KR0135708B1 - Novel mab against human blood apolipoprotein b-100 and - Google Patents

Abstract

The present invention provides a novel monoclonal antibody that recognizes apolipoprotein B-100 in human blood, a recombinant hybridoma cell line producing them, and a method for measuring the concentration of plasma apolipoprotein B-100 using monoclonal antibodies.

Description

New Monoclonal Antibodies to Human Blood Apolipoprotein Non-100 (B-100) and Recombinant Hybridoma Cell Lines for Their Production

Figure 1 shows the results of analysis of blood samples taken from mice immunized with apolipoprotein B-100 before fusion reaction by enzyme immunoassay (ELISA).

FIG. 2A is a micrograph of one day after culture of a recombinant hybridoma cell line in a HAT selective medium. FIG.

FIG. 2B is a micrograph of colonies formed as a result of culturing recombinant hybridoma cell lines in HAT selective medium.

3A shows the results of polyacrylamide gel electrophoresis of plasma proteins to analyze the specificity of monoclonal antibodies of the present invention.

Figure 3 B is Western blotting analysis of the gel of Figure 3 A using the monoclonal antibody MabB9 of the present invention.

FIG. 3C shows Western blotting analysis of the gel of FIG. 3A using the monoclonal antibody MabB23 of the present invention.

4 is isotyping the H-chain and L-chain of the monoclonal antibodies MabB9 and MabB23 of the present invention using an enzyme immunoassay.

5 is a titration curve of the monoclonal antibodies MabB9 and MabB23 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100.

6 is a standard curve of the monoclonal antibodies MabB9 and MabB23 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100.

The present invention relates to novel monoclonal antibodies, hybridoma cell lines and their use for producing them.

More specifically, the present invention relates to a protein portion of a low-density lipoprotein (LDL), ie, apolipoprotein, which mainly carries cholesterol and triglycerides in plasma lipoproteins involved in lipid metabolism in the blood. A novel monoclonal antibody against apolipoprotein B-100, a hybridoma cell line for producing it and its use for measuring the concentration of apolipoprotein B-100 in human blood.

Lipids in the blood (triglycerides, cholesterol and cholesterol esters, phosphate fats) are transported together with apolipoproteins to form a complex called lipoproteins. Lipoproteins contain triglycerides (triacyl glycerol, TG) and cholesterol esters, which are non-polar lipids, in the center of which are surrounded by polar lipids such as phosphate and cholesterol. There is a film of which forms a water-soluble mold as a whole.

Apolipoproteins are proteins that have been freed from fat by treating lipoproteins obtained from plasma with organic solvents. About 10 kinds of apolipoproteins are known from different immunological and chemical characteristics. The in vivo function of apolipoproteins plays an important key role in lipid metabolism, including the transport and redistribution of lipids between lipids, the activity of enzymes involved in lipid metabolism, the maintenance of water-soluble structures of lipoproteins, and their role as target proteins for lipoprotein receptors.

In the meantime, many immunological methods for the measurement of apolipoproteins in the blood, such as radial immunodiffusion, electroimmuoassay, radioimmunoassay, and enzyme-linked immnosorbentassay: ELISA), immunoturbidimety (Labeur C., Shepherd J., and Rosseneu, M., Clin. Chem. 36, 591-597 (1990)).

Among the various apolipoproteins in plasma lipoproteins, especially low-density lipoprotein (LDL), apolipoprotein B-100 has recently been analyzed by conventional cholesterol analysis (low density lipoprotein-cholesterol) in connection with the diagnosis of circulatory diseases such as atherosclerosis and coronary artery disease. It is known as a more reliable positive magnetic field (Brustolin D. ed al., Clin. Chem. 37, 742-747 (1991): Dona V. et al., Giorn. It. Chim. 12, 205 -214 (1987)).

Therefore, circulatory disease can be diagnosed easily and accurately by measuring blood concentration of apolipoprotein B-100. For this purpose, it is essential to develop monoclonal antibodies specific for apolipoprotein B-100.

Accordingly, the present invention provides a monoclonal antibody MaBb9 having a structure of H-chain IgG2b and L-chain κ, which specifically reacts against apolipoprotein B-100 in human blood, wherein the H-chain is IgG2b and the L-chain is λ. It provides a monoclonal antibody MaBb23 of structure.

The present invention also provides a method for producing the monoclonal antibody using the hybridoma cell lines H-MaBb9 (KCTC 0104 BP) and H-MaBb23 (KCTC 0105BP) for producing the monoclonal antibody.

In another aspect, the present invention provides a method for measuring the concentration of lipoproteins including plasma apolipoprotein B-100 using the monoclonal antibody.

