Patent Application: US-58874605-A

Abstract:
the invention relates to an in vitro method for detecting the formation of endothelins during serious illnesses , especially cardiovascular diseases , inflammations , sepsis and cancer , in whole blood , plasma or serum of a human patient for medical diagnosis . using this method , relatively long - lasting peptide fragments , especially a c - terminal peptide fragment , of the processed primary prepro - or proendothelins that contain neither the actual biologically active endothelin nor its direct precursor , big endothelin , can be detected .

Description:
the method according to the invention relates in its most general aspect to the determination of a relatively long - lived peptide fragment of proendothelin - 1 which does not contain the amino acid sequences of endothelin - 1 or its precursor big endothelin , in whole blood , plasma or serum samples , i . e . in the circulation of patients , for the indirect determination of the formation of endothelins , in particular of endothelin - 1 , in serious diseases . according to a preferred embodiment , the peptide fragment determined is a c - terminal fragment to which two antibodies bind which bind to peptides having amino acid sequences which correspond to the positions 168 - 181 and 200 - 212 of preproendothelin - 1 . for the practical implementation of the invention , noncompetitive sandwich assays , for example of the type as used for the more far - reaching detailed investigations and described more exactly below , are particularly preferably provided . compared with competitive immunoassays , noncompetitive sandwich immunoassays ( two - sided immunoassays ) have a number of advantages , which include the fact that they can be better designed than solid - phase assays ( heterogeneous assays ), may be more rugged in terms of handling , can give measured results with a higher sensitivity and are also more suitable for automation and series measurement . moreover , they can also provide additional information compared with competitive immunoassays which operate with only one type of antibody , in that sandwich immunoassays recognize only those molecules or peptides with which both binding sites for the antibodies used in the sandwich formation are present on the same molecule . the antibodies which may be used may in principle be any desired suitable monoclonal and / or polyclonal antibodies , but affinity - purified polyclonal antibodies are currently preferred . particularly preferably , the antibodies are obtained by immunization of an animal , in particular of a sheep , with an antigen which contains a synthetic peptide sequence which corresponds to a short amino acid sequence of preproendothelin - 1 and an additional cysteine residue at the n - terminus . in the following experimental section , in particular antibodies which bind to the amino acid sequences 161 - 181 and 200 - 212 , and their use in an assay are described . however , in the course of the investigations , additional antibodies which accordingly bind to the positions 184 - 203 and 136 - 148 were also used . the additional results obtained with these further antibodies in measurements are discussed only globally in this application . in a preferred embodiment , the method is carried out as a heterogeneous sandwich immunoassay , in which one of the antibodies is immobilized on any desired solid phase , for example the walls of coated test tubes ( e . g . of polystyrene ; “ coated tubes ”; ct ) or on microtiter plates , for example of polystyrene , or on particles , for example magnetic particles , while the other antibody carries a residue which represents a directly detectable label or permits selective linkage to a label and serves for detecting the sandwich structures formed . delayed or subsequent immobilization with the use of suitable solid phases is also possible . in principle , all marking techniques which can be used in assays of the type described may be employed , including marking with radioisotopes , enzymes , fluorescent , chemiluminescent or bioluminescent labels and directly optically detectable color markers , such as , for example , gold atoms and dye particles , as are used , in particular for so - called point - of - care ( poc ) or accelerated tests for determination in whole blood samples . in the case of heterogeneous sandwich immunoassays , the two antibodies may also have parts of a detection system of the type described below in relation to homogeneous assays . it is therefore within the scope of the present invention also to design the method according to the invention as an accelerated test . the method according to the invention can furthermore be designed as a homogeneous method in which the sandwich complexes formed from the two antibodies and the peptide fragment to be detected remain suspended in the liquid phase . in such a case , it is preferable to mark both antibodies with parts of a detection system which permits signal generation or signal triggering when both antibodies are integrated into a single sandwich . such techniques can be designed in particular as fluorescence amplification or fluorescence extinction assays . a particularly preferred method of this type relates to the use of detection reagents to be used in pairs , as described , for example , in u . s . pat . no . 