METHODS FOR DETERMINING THE PRESENCE OF BRAIN PROTEIN S-100

Abstract: An assay method for determining the presence of the brain protein S-100 in a clinical sample which uses antibodies directed to epitopes in the region from ser1 to asn38 and from thr82 to glu93 of the amino acid sequence of the beta subunit of human S100B is provided.

Description: METHODS FOR DETERMINING THE PRESENCE OF BRAIN PROTEIN S-100 The present invention relates methods for diagnosis and follow-up of patients with cerebral dysfunction as well as melanoma cancer, by determining the presence of the brain protein S-100 The invention also relates to peptides comprising useful antigemc determinants from S-100 as well as monoclonal antibodies binding to these peptides Λs is known, the nervous system contains a number of proteins unique to its vaπous cellular elements The cellular disruption of nervous tissue and cells of neural origin, by any pathogenic process, trauma or by neurological diseases, results in the release of normal soluble endogenous cytoplasmic proteins into the cerebral extracellular fluid and ultimately to other body fluids including the cerebrospinal fluid (CSF) and blood (serum and plasma) Examples of representative soluble small molecule weight proteins of this type can be found in the SI 00 protein family Λ review of this family can be found in Zi mer et al . Brain Research Bulletin, Vol 37, pp 417- 429. 1995 Following disruption of cell membranes, these proteins are released into the extracellular fluid in accordance with a time course and in quantities relative to the pathogenesis of the disease process or the extent of the brain tissue damages The proteins diffuse into the CSF and then the blood or directly into the blood The above mentioned cell membrane disruption is reflected by the blood plasmaor serum levels of one or more of these antigens and markers These protein antigens have the advantage of being stable and specific, not only for the brain, but for the cellular components in the brain By following the relative release of the vaπous nervous system protein antigens, it is possible to deduce the kind of destructive process occuπng in the course of neurological diseases and/or the extent of possible brain tissue damages Information of this type permits the diagnosis, evaluation of seventy and rate of progression of the above mentioned dieases and damages It is previously known to determine the amount of S-100 polypeptides in a clinical sample US-A-4 654 313 discloses a ra oimmunological assay method for S-l 00 protein The patent document does neither mention anything about different kinds of Sl OO-polypeptides nor about on which epitopes the assay method is based The detection limit is declared to be 0 20 ng/ml but concentrations between 1 5 and 2 5 ng/ml is required in order to have less than 10% false positives This concentration is rathei high Moreover, in some countries it is not permitted to use radioactive methods in clinical assays It is also known to determine S-100 polypeptides by using ELISA-related methods GB-A-2 109 931 discloses a solid-phase immunoanaiysis method composing the use of enzyme-labelled antigens and particles coated with protein A on which antibodies are bound S-l 00 proteins are only mentioned in claim 8 and nothing is revealed about the sensitivity of the method JP-A-6/109 734 describes a method suitable for analysing S-100 polypeptides. using a first polyclonal antibody fixed to magnetic particles, and a second labelled polyclonal antibody The method requires two different enzymes, namely horseradish peroxidase and alkaline phosphatase, and it composes at least ten consecutive steps The minimum detection limit is stated to be 0 02 ng/ml for cerebrospinal fluid and 0 06 ng/ml for bovine brain The complexity of clinical samples is often a seπous problem An assay method may give excellent results with artificial samples in the laboratory but quite a number of unreliable results might be obtained when the method is tested under clinical conditions When it comes to immunological assays the problems are often caused by an improper selection of antigemc determinants One antibody in an assay composing the use of two different antibodies, may be a hindrance to the other antibody