METHOD AND MEANS FOR DIAGNOSTIC DETECTION OF ELASTASE DERIVED B beta -FIBRINOPEPTIDES

Abstract: Method for the in vitro diagnostication of diseases associated with fibrin(ogen)olysis and/or pathological elastase activity, characterized in that a sample taken from a patient and containing fibrinopeptides is subjected to quantification of at least one elastase induced B beta 14(+/-2) - 45(+/-3) fibrinopeptide(s). The value thus obtained is compared with a normal value for healthy individuals, and that if the value obtained is higher than said normal value the patient from whom the sample has been taken is classed as being diseased. The assay method provides a way of demonstrating the presence of free elastase activity in samples taken from patients. Furthermore, novel antibody preparations as well as novel elastase induced B beta 14(+/-2) - 45(+/-3) fibrinopeptide preparations are described which may be employed in the said method.

Description: Method and means for diagnostic detection of elastase derived B β-fibrinopeptides. The invention is concerned with a method for the diagnosis of diseases associated with fibrin (ogen) olysis where samples taken from patients are subjected to quantification of certain peptide fragments therein which have been formed due to fibrin (ogen) olysis. The invention is useful as a prognostic in cases of disease involving fibrin formation and elastase digestion of fibrin, and provides possibilities, for the first time, of diagnosticating results of free elastase activity in vivo. Fibrinogen is composed of six peptide chains matching each other pairwise and called At , &A and y chains. They are held together by disulfide bonds. When fibrin is formed A and B fragments are cleaved off initially, and consequently fibrin contains o , Δ and ~& chains. For a general survey see Blomback, B (1) . Acute pulmonary insufficiency following after trauma and sepsis (ARDS) is a frequently occurring, serious condition. This syndrome is found in 150 000 patients/year in the U.S. alone, resulting in the death of half of the patients. A number of investigations have shown that this condition is associated with a considerable accumulation of fibrin in the lungs and that in some manner, hitherto unelucidated, this fibrin has a role in the pathogenesis, Elastase too is found to be abundant in the lungs. According to one hypothesis therefore, some peptide (s) released by the action of elastase on fibrin may be involved in the pathogenesis. There has thus been a demand, in numerous fields of activities, that methods should be made available to provide specific procedures by which elastase induced fibrinopeptides are quantified in the plasma in cases of ARDS, sepsis, pulmonary embolism, cancer of the lungs, leukemia, renal diseases, rheumatoid arthritis and other pathologic conditions. It has been reported earlier that major fragments obtained in vitro from fibrin(ogen) with elastase from leucocytes differ markedly from those obtained with plasmin (2, 3, 4) . We ourselves conjointly with various collaborators have previously isolated and studied a vasoactive peptide which is formed upon the in vitro digestion of fibrin with elastase. This peptide corresponds to amino acids 30-43 in the B chain of fibrinogen (5, 6 , 7, 8, 9) . In the blood from a leukemia patient Sterrenberg et al. have demonstrated - in a manner that is not convincing in our opinion - the presence of elastase activity and fibrinogen degradation products dissimilar from those obtained with plasmin (10, 11) . With only a few exceptions, no methods for assaying fibrino¬ peptides in plasma have been published up to now. Fibrinopeptides A and B are cleaved off when thrombin reacts with fibrinogen. Methods for determining these fibrino¬ peptides have been described (12, 13) . The occurrence of these fibrinopeptides is indicative of the amount of blood coagulation that may have occurred in the patient. A method for assaying a fragment B& 15-42 released from fibrin through the action of plasmin has been described earlier (14) . Drawbacks of these methods are that the antibodies employed cross react with several other peptides and also with fibrinogen, and that plasma samples have to be precipi¬ tated with ethanol prior to analysis. Recently a method has been described which will selectively measure a peptide B^ » 1-42 -cleaved off by plasmin-fibrinogen interaction (15) . Drawbacks of this method are that it fails to detect peptides released from fibrin, that the antibody cross reacts with fibrinogen, and that the samples have to be precipitated with ethanol prior to analysis. There are also other publications that have been considered to be within the field of the present invention (22-26) . Plow et al (23) has indicated that elastase degradation of fibrinogen results in the expression of epitopes that are exclusive. Also according to a recent report (16) there is a method designed for measuring a peptide that corresponds to Aαl 1-21 and is obtained upon degradation of fibrinogen with elastase in vivo. Elevated levels have been correlated with deficiency of oζ. proteinase inhibitor. The method such as presented is complicated and relies on HPLC. The peptide detected by this method can be