MONOCLONAL ANTIBODY SPECIFIC TO HEART-TYPE FATTY ACID BINDING PROTEIN, HYBRIDOMA CELL PRODUCING SAME, AND PRODUCTION METHOD THEREOF

The present invention non-limiting in the embodiment hereinafter through more detailed as follows. The present invention is to end in the embodiment of the present invention range in the embodiment described as intended is exemplified that the number represented by one interprets the holes which are disclosed.

In the embodiment 1: Recombinant h -FABP Number bath

IRB B hospital patient sample in the table 1 with cardiac related security protection material through 95 °C 30 seconds for primer using, 53 °C 30 seconds, 72 °C 1 to 35 minutes (USA) yarn conducting PCR using Biorad circulation device.

PCR execution Hot Start Taq PCR MasterMix (Bioquest, USA) is 0. 4UM embodiment using primer and, in which is obtained a target DNA agarose gel electrophoretic about 400bp size has been confirmed. BL21 (DE3) after cloning using pGEX-a 4T 1 vector having same GST fusion persimmons transformed (codon +) after selecting his FABP3-a pGEX-a BL21 codon plus.

A recombinant strain transformed with 50 micro g/ml LB containing [aym the phosphorus which will bloom (FABP3-a pGEX-a BL21 (codon +)) of liquid medium is provided which reduces, freely cultivation-gate 37 °C 16 hours or more. Similarly [aym the phosphorus which will bloom LB medium containing liquid medium having an amount reference 2 times to 1/50 volume ratio 37 °C to 600 nm while cultured in the culture of said inoculated flasks absorbance at 0. 7 Reaches a final concentration of 1 mm IPTG induction conditions respectively have agents embodiment, induction during a period from 4 by him as further each time. 4 °C culture expressed, in centrifugal separator 16, 000xg 3 minutes by repeating the process that were unsafe. Cells (Novagen, USA) per 5 ml of bug 1g contact portions may not be separated by adding protein extract solution using a supernatant after FPLC suspended in a positive number which gives rise to raw material upon him.

In the embodiment 2: Mouse of immunizing

Number hybridoma cells required to obtain the connection immunization of the bath, a phosphate buffer solution (phosphate buffered saline; PBS) to 100 micro l recombinant h a-FABP 100 micro g at the same dilution (complete Freund's adjuvant, Sigma) emulsion was mixed with a high pressure liquid coolant volume of CFA number. 5 Female BALB/c mouse abdominal cavity with said emulsion by using immunization work conducting 1 week zero difference. In order to enhance the immunity of a mouse after immunization such as behind a 14 1 difference amount of recombinant h a-FABP IFA (Incomplete Freund's adjuvant, Sigma) total 3 times 2 mouse abdominal cavity after said number tank emulsion by mixing it with main scanning intervals stored in the immune response. 3 Cell line a phosphate buffer solution (phosphate buffered saline; PBS) recombinant h a-FABP 100 micro g number tank before the mouse tail vein to a scanning frequency of 100 micro l dilution to him.

In the embodiment 3: H -FABP Producing specific monoclonal antibodies Hybridomas Cell number bath

The SP2/0 mouse myeloma cells (myeloma) cell lines for supplying life science and biochemical laboratory cell fusion before university completed) him as first. After immunized mouse spleen cells in spleen SP2/0 mouse myeloma cell line is therefore of 2:1 is first prepared by extracting so mixed. Then, the supernatant after centrifuging the mixed cell misfortune stand-alone a number added to a 1 ml 1 minutes PEG-a 1500 (polyethylene glycol-a 1500) was embodiment fusion cells. Fused cells cultured in 96 well plate have HAT selection, cell colony was cultured in HT heating is formed.

In the embodiment 4: Monoclonal antibodies Producing Hybridomas Cell sorting and Cloning

In high pressure liquid coolant in the embodiment 3 number hybridoma cells microscopes cells continue observing the bottom of each well 10 - 20% NaOCl NaOCl degree, in order to identify whether h a-FABP reacts specifically only antibodies of hybridoma cells was by performing ELISA (Enzyme non-linked immunosorbent assay).

96 Well plate cell line culture into 100 micro l/well less attention then h a-FABP antigen by reacting, anti mouse IgG HRP (horse raddish peroxidase) on with respect to the reaction product. Then adding substrate solution, ELISA reader (BioRad) in 450 nm absorbance using cell lines was measured at least selecting SnO 1 1 absorbance. 1 Difference number selected for hybridoma cells after dilution ELISA performed identically to a test object a method performing hybridoma cells exhibiting high absorbance difference selection result 2 2 a method of selecting his species. Finally selected for hybridoma cells culture is transferred 75 T/C culture flasks, each 1G 4, 3F 2 was referred to.

