Patent Application: US-201515114682-A

Abstract:
monoclonal antibodies , which can be produced in vitro , against cardiac epitopes of the human my - c are produced by generating myeloma cell clones that produce such specific antibodies having epitope specificity . these monoclonal antibodies allow , among other things , the creation of an enzyme - linked immunosorbent assay for the specific , cross - reactivity - free quantitative determination of my - c in serum , plasma , whole blood or other body fluid . specifically , a hybridoma cell clone producing a monoclonal antibody that detects and binds a cardiac epitope in the my - c is produced , which has no cross - reactivity with respect to the myosin - binding proteins of the skeletal muscles . the hybridoma cell line can be obtained by fusing myeloma cells with spleen cells of a test animal , in particular a mouse , immunized against recombinant my - c . the invention furthermore relates to epitope - specific antibodies produced by the hybridoma cell line , and to the use thereof .

Description:
the invention will be described in more detail hereafter by way of exemplary embodiments . the spleen of a mouse immunized in the known manner with cc0c2 of the my - c is removed under sterile conditions , and the spleen cells are flushed out of the spleen capsule using rpmi 1640 medium ( life technologies ™, karlsruhe ) with a syringe and isolated . the spleen cells are pelletized ( 10 minutes at 300 × g ), washed three times with rpmi 1640 medium , and resuspended in rpmi 1640 medium . they are then fused with myeloma cells of the line p3x63ag8 . 653 ( attc cpl 1580 ). for this purpose , cultivated myeloma cells , which are in the log phase of growth , are likewise pelletized and washed three times . 1 × 10 8 spleen cells and 5 × 10 7 myeloma cells are pipetted into a centrifuge tube , mixed intensively and centrifuged ; 1 . 5 ml preheated 50 % polyethylene glycol 1500 ( roche , basel ) is added dropwise to the cell sediment within one minute , while the tubule is continuously rotated at 37 ° c . the fusion batch is then incubated for another minute at 37 ° c . in the following three minutes , preheated medium ( rpmi 1640 ) is added dropwise , 1 ml being added in the first minute , 3 ml in the second minute , and then 18 ml . centrifuging is carried out immediately thereafter at 200 × g for 10 minutes . the cell pellet is placed in rpmi 1640 medium comprising 10 % fcs and hat . a portion of the pellets are seeded in 96 - well culture plates , and the remainder is frozen in liquid nitrogen at − 196 ° c . mouse peritoneal macrophages , which were cultivated 1 day prior to the fusion ( 1 × 10 4 macrophages per well in hat medium ), are used as feeder cells during cultivation . the cells are incubated in a co 2 incubator at 37 ° c . the medium is replaced after 3 to 5 days , respectively , with fresh rpmi 1640 hat medium , and , depending on the growth of the fused cells , the culture supernatants are tested after approximately 2 weeks for the reactivity thereof with respect to the antigen ( my - c ) using an elisa . all growing clones or the antibodies thereof were tested for reactivity using an enzyme - linked immunosorbent assay ( elisa ). the immunosorbent was the immunogen , this being the recombinant cc0c2 domain of the my - c ( approximately 2 μg / ml ). 1 . coat each of the microtiter plates ( costar , high binding ) with 50 μl immunogen solution per well at 4 ° c . over night ; 2 . wash the microtiter plates ( mtp ) 3 times with tris - buffered saline ( tbs ), ph 7 . 4 ; 3 . block the mtp using 200 μl blocking reagent ( boehringer , mannheim ) per well , 1 hour at 37 ° c . ; 4 . wash the mtp 3 times with nacl tween 20 ; 5 . incubate with culture supernatant of the hybridoma cultures ; 50 μl per well , respectively , diluted approximately 1 : 2 with tbs tween 20 ; 6 . wash the mtp 3 times with nacl tween 20 ; 7 . incubate with anti - mouse ig antibodies , coupled to peroxidase , 50 μl per well , 1 hour at room temperature ; 8 . wash the mtp 3 times with nacl tween 20 ; 9 . incubate with abts solution ( 100 mg abts per 100 ml substrate buffer [ citrate , sodium perborate , ph 4 . 4 ]), 50 μl per well ; 10 . measure at 405 nm after an incubation time of 60 minutes at room temperature using a microplate reader ( slt ). epitope mapping for the monoclonal antibody 3h8 in the human cardiac my - c the binding site of the monoclonal antibody 3h8 was identified by way of the peptide scanning method . for this purpose , the entire amino acid sequence of the human cc0c2 domain of the my - c that was used for the immunization is divided into a total of 111 overlapping amino acid sequences , each having a length of 15 amino acids . these sequences are synthesized as individual peptides in spots directly on a cellulose membrane . the membrane is incubated with the antibody - containing culture supernatants of the hybridomas , and the binding sites of the antibodies are rendered visible by way of incubation with a peroxidase - coupled anti - mouse ig antibody . for this purpose , after washing three times with tbs tween , the membrane is placed between copy film , and then incubated for 3 minutes with the ecl ™ ( enhanced chemiluminescent ) detection reagent ( amersham , braunschweig ). an applied film ( hyperfilm ecl ™ [ rpn 2103h amersham , braunschweig ]) is thereafter exposed to light , for between 30 seconds and 3 minutes . the identification of the sequences detected by the antibody takes place by assigning the spots 37 and 38 ( fig4 ) exposed on the film to the 15 - mer partial sequences of the immunogen ( cc0c2 domain of the my - c ) localized in the spots . the identified central sequence of the two partial sequences is the amino acid sequence — a149 - p - d - d - p - i - g - l - f - v - m -. this sequence is the detected epitope to which the antibody 3h8 binds in the human my - c . 1 . cooper a , timmis a , skinner j . assessment of recent onset chest pain or discomfort of suspected cardiac origin : summary of nice guidance . bmj . 2010 ; 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