released only from fibrinogen and not from fibrin. The method developed by us is highly specific and involves very little cross reaction v/ith other related peptides and fibrinogen. What is measured is essentially only fibrin (ogen) degradation with elastase and not degradation with plasmin, trypsin, chymotrypsin, collagenase or thrombin. Numerous peptide fragments can be formed theoretically when elastase is made to act on fibrin/fibrinogen. For this reason it is very difficult to predict the kind of fragments that might be detected in samples from patients. The exper- imentals below will demonstrate that elastase is capable of contributing to in vivo fibrin (ogen) olysis and that occurrence of unique fragments from the B^> chain of fibrin (ogen) can be detected in blood and correlated with certain pathologic conditions. It is the first time that this has been done. In the context of the present invention, fibrin/fibrinogen fragments which are specific for elastase digestion and originate from the fibrin (ogen) B/ι chain will be called "elastase induced B*> fibrinopeptides". According to one aspect the invention a sample taken from a patient is subjected to quantification of at least one elastase induced Ba fibrinopeptide. The value thus obtained is compared with a normal value, and if the value obtained is higher than said normal value the patient from whom the sample has been taken is classed as being diseased. The total number of pathologic conditions that may thus be indicated has not yet been worked out completely. Elevated values are pathological and are interpreted as being an indication of elastase activity in the patient. The term "normal value" refers to the value obtained in the case of healthy persons by means of measuring methods equal to or other than the method used for the value obtained. If the value obtained and the normal value have been measured by means of methods differing inter se then if necessary the values have to be converted to values of equal scale (magni¬ tude) . Our invention is based on our surprising discovery that the known BΔ 30-43 peptide appears to be unstable in vivo whereas a longer fibrinogen fragment immunologically similar to the Bβ> 30-43 peptide is more stable. This fibrinogen fragment therefore can be demonstrated in much higher levels than the Ε>£> 30-43 peptide. Thus, we have been able in plasma samples to successfully quantify a peptide (peptides) exposing at least one epitope which said peptide(s) have in common with the B^ 30-43 peptide. Elevated levels have been correlated with a number of pathologic conditions. This B /__ 30-43 type of epitope has not been demonstrable (cross- reactivity <5 %) in fragments B 1-42, Bf> 15-42, B^ 1-118, fibrinogen and B 30-43 NH (where "NH " indicates that the carboxy-terminal and is amidated) under conditions as set forth in the cross reactivity section of the experimental part of this specification. During the priority year data from a ino acid analysis have indicated that the major peptide(s) measured should consist of 33+4 amino acids. In terms of BA positions the terminals should be 12, 13, 14, 15 or 16 (amino terminal) respective 42, 43, 44, 45, 46, 47 or 48 (carboxy terminal) . The most probable candidates are BΛ 13-43 and B 14-46. Because the fragment(s) measured is (are) related antigeni- σally and demonstrable after elastase digestion of fibrin- (ogen) and apparently consist (consists) of 33+4 amino acids, peptides exposing the unique epitopes of this (these) fragment(s) will be called "elastase induced Bk 33+4 fibrinopeptides". Bk 30-43 is an example of this type of fibrinopeptides. Thus, the characteristic feature of the method of the invention resides in quantifying elastase induced Bft 33+4 fibrinopeptides associated with pathologic disorders. One aspect of the invention resides in an antibody preparation having the characteristic feature of being directed exclusively against one or more epitopes which are unique to one or more elastase induced B & fibrinopeptides. The expression "directed , exclusively" means that the preparation will not, in any way that is harmful in the context of the present invention, react with other substances contained in the type of patients' samples as here under consideration, such other substances being for instance other fibrinopeptides. The exclusiveness of the epitopes relates to the situation in vivo; it is not impossible that the epitope may be found to be present also on elastase induced Bβ fibrinopeptides produced in vitro. Examples of epitopes against which the preparation may be directed are such epitopes that are exposed and unique in the elastase induced B & fibrinopeptides of 33+4 amino acids length or in the B (3 30-43 fibrinopeptide. In the context of the antibody nomenclature used in this specification, X or its substitute in the expression "anti-X antibody" will refer to antibody specificity and not to the i munogen employed for raising the immune response. In the antibody preparation of the invention, the antibody- active components may be in a derivatized or fragmented form. Various chemicals may be added as required for certain uses (e.g. preservatives, salt, buffer etc) . The preparation may be freeze-dried. The