when bound to the antigen to be determined An improper selection of epitope for an antibody involved in the detection process may result in that the detection goup is completely or partially embedded in a protein complex and not av- ailable for detection Different proteins present in the sample might interfere Moreover, a method composing many consecutive steps may give uncertain results for complex clinical samples, as the interference possibilities increse with the number of steps and added extra components There is always a need for lmprovment of methods for analysing substances of medical intrest in clinical samples An ideal clinical assay method should be quick, accurate and possible to perform with all types of clinical samples without degeneration of the accuracy for certain types of specimen It should also require a minimum of extra components This applies to determination of S-1 0 polypeptides as well as other substances of medical interest Summary of the invention Now it has turned out that by using antibodies directed to epitopes in the region from serl to asn38 and from thr82 to glu93 of the amino acid sequence of human S-l OOβ polypeptide, an improved clinical assay method for determining S-100 polypeptides and particularly the β subunit or isoform thereof is obtained Hence, the main object of the present invention is an assay method using monoclonal antibodies directed to these epitopes Another object of the present invention relates to short peptides having sequences corresponding to parts of the amino acid sequence of the human S- I OOβ polypeptide from serl to asn38 and from thr82 to glu93 Yet another object of the present invention relates to analytical kits for carrying out the assay methods Detailed descnption of the invention As already mentioned above it is often very difficult to outline methods for analysing clinical samples It is neccesary that the method has a high sensitivity and gives accurate results It is also very important that known and unknown constituents of the sample other than the analyte do not influence the results The present invention relates to an immunological assay method for determining the presence and/or content of human S-l 00 polypeptide based upon a selection of suitable S-l 00 epitopes and corresponding antibodies which fulfil the above mentioned requirements It has turned out that the selected epitope combinations provides tests and test kits where a high sensitivity is achieved. 2 the antibodies of the kit binds equally strong to the internal standard as to the analyte in the clinical sample, the epitopes are chosen in such a way that the different antibodies do not interfere with each other when they bind to the analyte, I e that the epitopes are situated sufficiently distant from each other The epitopes of the present invention are all composed in the human S-l OOβ polypeptide Epitopes present within the amino acid sequences SELEK MVALIDVFHQYSGREGDKΗKLKKSELKELINN (SEQ ID NO 2) and TACHEFFEHE (SEQ ID NO 3) are preferred Particularly preferred are epitopes composed within the peptide AMVALLDVFHOYSGREGD HKLΓ KSEL ELINN (SEQ ID NO 4) and especially within the peptides REGDKHKLKKSELKEL (SEQ ID NO 5) and EFFEHE (SEQ ID NO 6) The disclosed epitopes are. among all, used to construct peptides for inducing the formation of suitable antibodies on which the claimed assay method is based These peptides mostly consist of up to 38 amino acids The whole amino acid sequence of a peptide according to the present invention is denved from human S-l OOβ These peptides may compose vaoants wherein the ooginal amino acid sequence is modified or altered by insertion, adώtion. substitution, inversion or deletion which preferably show at least 90% homology with the sequence of SEQ ID NO 2 and SEQ ID NO 3 and retain essentially the same immunological properties The peptides may also compose multiples of certain epitopes. and in this case their sequence length may exceed 38 amino acids By the expression "sub-fragment" is meant a polypeptide sequence having a length of at least 6 amino acids The epitopes can also be used to construct fusion peptides composing at least two distinct epitopes which, among all. can be used as internal standard in immunoassays Abbreviations 