4 , 822 , 733 , ep - b1 - 180 492 or ep - b1 - 539 477 and the prior art cited therein . they permit a measurement which selectively detects only reaction products which contain both marking components in a single immune complex , directly in the reaction mixture . the technology which is available under the brands trace ® ( time resolved amplified cryptate emission ) and kryptor ® and which implements the teachings of the above - mentioned application may be referred to as an example . in the investigations by the applicant , it was found that the determination , according to the invention , of the c - terminal peptide fragment of preproendothelin - 1 gives highly interesting and relevant results . as will be shown below , this statement applies not only to the sepsis diagnosis but also to cardiac diagnosis and cancer diagnosis . it is furthermore assumed that the assays according to the invention can also be particularly advantageously carried out as part of a so - called multiparameter diagnosis , in particular both in the area of cardiac diagnosis and in sepsis and cancer diagnosis . further parameters determined thereby are , for example , the cardiac parameters anp , bnp , proanp , proadm and probnp or sepsis parameters which are selected , for example , from the group which consists of anti - ganglioside antibodies , the proteins procalcitonin , ca 125 , ca 19 - 9 , s100b , s100a proteins , lasp - 1 , soluble cytokeratin fragments , in particular cyfra 21 , tps and / or soluble cytokeratin - 1 fragments ( scy1f ), the peptides inflammin and chp , other peptide prohormones , glycine - n - acyltransferase ( gnat ), carbamoylphosphate synthetase 1 ( cps 1 ) and c - reactive protein ( crp ) or fragments thereof . in said multiparameter assays , it is intended to determine the measured results for a plurality of parameters simultaneously or in parallel and to evaluate them , for example , with the aid of a computer program which also uses diagnostically significant parameter correlations . the invention is explained in more detail below by a description of the preparation of the preferred assay components , the procedure of a preferred embodiment of an assay of the sandwich type and the results of the determination of a c - terminal peptide fragment in edta plasmas of control persons and of sepsis , cardiac and cancer patients , obtained with the use of such an assay . derived from the known amino acid sequence of human preproendothelin - 1 ( seq id no : 1 ), three ranges were selected ( pos . 168 - 181 , 184 - 203 , 200 - 212 ). in each case supplemented by an n - terminal cysteine residue , these ranges were chemically synthesized as soluble peptides by standard methods , purified , quality - controlled by means of mass spectrometry and reversed phase hplc and lyophilized in aliquots ( jerini ag , berlin , germany ). the amino acid sequences of the peptides are : furthermore , the following peptide was synthesized as a standard for calibrating the assays : the peptides pct15 and pcw14 were conjugated with the carrier protein klh ( keyhole limpet hemocyanine ) by means of mbs ( m - maleimidobenzoyl - n - hydroxysuccinimide ester ) ( cf . operating instructions “ nhs - esters - maleimide crosslinkers ”, from pierce , rockford , ill ., usa ). sheep were immunized with these conjugates according to the following scheme : each sheep initially received 100 μg of conjugate ( stated mass based on the peptide fraction of the conjugate ) and then 50 μg portions of conjugate every 4 weeks ( stated mass based on the peptide fraction of the conjugate ). beginning with the fourth month after beginning of the immunization , 700 ml of blood per sheep were taken every 4 weeks and antiserum was obtained therefrom by centrifuging . conjugations , immunizations and recovery of antisera were carried out by micropharm , carmarthenshire , uk . the peptide - specific antibodies were prepared in a one - step method from the antisera which had been recovered beginning with the fourth month after immunization . for this purpose , the peptides pct15 and pcw14 were first coupled to sulfolink gel ( cf . operating instruction “ sulfolink kit ”, from pierce , rockford , ill ., usa ). in each case 5 mg of peptide per 5 ml of gel were offered for coupling . the affinity purification of peptide - specific antibodies from sheep antisera against both peptides was carried out as follows : the peptide columns were first washed three times alternately with 10 ml each of elution buffer ( 50 mm citric acid , ph 2 . 2 ) and binding buffer ( 100 mm sodium phosphate , 0 . 1 % tween , ph 6 . 8 ). 100 ml of the antisera were filtered with 0 . 2 μm , and the column material present was added . for this purpose , the gel was quantitatively rinsed from the column with 10 ml of binding buffer . the incubation was effected overnight at room temperature with swirling . the batches were transferred quantitatively into empty columns ( nap 25 , pharmacia , emptied ). the runnings were discarded . the columns were then washed protein - free with 250 ml of binding buffer ( protein content of the wash eluate & lt ; 0 . 02 a280 nm ). elution buffer was added to the washed columns , and 1 ml fractions were collected . the protein content of each fraction was determined by means of the bca method ( cf . operating instructions of pierce , rockford , ill ., usa ). fractions having protein concentrations & gt ; 0 . 8 mg / ml were pooled . after protein determination of the pools by means of the bca method , yields of 97 mg for the anti - pct15 antibody 0407 - pak and 60 mg for the anti - pcw14 0410 - pak antibody were obtained . 500 μl of the purified antibody were rebuffered in 1 ml of 100 mm potassium phosphate buffer ( ph 8 . 0 ) according to the operating instructions over an nap - 5 gel filtration column ( pharmacia ). the protein concentration determination of the antibody solution gave a value of 1 . 5 mg / ml . for chemiluminescence marking of the antibody , 10 μl of ma70 acridinium - nhs - ester ( 1 mg / ml ; from hoechst behring ) were added to 67 μl of the antibody solution and incubated for 15 minutes at room temperature . thereafter , 423 μl of 1 m glycine were added and incubation was effected for a further 10 minutes . thereafter , the marking batch was rebuffered according to operating instructions over an nap - 5 gel filtration column ( pharmacia ) in 1 ml of mobile phase a ( 50 mm potassium phosphate , 100 mm nacl , ph 7 . 