essential feature of the antibody preparation of this invention does not reside in any such additives or modifications of the antibodies contained in the preparation - rather, the essential feature is the unique specificity of the preparation. Examples of various derivatized forms of antibodies are the following: antibody- active components bound to phases which are insoluble in aqueous media (covalently or adsorptively bound forms of antibodies) ; antibodies provided with one or more of such analytically detectable groups as are well known within the field of immunochemical techniques (radioactive, enzymatically active, fluorogenic (fluorescent) , σhemiluminogenic (chemi- luminescent) r biotinyl etc group etc) . As examples of fragmented antibody-active components may be mentioned the well-known fragments Fab, Fab' and F(ab')^ which may also be derivatized as stated above. As regards cross reactivity of an anti- (BA 30-43) antibody preparation with respect to fibrinopeptides formed on digestion of fibrin(ogen) with enzymes other than elastase, and with respect to chemical degradation products , the following values will apply (based on equimolar biological concentration of cross reacting substance and B b 30-43 peptide) BP> 1-42, <5 % Bb 15-42, <5 % fibrinogen, <0,005 % These values apply to the conditions and methods of calculation as set forth in the experimental part of this specification. An antibody preparation according to the invention is prepared in that cells potentially capable of producing antibodies having a specificity as according to this invention are caused to secrete said antibodies, whereupon these antibodies (i) are selected and optionally isolated and purified, and (ii) if required are subjected in a manner know per se to derivatization and/or fragmentation. In a vertebrate (e.g. mammal such as mouse or rat) secretion may take place in vivo following immunization with a suitable immunogen, for example an elastase induced B$ 30-43 fibrino- peptide conjugated to an immunogenic carrier. In a similar way the elastase induced B fibrinopeptides of 33+4 amino acids length may also be used. The immune response thus obtained is polyclonal, meaning that the desired antibody/ antibodies will be found in admixture with antibodies directed against other determinants of the immunogen. For selection purposes and removal of said last-mentioned antibodies recourse may be had to so-called immunosorbent purification (IS purification) , for example on solid-phase- bound forms of the aforesaid elastase induced fibrinopeptides. Depending on the immunosorbent employed, the antibodies desired will be found either in the eluate or on the adsorbent, The selection may also be based on monoclonal techniques. In accordance v/ith the invention, assaying is preferably done by means of so-called immunochemical techniques although it is possible also to determine the analyte in some other manner, using methods which provide satisfactory specificity, precision and sensitivity. Immunochemical assays comprise a large number of general methods which are known per se and to which the invention is applicable. They utilize as a rule an antibod}' preparation that is specific for the species to be assayed. This means in the case of an elastase induced fibrinopeptide (= analyte) that e.g. anti- (elastase induced Bø 33+4 fibrinopeptide) antibody active components contained in the antibody preparation of the present invention are allowed to react (or alternatively contacted) with a sample containing elastase induced B0 33+_4 fibrinopeptide (s) , to thus form an immune complex the formation and amount of which will give a quantitative indication and quantitative indication respectively of the fibrinopeptide present in the sample. Further immune reactants or other reactants reacting in biospecifiσ affinity reactions with components that can be incorporated into the immune complex may be added in order to facilitate quantification. A usual expedient consists in adding a reactant provided with a marker group, a so-called analytically detectable group. The amounts of reactants employed are chosen such that the amount of labeled reactant incorporated in the complex or remaining unincorporated will be a measure of the analyte present in the sample. Immunochemical methods may be classified as being either homogeneous or heterogeneous methods. In the case of homo¬ geneous methods, a labeled reactant is determined and correlated with the analyte without any physical separation of complexed reactant from the non-complexed form of the reactant. In the case of the heterogeneous methods, the two forms of labeled reactant are separated physically from each other before the labeled reactant is assayed in one or both of the two forms. In order to facilitate separation, it is convenient if one of the reactants is insoluble or insolubilizable in the test medium (but not the labeled reactant) . According to a second classification system, the methods may be classed as being either competitive or noncompetitive methods. In a competitive method the arrangement is such that two immune reactants having a common epitope (determinant) are made to compete for an insufficient number of homologous binding