10 The following abbreviations are used S100 -Sl OOβ RT -Room Temperature BSA -Bovine Serum Albumin i Mab(s) -Monoclonal antιbody(ιes) D -kiloDalton CCL -Enhanced Chemiluminescent Assay CBB -Commassie Bolliant Blue LIA -Luminometπc Immuno Assay 0 1RMA -Immuno Radio Metric Assay ELISA -Enzyme Linked ImmunoSorbent Assay SDS-PAGE -SodiumDodecylSulfate - PolyAcrylamideGelElectrophoresis PBS -Phosphate Buffered Saline RLU -Relative Light Units 25 NHS -N-HydroxySuccimmide EDC -N-ethyl-N 1 -(dιmethylamιnopropyl)-carbodnmιde RAMFc -RabbitAnti-MouseFc antibody EDTA -EtylenDiamineTetraAcetic acid NaCl -Sodium Chloπde 30 NaNi -Sodium azide lv -intravenously aa -amino acid The peptides were prepared by the methods disclosed in Merrifield (1963), J. Am. Chem. Soc, vol. 85, p 2149; Gutte et al.( 1971 ), J. Biol Che vol. 246, p. 1922; and Carpino et al. ( 1970), J Am Chem Soc vol. 92. p. 5748. The monoclonal antibodies were prepared by the method according to Kόhler et al.( 1975). Nature vol. 256, p. 495; and Harlow et al.( 1 88). Antibodies. A Laboratory Manual. Cold Spring Harbor, p. 139. Antisen and Standard preparations Procedure for preparation and purification of SI 00 antigen prior to immunisation of Balb/c mice was according to Moore (Biochim. Biophys. Res. Comm. 1965, 19; 739 - 744) with a slight modification according to Haglid & Stavrou ( J. Neurochem. 1973, 20: 1523-1532). Briefly, bovine brain was homogenised in Tris buffer, pH 7.2. The homogenate was centrifuged at 10.000 r.p.m. and the clear supernatant was used for further purification by ammonium sulphate precipitation. The fraction still soluble after saturation by ammonium sulphate was dialysed and purified by separation on a Sephadex Gl 50 Sepharose (Pharmacia Biotech AB, Uppsala Sweden )chromato graphic column followed by separation on a DEAE-sephadex (ionic exchange) column (Pharmacia Biotech AB, Uppsala Sweden). The fraction eluted by 0.3 - 0.4 M NaCl was collected, desalted, lyophilised and used for further expenments. Hybodoma construction Balb/c mice were immunised with puofied Sl OOββ intrapeπtonially in Freund s complete adjuvant and were given booster iv injection 6 weeks later duπng 3 consecutive days The spleen was removed on the fourth day after last injection and prepared for fusion The myeloma cell line Sp2/0-Agl 4 was used for fusion of Balb/c spleen cells Antibody puofication and subclass determination Monoclonal antibodies were identified, extracted and puofied from hybodoma supernatant according to Harlow & Lane Eds in ANTIBODIES A LABORATORY MANUAL. Cold Spπng Harbour Laboratory Press. New York 298-299 & 31 1 Boefly. positive hybodoma clones carrying supernatant with specific antibodies were identified using ELISA with microtitreplate wells coated with S I OOββ Immunoglobulins were precipitated using saturated ammoniumsulphate and dialysed against 1 5 M Glycine, 3 M NaCl, pH 8 9 Dialysed mateoal were affinity chromatography puofied on an protem-A Sepharose (Pharmacia Biotech AB. Uppsala Sweden) column Fiactions were neutralised by addition of small volumes of l M Tos pH 8 0 Epitope mapping. l OOβ (monomer) epitopes for respective antibody was investigated by use of a synthetic peptide library Peptides were linked to nitro-cellulose filter membrane via an amide link, according to the manufacturer (Research Genetics ' . USA) and covers all ninety-one aa in the protein In total the library consisted of thirty-one, all except one being ten aa-residues long synthetic peptides Each peptide was consecutively shifted three aa towards the -COOH terminal end of the protein Positive antibody- binding was indicated by the use of a second anti-mouse antibod conjugated with HRP and detected using an ECL assay (Amersham, UK) Results Two binding sequences were found Epitope 1 AMVALl FHQYSGREGDKf-KLKKSELKELINN (residues 6-38)(SEQ ID NO 4) and Epitope 2 EFFEHE (residues No 86-91 ) (SEQ ID NO 6) Antibody reactivity Puofied antibodies reacting with the epitopes were checked for reactivity and affinity using the BIAcore r system (Pharmacia Biosensor AB, Uppsala Sweden) Boefly. in order to test the specificity of the antibodies, the RAMFc was immobilised onto the sensor chip CM5 NHS-ester activated surface, according to standard procedure, to