reactivity of antibodies with the natural N-terminal proBNP in different groupings of patient sera. METHOD TO IDENTIFY N-TERMINAL proBNP \ SUMMARY OF THE INVENTION The invention relates to a method for identifying the N-terminal proBNP of a sample with at least two antibodies that detect different epitopes of the N-terminal proBNP. The method is used to differentiate or classify samples from healthy individuals and samples from NYHA patients types I to IV. The invention also relates to the N-terminal proBNP Recombinant, its use as a model in a method to identify the N-terminal proBNP, to antibodies that detect the recombinant N-terminal proBNP and to its production.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE19903489.3 | 1999-01-29 | ||
DE19911044.1 | 1999-03-12 |
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