sites on an immunological counterpart. In the case of the present invention; the elastase induced fibrinopeptide (= analyte) present in the sample will compete with the corresponding labeled fibrinopeptide or corresponding solid-phase-bound fibrinopeptide for antibody active compo¬ nents present in the antibody preparation of this invention. The amount thus reacting with the antibody active components will be a measure of the content of the said fibrinopeptide in the sample. Competitive methods are also referred to as "inhibition methods". In a noncompetitive method arrangements are such that no competition can take place. According to a third classification system, the methods may be classed as being either precipitation or nonprecipitation methods. In the case of the present invention, when the first of these two methods is utilized the antibody preparation of the invention is chosen such that its reaction with the fibrinopeptide to be assayed will give a precipitate or the resultant immune complex is precipitated afterwards with an addition of an excess of precipitating agent such as polyethylene glycol, anti-antiserum, solid-phase-bound anti-antibody etc. If a labeled reactant is employed the arrangement has of course to be such that the anti-antiserum or the solid-phase-bound anti-antibody is not directed against the labeled reactant. In a fourth classification system, the methods are classed according to the marker group, the methods then being such as radio-, enzyme, fluorescence-, chemiluminescenσe-, enzyme-substrate-, immunochemical methods etc. Among immunochemical methods may be mentioned also immuno- electrophoresis, particle agglutination, immunodiffusion and microscopy with labeled antibodies. The most preferred techniques for elastase induced fibrino¬ peptides of molecular weights <5 000 daltons are competitive techniques of either the homogeneous or the heterogeneous type. As has been explained above, the invention permits using preparations of elastase induced Δ 33+4 fibrinopeptides. These peptide preparations may contain derivatized forms of the said fibrinopeptides, e.g. B0 30-43 or one of those elastase induced β 33+_4 fibrinopeptides that can be detected in body fluids. The peptides can be bound adsorp- tively or covalently to a so-called "solid phase" (that is, a phase that is insoluble in the test medium) , or can be provided with an analytically detectable group of the kind referred to above. The preparation may comprise the pure derivative (e.g. having a degree of purity exceeding 90 % w/w based on proteins and peptides from the source of raw material) or may comprise the derivative together with chemicals required for a given type of use (such as e.g. aqueous solvents, buffer components, bacteriostats etc). The preparations may be in a dry state, to be dissolved or resuspended when they are to be used. In cases where a preparation is to be used for immunoassays in contact with blood or plasma samples it is of importance that the peptide derivative should be one that is stable. Thus, if merely and solely a stability point of view were to apply in respect of the elastase induced BQ fibrinopeptide preparations what would be preferred would be to choose the naturally occurring elastase induced BA 33+4 fibrinopeptides, rather than the BA 30-43 peptide. It may be noted that preparations containing an elastase induced Bβ 33+4 fibrinopeptide are also comprised by the scope of the invention. In these preparations it is not necessary for the fibrinopeptide to be derivatized as indicated in the preceding paragraph, although it may be thus derivatized. This fibrinopeptide may be prepared by means of elastase digestion of fibrin(ogen) , see the exper¬ imental part of this specificaiton, or by the use of its amino acid sequence it may be prepared by way of peptide synthesis, for example in a manner analogous to that used for the BΔ 30-43 peptide. The fibrinopeptide preparations as aforesaid comprise one aspect of the invention. Another aspect resides in a test kit containing at least one of the aforesaid peptide or antibody preparations. The immunochemical reaction(s) contemplated will be carried out under conditions such as are commonly employed in each particular assay context. The temperature thus is selected in the range of 0-40 °C, especially 15-40 °C. The pH is selected from 4.5 up to 9.0, being preferably within the range of about 5-8.6. Reaction solutions are buffered in a manner known per se with the aid of buffer components having a pKa within or close to the pH range in which the immune reaction to be performed will be practically independent of the pH. It is reσommendable to add protease inhibitors and/or metal ion complexing agents like EDTA. Samples from patients as employed in accordance with the invention will in the first place be blood samples, as e.g. serum sampels or preferably plasma samples, although it is potentially feasible to employ other samples containing the aforesaid fibrinopeptides - for instance urine, saliva, laσrimal fluid and CSF. The invention is defined in the attached claims which form an integral part of this specification. The invention will now be illustrated by means of a number of examples which however