provide approximately 600 RLU Then each Mab was bound to the RAMFc surface to approximately 300RLU. followed by the Sl OOαα and the SI 00 standard (consisting of 50% Sl OOαβ and 50% Sl OOββ) in separate expeoments All reactions were earned out in continuos flow of the phosphate buffer The kinetics between antibodies and antigen was done similarly SI 00 antigen was added to the chips at 200-450nM for reactivity measurements of the antibody intended for the solid phase and at 1000- 1500nM for measurements of the antibody intended for tracers Kinetics was determined using the BIAcore 1 Kinetic evaluation 2 1. software (Pharmacia Biosensor AB, Uppsala Sweden) It can be concluded from the reactivity profile that the antibodies reactive with the epitopes are specific for the β-containing forms of SI 00 and not the α-containing form Example 1 Development of an lmmunoluminometnc procedure Tracer antibody was conjugated with luminol Boefly. ABEI (Sigma. St Louis, Ms) was linked with a diactivated ester (Etylenglykolbis-succimidyl succinat. EGS) The ABEI-EGS-conjugate was next mixed with monoclonal antiSl OO-antibody in an approximately 50 5 molar ratio in l OOμl of PBS pH 7 4, containing 15% acetonitnle and incubated 1 h at room temperature. The ABEI-conjugated antibody was puofied on a Sephacryl ® S 300 HE. (Pharmacia Biotech AB, Uppsala Sweden) gelfiltration column, and appropoate fractions were pooled and diluted in phosphate-buffer Preparation of antibody coated tubes for LIA Polystyrene tubes (Greiner, Germany) were incubated overnight at room temperature with 3μg of S I 00-antι bodies in 300 μl of PBS pH 7 5 The tubes were washed with 0 1 % Tween20 ® in PBS Next, tubes were blocked with a solution containing 0 9%BSA and 4% Saccarose and incubated for 24h The solution was aspirated and the tubes allowed to dry LIA test procedure The test was conducted in a two step procedure by incubating l OOμl of patient body fluid in antibody coated tubes, or SI 00 standard with 100 μl of diluent (PBS + 5%BSA) and incubated at room temperature After washing 200 μl of the luminol- labelled antibody was added and a further 2 h of incubation was performed before measurement After another washing the luminescence was developed using the LIA-mat starter service kit ( Byk-Sangtec, Diezenbach Germany) and immediately measured as integrals over a peπod of 5 sec in luminometer (Berthold. Germany) In order to convert the obtained liglit signal into concentration of SI 00 measurements on patient samples were compared with measurements on solutions with known concentrations of SI 00 (standards) The detection limit (zero standard +3 standard deviations) was approximately 0 01 μg/1 Preparation of a Standard curve S100B protein was obtained from Medisera. Lund. Sweden, and diluted in PBS + 5% BSA Dilutions contained 0 10,0 40, 2 00, 8 00 and 20 00 μg/1. of an SI 00 preparation consisting of 50 % of the ββ form and 50 % of the αβ form PBS + 5 % BSA was used as standard 0 Three measurements were caroed out for each dilution The measured results as well as statistical calculations are presented in table 1 below Table 1 Concentration Counts Average Calculated cone Average μg/l μg/l 0 0019 μg/1 2024 O μg l 0 135 μg/1 0 0647 μg/1 0 105 μg/1 2963 0 10 μg/1 0 394 μg/1 0 405 μg/1 0 401 μg/1 5564 0 40 μg/1 2 049 μg 1 1 988 μg/1 1 966 μg/1 21 109 2 00 μg/1 Average Calculated cone Average 7 823 μg/1 7 677 μg/1 8 494 μg/1 70064 8 00 μg/1 22 12 μg/1 18 54 μg/1 19 51 μg/1 16391 8 20 00 μg/1 The lower detection limit was defined as three standard 0 determinations plus 3 X the standard deviation value For this measurement, it was calculated to be 0 006 μg/1 Clinical determinations of SI 00 in serum The SI 00 concentration was determined in serum from patients receiving heart bypass surgery and being connected to a heart-lung machine The results are presented in table 2 below Table 2 'atient Counts Average Concentration Average 3780 0 23 μg/1 Patient Counts Average Concentration Average 4 10 μg 1 _» 0 65 μg/1 0.77 μg/1 Example 2 Development of an ELISA test procedure. As tracer antibody was used monoclonal antiSl OO antibody conjugated with β- galactosidase according to Harlow & Lane Eds. in ANTIBODIES A LABORATORY MANUAL. Cold Spring Harbour Laboratory Press, New York page 351 Preparation of antibody coated microtiter wells for ELISA. Microtiteφlatewells (Coming. Denmark) were incubated overnight at +4°C with 2.5μg of microtiter wells were finally washed three times with 0.05% Tween20 ® and air doed before use. ELISA test procedure. The ELISA was conducted in a multiple step incubation procedure. 