In the embodiment 5: Selected for Hybridomas H - fromFABP Specific Monoclonal antibody Production

H a-FABP in the embodiment 4 to obtain specific monoclonal antibodies selected for hybridoma cells in culture number mouse abdominal cavity and high pressure liquid coolant, antibodies was separating therefrom.

A plurality liquid to obtain first antibodies (ascites) in advance before the BALB/c mouse hybridoma cells intraperitoneally (pristane) compositions comprising scanning a column scanning micro l/mouse was 500. In the embodiment 4 in 7 - 10 days post-administration selected for hybridoma cells in 75 T/C free compositions comprising mouse intraperitoneally was discharged through a scanning frequency of the culture flasks. 7 - 14 Days post-within the mouse abdominal cavity (ascites) are respectively determined, after the mask it boils each mouse using a cervical syringe plurality that were unsafe. Retrieving the plurality to separate and recover only supernatant by centrifuging to produce his -20 °C for archiving.

In the embodiment 6: H -FABP Specific Of the monoclonal antibodies Positive number

In the embodiment 5 (ascites) is recovered using affinity column chromatography method was positive to a plurality protein G column number. Ammonium sulfate fractionation (ascites) precipitating a plurality first recovered before positive number (GE health care, HiTrap protein G HP) after passing the epithelial eluent buffer Protein G column (elution buffer, 100 mm glycine a-HCl, pH 2. 7) Are separated from each other using, finally monoclonal antibody positive number was complete. (2 Also)

In the embodiment 7: Hybridomas Produced from cells Of the monoclonal antibodies H -The FABP Binding specificity for identifying

Of the present invention hybridoma cells (1G 4, 3F 2) produced from h a-FABP angptl4 selectively recognizing in order to identify binding specificity is confirmed to ELISA (Enzyme non-linked immunosorbent assay).

H a-FABP voice controls include BSA and GST fusion protein coating on recombinant buffer (0. 05M Na-a cabonate, pH 9. 5) 96 Well ELISA plate to 1 ug/ml concentration after each [...] mixed and are expanded, 0 same. Buffer solution including 5% phosphorus was to block the car number.

After this, a stand-alone solution after said number of the present invention hybridoma cells (1G 4, 3F 2) by reaction in the embodiment 4 monoclonal antibody produced from recombinant antigens against the method identical to the monoclonal antibody recognition ability of h a-FABP were measured.

As a result, hybridoma cells of the present invention (1G 4, 3F 2) voice matching of the troop BSA and GST would monoclonal antibody fusion proteins which comprise a little recognition by screening for antigen recognition capability without recombinant h-a FABP3 high shown. (3 Also)

Table 2 and 3 is monoclonal antibody (1G 4, 3F 2) of h a-FABP identifying for binding specificity and titer

{Antigen: ng/ml}

In the embodiment 8: Monoclonal antibodies Diagnostic use Kit Number bath and test

H a-FABP in high pressure liquid coolant in the embodiment 6 a number specific monoclonal antibody (1G 4) 1 mg/ml concentration by utilizing dividers nitro cellulose membrane on a mouse IgG anti - 37 °C after 10 in a continuous division number was dried and generates a high pressure liquid coolant antibody fastening strips.

Other monoclonal antibody (3F 2) is 0. Concentration 1 mg/ml to 30 minutes at room temperature with respect to the gold particle and reaction. 30 Minutes into a reaction solution 1% BSA to further reacted with 1% BSA buffer solution and then centrifuging the precipitate dissolves gold particle conjugates was reconstituted with a stand-alone number number antibody - high pressure liquid coolant. The high pressure liquid coolant - gold particle conjugates number antibodies fixing strip antibody immune chromatography palladium number was a high pressure liquid coolant.

H a-FABP is detected using a voice recombinant h a-FABP on serum results to identify limit detection sensitivity can be about 7 ng/ml has been. (4 Also)

In the embodiment 9: Anger misfortune of stable (hereinafter R1) Number bath

75 Mm HEPES buffer (pH 8. 0) BSA to a 0. R1 to a dicarboxylic acid 5% are obtained.

In the embodiment 10: Insoluble Carrier Fixing antibodies Sensitization Reagent (hereinafter R2) number of bath

10 Mg of 2% latex solution latex of the present invention monoclonal antibodies per 750ug MES buffer (pH 5. 8) 3 In stirring time-gate. 2% BSA blocking time after adding 1 is so agitating centrifuging the latex Tris a-HCl buffer (pH 8. 5) Is suspended in R2 are obtained.

In the embodiment 11: A h - latex reagentsOf FABP Quantitative

In the embodiment 9 10 via a number of R1 and R2 on high pressure liquid coolant using solution of automatic analyzer (Beckman Coulter AU680) were measured to agglutination reaction with h a-FABP. 450 Nm wavelength to the livestock products was also a result of the determination such as 5 in Figure 0 160 ng/mL h a-FABP measurable.