are not intended to limit the invention in any way. Materials Peptides B£> 1-42, 15-42 and 1-118 were obtained from Dr Birgit Hessel, Stockholm, Sweden. Peptide f> 30-43 NH 2 was synthesized by Ferring AB, Malmδ, Sweden. β 15-42 RIA and purified fibrinogen from IMCO Corp Ltd, Stockholm, Sweden. Human fibrinogen (Grade L) , trans-4-aminomethyl-cyclo- hexane-carboxylic acid (AMCA) and plasmin from AB KabiVitrum, Stockholm, Sweden. Human leucocyte elastase from Dr Kjell Ohlsson, Malmδ, Sweden. SP-Sephadex ® G-25, Sephadex G10, G15, G25, G25F, Dextran T-70, Lysine Sepharose ® 4B, Mono Q from Pharmacia AB, Uppsala, Sweden. Bio Gel P6 ® from Bio Rad Richmond, Ca, USA. DIAFLO PM10 Membrane, Amicon, Lexington, Mass., USA. Bovine serum albumin RIA-grade (BSA) and activated 125 carbon (250-350 mesh) from Sigma, St Louis, USA. Na I from Amersham International pic, Amersham, UK. Freund' s complete and incomplete adjuvants from Difco Laboratories, Detroit, Mich., USA. Aprotinin (Trasylol^) Bayer AB, Leverkusen, F.R.G. Evacuated Blood Collection Tube, EDTA, heparin, citrate from Becton Dickinson Vacutainer System Europe Meyland Cedex, France. All the other chemicals employed were of analytical reagent grade. White New Zealand male and female rabbits weighing 1.5-2.5 kg were used for immunizations. Buffer I: 0.5 M a 2 HP0 4 , pH 7.5. Buffer II: 0.05 M Tris, 0.15 M NaCl, 0.1 % BSA, pH 7.5. Buffer III: 0.05 M Tris, 0.15 M NaCl, 1 % BSA, pH 7.5. Buffer IV: 0.04 M Tris, 0.11 M NaCl, 0.01 % EDTA, 0.02 % a 3 , 20 KIE/ml Trasylol ® , 0.1 % BSA, pH 7.4. In the experimental part of the specification, the term "B/> 30-43" will refer (unless otherwise stated) to the peptide of 14 amino acids having the corresponding amino acid sequence; the term n & 30-43 RIA" refers to an assay detecting epitopes present in this peptide. Methods Preparation and purification of A 30-43 fragment: This peptide was synthesized by the Merrifield solid phase technique (17, 18) . The crude lyophilized peptide was dissolved in a minimum amount of 9 M acetic acid. It was then desalted on Sephadex G-15 in the same buffer and lyophilized. The desalted peptide was dissolved in distilled water and chromatographed on a SP-Sephadex G-25 ion exchanger column (2.6 x 34.5 cm, gel volume 185 ml) . The eluant buffer was 0.33 M ammonium acetate, pH 7.0 and the gradient system was 0.33-0.83 M ammonium acetate, pH 7.0. Pure peptide eluted at about 0.5 M ammonium acetate. The material was then lyophilized and desalted on Sephadex G-10 with 0.2 M acetic acid as eluant buffer and again lyophilized. Production of antiserum: Purified Bfi 30-43 was conjugated to bovine serum albumin with the aid of carbodiimide. 46 mg of bovine serum albumin were dissolved in 1.00 ml water. The pH was adjusted to 6.10. 10 mg of BA 30-43 were added to the solution at room temperature, whereupon 10 mg of 1-ethyl- 3- (3-dimethylaminopropyl)-carbodiimide were added to the albumin-peptide solution. After 6 h another 10 mg portion of carbodiimide was added. After a further 16 h period the reaction mixture was desalted on a Sephadex G-25 column (2.0 x 90 cm) in 0.25 M ammonium acetate, pH 5.5, and then again lyophilized. The immunogen was resuspended in saline (0.9 % NaCl) to a concentration of 6.67 mg/ml and mixed thoroughly with 1 part of Freund's complete adjuvant until a thick emulsion formed. 5 white male or female rabbits were immunized with 333 ,ug (100 ,ul) of the conjugated peptide in a lymphatic gland of each hind leg. Every two weeks after the primary injection, each rabbit was boosted with 333 ,ug of the immunogen + 1 part of Freund's incomplete adjuvant given intramuscularly into the back. Blood samples were taken after 8-10 weeks and the immune response was determined. Two rabbits with positive immune response were killed after 12 weeks and their sera were collected. Stored at -70 °C. Preparation of labeled 30-43 (= tracer) : The peptide contains a tyrosine residue at position B^_> 41. I labeling was done by the chloramine method (19) . 5 ,ug 30-43 peptide were dissolved in 25 ,ul of buffer I and mixed with 10 ,ul of carrier-free Nal (10 ,uCi) . 10 ,ul of chloramine-T (50 ,ug) were added, and after mixing (30 sec) the reaction was terminated with 100 ,ul of sodium metabisulfite (100 ,ug) . (Chloramine-T and sodium metabisulfite were dissolved in water just before the use.) In order to minimize adsorption loss of the tracer during desalting, the reaction mixture was then immediately diluted with 200 ,ul of buffer III. The reaction mixture was then passed over a Sephadex G-10 column (1.5 x 5 cm) equilibrated and eluted with buffer II and at a flow rate of 0.25 ml/min. Fractions of 0.5 ml were collected. The protein-peptide peak contained most of the radioactivity (90 %) . Fractions containing the tracer were pooled, diluted with 4 volumes of buffer III and dispensed in 0.2 ml aliquots which were stored frozen at -25 C until used. The tracer is stable for at least 4 mc without loss of any of its immune reactivity. BΔ 30-43 RIA: Dilutions of standard, test samples, antiserum and tracer were made in buffer IV. Each assay tube contained 100 .ul of standard concentration of B^d 30-43 or test sample (undiluted plasma, urine etc) + 50 ,ul of