4 ) and freed from low molecular weight constituents . a gel filtration hplc was carried out for separating off final residues of labels not bound to antibodies ( column : waters protein pak sw300 ). the sample was applied and was chromatographed at a flow rate of 1 ml / min with mobile phase a . the wavelengths 280 nm and 368 nm were measured using a flow photometer . the absorption ratio 368 nm / 280 nm as a measure of the degree of marking of the antibody was 0 . 10 at the peak . the fractions containing monomeric antibodies ( retention time 8 - 10 min ) were collected , and were collected in 3 ml of 100 mm sodium phosphate , 150 mm nacl , 5 % bovine serum albumin , 0 . 1 % sodium azide , ph 7 . 4 . irradiated 5 ml polystyrene tubes ( from greiner ) were coated with purified antibody as follows : the antibody was diluted to a concentration of 6 . 6 μg / ml in 50 mm tris , 100 mm nacl , ph 7 . 8 . 300 μl of this solution were pipetted into each tube . the tubes were incubated for 20 hours at 22 ° c . the solution was filtered with suction . each tube was then filled with 4 . 2 ml of 10 mm sodium phosphate , 2 % karion fp , 0 . 3 % bovine serum albumin , ph 6 . 5 . after 20 hours , the solution was filtered with suction . finally , the tubes were dried in a vacuum drier . an assay buffer of the following composition was prepared : 100 mm sodium phosphate , 150 mm nacl , 5 % bovine serum albumin ( bsa ), 0 . 1 % unspecified sheep igg , 0 . 1 % sodium azide , ph 7 . 4 the above - mentioned chemically synthesized peptide ( peptide psw44 ) which corresponds to the positions 169 - 212 of preproendothelin - 1 serves as standard material . this was serially diluted in horse normal serum ( from sigma ). concentrations according to the weight of the peptide taken were ascribed to the standards thus prepared . measuring samples were edta plasmas of apparently healthy persons , of patients with sepsis and of patients with various cardiovascular diseases . 50 μl of standards or samples and 200 μl of assay buffer were pipetted into the test tubes . incubation was effected for two hours at 22 ° c . with shaking . thereafter , washing was effected 4 times with 1 ml of wash solution ( 0 . 1 % tween 20 ) each time per tube and the latter were allowed to drip off . 200 μl of assay buffer , containing 1 million rlu ( relative light units ) of the ma70 - marked antibody , were then pipetted . incubation was effected for two hours at 22 ° c . with shaking . thereafter , washing was effected 4 times with 1 ml of wash solution ( 0 . 1 % tween 20 ) each time per tube , the latter were allowed to drip off and the chemiluminescence bound to the tube was measured in a luminometer ( from berthold , lb952t ; base reagents brahms ag ). using the multicalc software ( spline fit ), the concentrations of the samples were read from the standard curve . the analyte measurable using the sandwich immunoassay developed ( antibody against the positions 168 - 181 and 200 - 212 ) is referred to below as c - terminal proendothelin or ct - proendothelin . a typical standard curve for the test developed is shown in fig1 . by means of the test , it is also possible to determine ct - proendothelin concentrations substantially below 50 pg / ml . in order to examine the question as to whether problems were to be expected in a measurement of the c - terminal peptide fragment owing to insufficient stability in a sample or measuring solution , 5 sepsis plasmas were measured in each case fresh and after storage for 12 hours at room temperature . the results are summarized in fig2 . they show that , after storage for 12 days , the immunoreactivity was virtually unchanged at about 93 % of the initially measured immunoreactivity . this stability detected is a major advantage for diagnostics from points of view relating to handling . by means of the test , plasmas of cardiological and sepsis patients were measured . the results obtained are shown in fig3 a and 3 b . for all cardiological clinical pictures investigated , increased values were found compared with normal controls . increased values were also found for patients with sirs ( systemic inflammatory response syndrome ) and septic conditions . the diagnostic sensitivity ( at given 100 % specificity , based on healthy controls ) increased with the severity of the disease : sepsis 32 . 3 %, severe sepsis 65 . 5 % and septic shock 75 %. when the samples were measured using a modified assay in which one of the antibodies of the above - mentioned sandwich assay was replaced by an antibody which detected the amino acids 184 - 203 of preproendothelin - 1 , substantially identical results were obtained , as expected . on the other hand , when one of the antibodies used recognized an amino acid sequence which is localized more closely to the n - terminus of the preproendothelin ( 32 - 52 or 136 - 148 ), it was not possible to obtain measured values raised compared with healthy persons . this indicates that proendothelin as such was not present in the plasma samples measured and is not proteolytically processed only with formation of big endothelin , but that the c - terminal sequence 93 - 212 liberated is also further cleaved , it being necessary for at least one such cleavage point to be present in the range of the amino acids 149 - 167 . the statement applies to the plasmas of patients with the diseases investigated . however , it cannot be ruled out that , for example , the entire c - terminal fragment 93 - 212 is retained in other patient groups and its selective measurement can deliver diagnostically relevant results . 1 . agapitov a v , haynes w g . role of endothelin in cardiovascular disease . j renin angiotensin aldosterone syst 2002 ; 3 : 1 - 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