100 μl of 1 : 1 diluted patient sample or 10Oμl of SI 00 standard (0 - 20 μg/ml ) was added to the wells. The plate was incubated for 1 5h at RT under shaking. The plates were washed three times with 3 OOμl 0 05% Tween20 ® in PBS 100 μl of alkaline phosphatase conjugated tracer antibody was added and a further 1 5h of incubation on a shaker was performed The wells were then washed three times with 0 05% Tween20 in PBS 1 OOμl of a 5% o-nitro-phenyl-β-galactoside substrate solution was added and the plates were incubated with substrate for another forty-five minutes and colour is developed The colour development was stopped by the addition of 1 OOμl 0 66M Na 2 C0 3 Each well of the plate was read at 405nm in a standard microtiteφlate reader In order to convert the obtained colour signal into concentration of SI 00, measurements on patient samples were compared with measurements on solutions with known concentrations of SI 00 (standards) The detection limit (zero standard +3 standard deviations) was approximately 0 2 μg/1 Result Standard (μg/1) 0 0 5 1 5 5 15 A 405 0 088 0 147 0 244 0 675 196 Example 3 Development of an lmmunoradiometnc (IRMA) test procedure IRMA tracer antibody coniugation A monoclonal antiSl OO antibody was conjugated with Iodine using the Chloramine T method according to Greenwood et al (Biochem J 1963. 89 1 14-123) The specific activity was determined to be 520±80 MBq/mg Preparation of antibody coated to polystyrene beads Monoclonal anti SI 00 antibodies were coupled to polystyrene beads by the Glutaraldehyde coupling method according to Harlow & Lane Eds in ANTIBODIES, A LABORATORY MANUAL, Cold Spong Harbour Laboratory Press. New York. 533 & 536-537 Final blocking was by 1 % BSA, 0 1 % NaN 3 in PBS pH7 5 IRMA test procedure 1 OOμl of patient sample or standard was added to polystyrene tubes together with 1 OOμl PBS diluent One polystyrene coated bead was added to each tube followed by incubation for 1 h at RT on a shaker Next the beads were washed once with 2ml of derruneralised water and 200μl of 1-125 labelled tracer antibody was added and the tubes were incubated a further 2h on a shaker After washing the radioactive signal on the bead was measured in a standard γ-counter In order to convert the obtained radioactive signal into concentration of SI 00. measurements on patient samples were compared with measurements on solutions with known concentrations of S100 (standards) The detection limit (zero standard +3 standard deviations) was approximately 0 1 μg/1 Example 4 Use of IRMA test procedure for assay of SI 00 in serum from melanoma patients The SI 00 based test procedure was applied on clinical questions relating to melanoma Blood samples from patients with melanoma of vaoous stages of cancer progression were collected in serum collecting tubes Samples were then frozen and treated according to the test procedure descobed above in Example 3 Results Relationship to staging Clinical Stage 1 vs Clinical Stage II In a study of 577 patients the geometoc mean for Stage I was found to be 0 12 μg/l and for Stage II the geometoc mean was found to be 0 33 μg/1 p-value < 0 001 Example 5 Use of IRMA test procedure for assay of SI 00 in serum from melanoma patients The SI 00 based test procedure was applied on clinical questions relating to melanoma Blood samples from patients with melanoma of vaoous stages of cancer progression were collected in serum collecting tubes Samples were then frozen and treated according to the test procedure descobed above in Example 3 Results Relationship to survival Clinical Stage I vs Clinical Stage II and III In a study with respect to survival performed on 643 patients the lelative hazard and 95% confidence interval was calculated The relative hazard was found to be 12 3 and the confidence interval 5 6- 27 2 with a p-value of <0 001 Example 6 Use of the SI 00 LIA-method for evaluation of the influence of extra