tracer + 100 ,ul of antiserum in a dilution sufficient to bind 35 % to 45 % of the total bindable tracer. After each step the tubes were shaken thoroughly. After a 16 h incubation period at +4 C, free tracer and tracer bound to antibody were separated by adding 500 ,ul of dextran-coated active carbon (consisting of 1 % carbon and 0.1 % Dextran-T 70) (final cone.) at 0 C. The tubes were shaken thoroughly and left on ice for 5 min. They were then centrifuged for 10 min at +4 C and 1 500 g 600 ,ul of the supernatants were transferred to new tubes, and radioactivity was counted on a gamma counter. Bfr 15-42 RIA: The method employed was that of Kudryk et al. (20) . It can only be applied to heparin-trasolyl plasma precipitated with ethanol. Blood collection and processing: Blood samples were taken from normal healthy subjects and patients by means of venipuncture. The blood was collected in 4.5 ml tubes containing either 0.5 ml of 0.129 M sodium citrate or 71 USP units of heparin or 0.054 ml of 0.34 M EDTA. To all tubes containing heparin 0.2 ml of Trasolyl ® (2 000 KEI units) was added. The tubes were centrifuged at 1 500 kg, +4 C, 20 min. The plasma samples were analyzed directly or were stored at -70 C. Frozen plasma samples were quickly thawed at +37 C and then analyzed immediately or processed as stated below. Ethanol treated samples: 1 ml of ice-cold ethanol was mixed with 1 ml of plasma in a centrifuge tube. After mixing and incubation in an ice bath for 30 min, the samples were centrifuged at 1 500 g, +4 C , 20 min. The supernatant was transferred to a new centrifuge tube and kept in the ice bath for an additional period of 30 min, and was then recentrifuged. To some of the ethanol treated samples, 4 volumes of xrater were added whereupon they were freeze- dried. Cross reactions of the antibody preparation: Some closely related peptides were examined. They were added in a pure form (0-80 000 pmol/L) to buffer IV and analyzed directly. Pure fibrinogen was added to either buffer IV or normal plasma in a concentration varying from 2 to 16 mg/ml (5.8-46.4 ,umol/L) . Normal value 2.4-4.2 mg/ml (7-12.3 ,umol/L) in human plasma. Fibrinogen: Its concentration in citrated plasma was deter¬ mined by the method of Nilsson and Olow (21) . Determination of in vitro behavior of synthesized Bp 30-43 peptide in plasma: 0.45 ml of (1) citrate-, (2) heparin + trasolyl- and (3) EDTA-plasma was incubated each by itself with 0.05 ml of B 30-43 peptide (1 ,umol/L) . The incubations were performed at 0 C, +22 C and +37 C. 50 ,ul samples were taken after 0, 15, 30, 45 and 60 minutes. Incubation was terminated by adding 9 volumes of ice-cold buffer IV. The experiments have shown that the peptide is almost completely stable in ice-cold buffer IV despite the presens of plasma proteins . Recovery of BΛ 30-43 in plasma: To 11 tubes containing 1.35 ml of normal EDTA-plasma was added 0.15 ml of synthesized B 30-43 at different concentration; incubation was carried out at +22 °C for 15 minutes. BΛ 30-43 concentrations varied between 0 and 25 000 pmol/L. _ _ Correlation between B & 30-43 RIA values and BO 15-42 RIA values: 48 heparin-trasylol patient-plasma samples were tested according to each of the two methods. In B 30-43 RIA they were analyzed directly and in BΔ 15-42 RIA they were analyzed after having been ethanol treated and freeze-dried. The values obtained in Bβ 30-43 RIA were subtracted by 60 pmol/L (influence of fibrinogen) and the values of BΔ 15-42 were calculated according to the instructions of the manufacturer. Purification of the elastase induced fibrin(ogen) opeptide having a length of 33—4 amino acids 1 g of human fibrinogen containing about 3 mg plasminogen was dissolved in 100 ml of H 2 0 and freed from plasminogen by affinity chromatography in batch, with Lysine Sepharose 4B. This chromatography was repeated twice. The solutions was then dialyzed against 20 volumes of a buffer, containing 0.15 M ammoniumacetate, pH 8.5, for 72 hours. The dialysis buffer was replaced six times. After dilution to 2 mg of fibrinogen/ml with the same buffer mentioned above, the batch was divided into two equal aliquots. One of the aliquots was admixed with 4 ,ug of elastase, from human leucocytes, per mg fibrinogen (molar ratio of substrate to enzyme 24:1) , and the other aliquot was treated in the same manner except that it was first "coagulated" with thrombin (1 NIH/mg) . After 24 h at +37 °C the lysis were terminated by addition of an elastase inhibitor (PMSF, final cone. 10 mM) . The lysates were then ultrafiltrated on a DIAFLO PM10 membrane. The retentates and diffusates obtained were lyophilized. The diffusates (containing peptides of molecular weight less than 10,000 daltons) contained all of the immunological activities. The desired peptide could be isolated from the diffusates, by means of a combination of gel chromatography on Bio Gel P6 ® in 0.1 M ammoniumacetate, pH 6.2 and HPLC on Mono Q in 0.02 M ethanolamine, pH 9.5. The active material was lyophilized and amino acid analyzed on a Durrum D-500 analyser. The active fractions obtained with digest from fibrin or fibrinogen were the same both in gel chromatography and HPLC. Labeled synthesized BΛ 30-43: The specific radioactivity of the tracer was 7.5 + 0.5 MBq/ ,ug BU 30-43 (n=3) . BA 30-43 RIA: For the labeled B2? 