coφoral circulation equipment on the brain The SI 00 based test procedure in Example 1 was applied on monitoong cerebral ιn)ury following extra coφoreal circulation (ECC) Blood samples from patients undergoing extra coφoreal circulation were collected in serum tubes and treated according " Test procedure" Results Before start of End of ECC 1 day after sur- 2 days after sur- ECC gery gery SI 00 levels μg/1 0 1 ,67 0,21 o,ι : In this group of patients the level of SI 00 in serum was elevated for at least 2 days after surgery Uncomplicated cases should return to normal levels within the first 24 hours (Ref 1 Johnson et al. J Cardiothor Vase Anaesthesia, 9:6 ( 1995) 694-99) Example 7 Use of LIA test procedure for assay of SI 00 in serum from melanoma patients The SI 00 based test procedure was applied on clinical questions relating to melanoma. Blood samples from patients with melanoma of vaoous stages of cancer progression and blood donors were collected in serum collecting tubes Samples were frozen and treated according to the test procedure descobed above in Example 1 Result Of 136 patients with vaoous stages of melanoma 25 had a level of SI 00 below 0 08 and of 100 blood donors tested on the same occasion 7 had a level equal to or above 0 08 μg/1 Example 8 The reliability of both the test and the SlOOβ polypeptide marker per se when diagnosing melanoma were investigated On 252 patients with melanoma, serum was drawn before treatment was started and determination of the level of SlOOβ polypeptide was performed by the assay method disclosed in example 1 When a cut-off value of 0.16 μg/1 was used, the medium survival time of patients having a Sl OOβ concentration above the cut-off value was 7 months, whereas the medium survival time was more than 120 months for patients having a Sl OOβ concentration below the cut-off value In a patient diagnosed with malignant melanoma, considered to show no evidence of disease and monitored by the immunoradiometoc assay method as ώsclosed in example 3, elevated levels of Sl OOβ were recorded 2 months poor to the appearance of skin metastases and 6 months before metastases in organs were found SEQUENCE LISTING ( 1 ) GENERAL INFORMATION (i) APPLICANT (A) NAME AB Sangtec Medical (B) STREET P O Box 20045 (C) CITY Bromma (E) COUNTRY Sweden (F) POSTAL CODE (ZIP) 161 02 (G) TELEPHONE +46 8 635 12 00 (H) TELEFAX +46 8 29 21 81 (ii) TITLE OF INVENTION Methods for determining brain antigens (m)NUMBEROF SEQUENCES 8 (IV) COMPUTER READABLE FORM (A) MEDIUM TYPE Floppy disk (B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS (D) SOFTWARE Patentln Release #1 0, Version #1 30 (EPO) (2) INFORMATION FOR SEQ ID NO 1 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 91 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (n) MOLECULE TYPE protein (m) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 Ser Glu Leu Glu Lys Ala Val Val Ala Leu He Asp Val Phe His Gin 1 5 ' 10 15 Tyr Ser Gly Arg Glu Gly Asp Lys His Lys Leu Lys Lys Ser Glu Leu 20 25 30 Lys Glu Leu He Asn Asn Glu Leu Ser His Phe Leu Glu Glu He Lys 35 40 45 Glu Gin Glu Val Val Asp Lys Val Asn Glu Thr Leu Asp Ser Asp Gly 50 55 60 Asp Gly Glu Cys Asp Phe Gin Glu Phe Met Ala Phe Val Ala Met He 65 70 75 80 Thr Thr Ala Cys His Glu Phe Phe Glu His Glu 85 90 (2) INFORMATION FOR SEQ ID NO 2 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 38 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE N-terminal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 2 Ser Glu Leu Glu Lys Ala Met Val Ala Leu He Asp Val Phe His Gin 1 5 10 15 Tyr Ser Gly Arg Glu Gly Asp Lys His Lys Leu Lys Lys Ser Glu Leu 20 25 30 Lys Glu Leu He Asn Asn 35 (2) INFORMATION FOR SEQ ID NO 3 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 10 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 3 Thr Ala Cys His Glu Phe Phe Glu His Glu 1 5 10 (2) INFORMATION FOR SEQ ID NO 4 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 33 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 Ala Met Val Ala Leu He Asp Val Phe His Gin Tyr Ser Gly Arg Glu 1 5 10 15 Gly Asp Lys His Lys Leu Lys Lys Ser Glu Leu Lys Glu Leu He Asn 20 25 30 Asn (2) INFORMATION FOR SEQ ID NO 5 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 16 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE, peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 5 Arg Glu Gly Asp Lys His Lys Leu Lys Lys Ser Glu Leu Lys Glu Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO 6 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 6 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 Glu Phe Phe Glu His Glu 1 5 (2) INFORMATION FOR SEQ ID NO 7 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 10 amino acids (B) TYPE ammo acid (C) STRANDEDNESS (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 7 Asp Lys His Lys Leu Lys Lys Ser Glu Leu 1 " 5 10 (2) INFORMATION FOR SEQ ID NO 8 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 10 amino acids (B) TYPE amino acid (C) STRANDEDNESS (D) TOPOLOGY linear (11) MOLECULE TYPE peptide (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Homo sapiens (xi) SEQUENCE DESCRIPTION SEQ ID NO 8 Lys Leu Lys Lys Ser Glu Leu Lvs Glu Leu 1 5 10

Claims: Claims 1 A peptide consisting of at least one sub-fragment of the human S-1 OOβ polypeptide composing from 6 to 38 ammo acids, where said sub-fragments show at least 90% homology with the sequence SELEKAMVALIDWHQYSGREGDKΗ 1K_KSELKELINN (SEQ ID NO 2) and or the amino acid sequence TACHEFFEHE (SEQ ID NO 3) and retain essentially the same immunological properties 2 A peptide according to claim 1 characterized in that the sub-fragments are deoved from the amino acid sequence SELEKAMVALIDVPHQYSGREGDKI J<^KKSEL ELINN (SEQ ID NO 2) 3 A peptide according to claim 2, which is REGD HKLKK (SEQ ID NO 5), D HKLKKSEL (SEQ ID NO 7), or F L KSELKEL (SEQ ID NO 8) 4 A peptide according to claim 1 , characterized in that the sub-fragments are deoved from the amino acid sequence TACHEFFEHE (SEQ ID NO 3) 5 A peptide according to claim 4, which is EFFEHE (SEQ ID NO 6) 6 A peptide according to claim 1 , characterized in that it consists of at least one sub-fragment deoved from the sequence according to SEQ ID NO 2 and at least one sub-fragment deoved from the sequence according to SEQ ID NO 3 7 A monoclonal antibody or a fragment of such an antibody specifically binding a peptide according to anyone of the preceeding claims 8 A monoclonal antibody or an antibody fragment according to claim 7, specifically binding a peptide according to claim 2 9 A monoclonal antibody or an antibody fragment according to claim 7. specifically binding a peptide according to claim 4 10 Use of a monoclonal antibody or an antibody fragment according to anyone of claims 7-9 in lmmunological assay methods 1 1 Use of a peptide according to anyone of claims 1 -6 for eliciting antibodies 12 Use of a peptide according to anyone of claims 1 - 6 in immunological assay methods 13 A method of determining the presence of human S- 1 OOβ polypeptide in a sample composing the steps of letting the sample to be analyzed immuno logically react with a first monoclonal antibody according to claim 8, said first antibody being coupled to a earner, letting the sample immunologically react with a second monoclonal antibody according to claim 9, said second monoclonal antibody being provided with detection means. Washing, and detecting the amount of S-l OOβ polypeptide in the sample 14 A method according to claim 13 where the detection means is a group having the ability of emitting luminescence 1 5 A method according to claim 14 where the caroer is a magnetic particle 16 A kit for determining the presence of human S-l OOβ polypeptide in a sample, composing a peptide according to anyone of claims 1 - 6 and/or an antibody according to anyone of claims 7 - 9 17 A kit according to claim 16 composing a first monoclonal antibody according to claim 8 and a second monoclonal antibody according to claim 9, said first monoclonal antibody being coupled to a caroer and said second monoclonal antibody being provided with a detection means 18 A kit according to claim 17 wherein said caroer is a magnetic particle and said detection means is a group having the ability of emitting luminescence, such as luminol

Inventors: BRUNDELL JAN, NYBERG LENA, BRUNDELL, JAN, NYBERG, LENA
Applicants: BRUNDELL JAN, NYBERG LENA, SANGTEC MEDICAL AB, AB SANGTEC MEDICAL, BRUNDELL, JAN, NYBERG, LENA
Application year: 1997
Main classification: C07K14
IPC codes: C07K14, C07K16, C12P21, G01N33