30-43 a dilution giving about 4 000 CPM/50 ,ul was preferable. In that case an antiserum dilution of 1/800 binds about 40 % of the total number of counts. Binding studies were repeated for each synthesized preparation of labeled Bfi> 30-43. Nonspecific binding in our procedure (without antibody) was about 3-5 %. Results from the final precipitation step were fairly constant in our procedure. Unlabeled B 0 30-43 progressively inhibited binding of labeled substance to the antiserum. 10 standard concentrations were used for construction of the standard curve, within the range of from 50 to 25 000 pmol/L. The sensitivity was about 30 pmol/L. Peptides measured by means of B 30-43 RIA: Gel chromatography (Sephadex G-25 Fine) was carried out with (1) urine samples, (2) plasma samples (EDTA) , (3) elastase digested fibrin and (4) synthetic Bfl» 30-43 in order to roughly determine the identity of the fibrin (ogen) fragments which are measured by means of the invention. In the case of (l)-(3) , measurable fragments could be detected by means of Δ> 30-43 RIA in the fractions corresponding to peptides comprising about 30 amino acids. (Amino acid analysis performed during the priority year has indicated that it should consists of 33+4 amino acids. See Table III.) When (l)-(3) were then further digested v/ith elastase, activity was obtained in fractions corresponding to the same peptide length as BΛ 30-43. No loss of immunological activity could be demonstrated. In the case of (4) , there was no loss of immunological activity, but the length of measurable peptides had been reduced to one corresponding to 8-11 amino acids. Cross reactivity of the antibody preparation: Cross reac¬ tivities of peptides BΔ 1-42, B> 15-42, BΔ» 1-118 and peptide ]_A 30-43 NH 2 with the anti-(B 30-43)-antibody were determined. Varying concentrations of fibrinogen were also studied. The results are summarized in Tables I and II. Table I Cross reactivity of some peptides with respect to B 30-43 RIA Conσen- BΛ * tration Peptide B* 1-42* %** BΔ 15-42* %** BΔ 1-118* %** 30-43 NH 2 %* 6250 pnol/L 0 0 0 0 0 0 0 0 12500 " 0 0 0 0 0 0 0 0 25000 " <30 <0.1 <30 <0.1 0 0 0 0 80 000 " 190 0.24 250 0.31 / / 0 0 The values set forth are the Bi 30-43 pmol values that would have given a corresponding inhibition. ** Cross reactivity in %. Table II Cross reactivity of fibrinogen in normal plasma (HNP) % cross reac¬ Cross reactivity tivity HNP + 3.02 g/L Fbg 8.9 .umol/L 20 pmol/L 0.0002 + 2.43 g/L Fbg = 16.0 ,umol/L 41 pmol/L 0.0003 + 4.48 g/L Fbg = 22.1 .umol/L 60 pmol/L 0.0003 + 9.54 g/L Fbg = 36.9 .umol/L 195 μnol/L 0.0005 + 17.78 g/L Fbg = 61.2 .umol/L 567 pmol/L 0.0009 Fibrinogen was , added to HNP (3,02 g/1) Fibrinogen cross reactivity of the same magnitude was . demonstrated when the substances were added to buffer IV. We decided that 60 pmol/L was a background value to be subtracted from analyzed values. This background value included all the cross reacting peptide and normal fibrinogen values. In 15 healthy individuals of our laboratory the measured value was 60 —+ 42 pmol/L (Mw —+ S.D.) (measuring range 0-120 pmol/L) . So we decided that patients whose values exceeded 140 pmol/L were to be deemed to have elevated levels of elastase induced B fi- 30-43 fibrinopeptides. Reproducibility: By BA 30-43 RIA measurements it could be shown that synthesized BΛ 30-43 quickly lost its immunological reactivity in citrate-plasma and heparin-plasma but not in EDTA-plasma. The conclusion to be drawn from this is that complexing agents should be added to plasma samples which are to be incubated with derivatives of the peptide B 30-43. When synthesized BN 30-43 was measured in EDTA-plasma the results v/ere independent of whether measurements were performed (i) directly, (ii) after ethanol precipitation, or (iii) after ethanol precipitation plus freeze-drying. The values obtained from patients ' plasma samples were higher when analysis was performed directly than when the samples were first ethanol precipitated, or ethanol precipitated + freeze-dried. Correlation between B 30-43 RIA values and BA 15-42 RIA values: Samples from 48 patients were studied with these different methods. The correlation was 0.46. The invention has been applied to a clinical material. What we found was that elevated values of elastase induced Bl» fibrinopeptides can be demonstrated in i.a. inflammatory conditions, for example in cases of som diseases of the lungs (pulmonary congestion, pulmonary embolism) and diseases of the kidneys (probably associated with glomerular lesions) . Very high levels have been demonstrated in particular in cases of ARDS and septic patients. Purification of elastase induced fibrinopeptide: By chromatography on Bio Gel P6, the fractions with most of the immunological activity were found between Kav 0.00-0.10. Less than 1 % of the peptide material applied on the column was found in these fractions. For HPLC, all of the activity was found in the fraction which passed throw the column without any delay. About ten other inactive peaks were separated from the active one in this way. Amino acid analysis showed pure peptide and the composition of the peptide is given in table III. The active peptide was found in digest from fibrinogen and fibrin. Table III Amino acid compo,sition Component observed A B A B Aspartic acid 10.27 1.34 1 1.26 1 Threonine (3.39) Serine 17.88 2.33 2 2.19 2 Glutamic ac :id 17.18 2.24 2 2.11 2 Proline 47.47 6.20 7 5.82 6 Glycine 30.66 4.00 4 3.76 4 Alanine 28.80 3.76 4 3.53 4 Valine (1.61) Isoleucine 7.52 0.98 1 0.92 1 Leucine 16.74 2.19 2 2.05 2 Norleucine 10.00 Internal standard Tyrosine 6.36 0.83 1 0.78 1 Phenylalanine (0.80) Histidine 7.06 0.92 1 0.87 1 Lysine 18.92 2.47 __-. 2.32 2 Ammonia 0.00 Arginine 43.96 5.74 6 5.39 5 Sum 252.82 33 31 A. Calculated number of amino acids based on the analysis. B. Number of amino acids known from the sequence. References 1. Blomback, B. : In: Plasmaproteins, John Wiley & Sons: 223-53, 1979. 2. Plow, E.F. et al. : J. Clin. Invest. 56: 30, 1975. 3. Bilezikian, S.B. et al.: Blood 50: 21, 1977. 4. Plow, E.F. et al. : 330, 1978 In: Neutral proteases of human polymorphonuclear leukocytes. Ed: Haseman, K and Janoff, A. Urban & Schwarzenberg, Inc. r Baltimore, Munich. 5. Wallin, R. et al. : Thrombos Haemost 46: 52, 1981. 6. Saldeen, T. : Fibrinogen-Structure, Functional Aspects, Metabolism 2: 315-22, 1983. 7. Wallin, R. et al. : Fibrinogen Structural Variants and Interactions 3: 289-300, 1985. 8. Saldeen, K. et al. : Thromb. Res. 37: 85-9, 1985. 9. Nichols, W.W. et al.. Thromb. Res. 38: 223-9, 1985. 10. Sterrenberg, L. et al. : Thrombos. Res. 31: 719, 1983. 11. Sterrenberg, L. et al. : Haemostasis 15: 126, 1985. 12. Nossel, H.L. et al.: Proc. Natl. Acad. Sci. USA 68: 2350-53, 1971. 13. Bilezikikian, S.B. et al. : J. Clin. Invest. 53: 438-45, 1975. 14. Kudryk, B. et al.: Thromb. Res. 25: 277-91, 1982. 15. Weitz J.I. et al.: Blood 67: 1014-1022, 1986. 16. Weitz, J.I. et al.: J. Clin. Invest. 78: 155, 1986. 17. Merrifield, R.B.: J. Am. Chem. Soc. 85: 2149, 1963. 18. Erickson, B.W. et al.: In: The Proteins (Neurath, H., Hill, R. & Boeder, C.L., eds. ) , vol. II, 3rd edn, pp. 255-527, Academic Press, New York, 1976. 19. Hunter, W.M. et al.: Nature 194: 495, 1962. 20. Kudryk, B. et al. : Measurement in human blood of fibrinogen/fibrin fragments containing the B 15-42 sequence. Thrombos. Res. 25: 277, 1982. 21. Nilsson, I.-M. et al. : Thromb. Diath. Hae orrh. 8: 297, 1962. References added after the priority application 22. Eckhardt, T. : Blut 53:39, 1986. 23. Plow, E.F. et al.: J. Lab. Clin. Med. 102: 858, 1983. 24. Sterrenberg, L. et al.: Thromb. Haemostas. 51:398, 1984. 25. EP-A2-151,239; New York Blood Center Inc. 26. EP-A2-152,6I2: Max Planck Gesellschaft zur Forderung der Wissenschaften.

Claims: Claims 1. Method for the in vitro diagnostication of diseases associated with fibrin(ogen) olysis and/or pathological elastase activity, characterized in that a sample taken from a patient and containing fibrinopeptides is subjected to quantification of elastase induced B f> 14 (+2) - 45( -3) fibrinopeptide(s) , that the value thus obtained is compared with a normal value for healthy individuals, and that if the value obtained is higher than said normal value the patient from whom the sample has been taken is classed as being diseased. 2. Method according to claim 1, characterized in that said quantification is effected immunochemically by the use of an antibody preparation directed against said elastase induced fibrinopeptides. 3. Method according to claim 2, characterized in that the antibody preparation is an anti-(B > 30-43) antibody preparation. 4. Method according to claim 3, characterized in that said quantification is performed by means of a competitive immunochemical method. 5. Anti-(elastase induced B b fibrinopeptide) antibody preparation. 6. Antibody preparation according to claim 5, characterized by being directed preferentially against at least one epitope that is unique for an elastase induced BΛ 14 (+2) - 45(+_3) fibrinopeptide. 7. Antibody preparation according to claim 6, characterized in that at least one of said at least epitopes can be retrieved on the B A 30-43 peptide. 8. In vitro assay method for determining in vivo free elastase activity in a human, characterized in that a sample being taken from a human individual and containing fibrinopeptides is assayed for at least one elastase induced B b 14(+_2) - 45 (+3 ) fibrinopeptide. 9. Method according to claim 8, characterized in that the assay is performed immunochemically by the use of an antibody preparation directed against said elastase induced fibrinopeptides. 10. Method according to claim 8, characterized by the antibody preparation being an anti-(Bfc> 30-43) antibody preparation. 11. Elastase induced Bfe fibrinopeptide in substantially pure form having at least one unique epitope in common with the BA 30-43 fibrinopeptide (s) , with the provision that when the elastase induced B-beta fibrinopeptide is the B 30-43 fragment it only occurs in derivatized form.

Inventors: SALDEEN TOM GUSTAF PER, WALLIN ROLF KENTH SIGVARD, SALDEEN, TOM, GUSTAF, PER, WALLIN, ROLF, KENTH, SIGVARD
Applicants: SALDEEN TOM GUSTAF PER, WALLIN ROLF KENTH SIGVARD, SALDEEN, TOM, GUSTAF, PER, WALLIN, ROLF, KENTH, SIGVARD
Application year: 1988
Main classification: G01N33
IPC codes: G01N33