Hereinafter, the present invention will be described in more detail.

Apolipoprotein antigens to be used for immunoinjection for the production of monoclonal antibodies from human blood are isolated purely by methods such as KBr density chromatography, a conventional method (Methods Enzym, 128, Section IIIII). The isolated apolipoprotein B-100 was well mixed with Freund's complete adjuvant and dead Salonella cells and intraperitoneally injected into mouse Balb / c females. After 20 days, the apolipoprotein B-100 was supplemented with Freund's complete adjuvant. inject additionally by mixing with incomplete adjuvant). Afterwards, a further injection of apolipoprotein B-100 dissolved in PBS buffer (f10 mM sodium phosphate, 150 mM NaCl, pH 7.2) is carried out every 10 days until the experimental serum from mice is positively tested for apolipoprotein B-100. . The final injection is administered intravenously, and on the third day after the last injection, the rat is opened and the body length is taken out and comminuted. Massed pancreatic cells were treated with mouse myeloma by 1/10 of the cell count in the presence of 50% polyethylene glycol (PEG) according to known methods (Galfre G. et al., Nature 266, 500-552 (1977)). After fusion reaction with the cells (Sp2 / 0), incubate for one week in HAT (Hypoxanthin, Aminopterine, Thymidine) selection medium. The resulting colonies are diluted and cultured in 96-collective plates by limiting dilution and the antibodies are analyzed in antigen-coated titers according to enzyme immunoassay to finally select hybridoma cell lines producing monoclonal antibodies.

The selected cell lines were cultured in a suitable medium, and the obtained culture was passed through an antigen-immobilized column or Protein-a column to monoclonal antibodies MaBb9 and MaBb23 against apolipoprotein B-100. Purify and use.

The Shoso-linked immunoassay (ELISA) to analyze the concentration of apolipoprotein B-100 in blood using monoclonal antibodies is carried out as follows.

Place an appropriate amount of epitope portion of apolipoprotein B-100, low density lipoprotein (LDL), or apolipoprotein molecule produced by chemical synthesis or genetic recombination, or a combination thereof on a 96-microtiter plate or a suitable solid surface The reaction is carried out at 4 ° C. to a suitable temperature for one day to cover the adsorption.

Dilute the sample to be measured at about 200-fold to an appropriate ratio, add the monoclonal antibody MaBb9 or MaBb23 (primary antibody) to the assay plate, and react and wash at room temperature to 37 ° C. for a day. According to the assay (ELISA), the enzyme-bound secondary antibody and enzyme substrate are added to the assay plate and developed. The absorbance is read with an ELISA reader to determine the concentration of apolipoprotein in the sample. As a secondary antibody, an antibody combined with a peroxidase or alkaline phosphatase is used.In this case, a secondary antibody is used directly by combining the primary antibody with the enzyme instead of a secondary antibody bound to the enzyme, thereby not using a secondary antibody. The analysis step can be reduced.

The enzyme-linked immunoassay of the present invention can be used for the production of a diagnostic card for measuring the concentration of apolipoprotein B-100 in human blood in connection with the diagnosis of circulatory diseases, and can be used for the early treatment of atherosclerosis, coronary heart disease and stroke. It can be usefully applied for prevention and early treatment through diagnosis.

The accompanying drawings will be described in detail as follows.

Figure 1 shows the results of analysis of blood samples taken from mice immunized with apolipoprotein B-100 before fusion reaction by enzyme immunoassay (ELISA). Antiserum was diluted every 10 times (dilution rate for each number: 1, 10 ¹ : 2, 10 ² : 3, 10 ³ : 4, 10 ⁴ : 5, 10 ⁵ : 6, 10 ⁶¹ ), and N was immunized. It is a negative control using the blood of mice collected before the following.

A of FIG. 2 is a micrograph 1 day after culture of a recombinant hybridoma cell line in HAT selective medium. After fusion reaction between pancreatic cells and myeloma cell line Sp2 / 0 of mice immunized with apolipoprotein B-100, the cells were cultured in HAT (hypoxanthin, aminopterine, thymidine) selective medium and observed at 100-fold magnification after 1 day of culture. In contrast, relatively large, round-shaped lymphocytes are shown in the background of reelfossite and dead cell remnants that are not fused with macrophage.

B of FIG. 2 is a micrograph of colonies formed as a result of culturing recombinant hybridoma cell lines in HAT selective medium.

A of FIG. 3 shows the results of polyacrylamide gel electrophoresis of plasma proteins to analyze the specificity of monoclonal antibodies of the present invention. 1,2 is protein molecular weight standard, 3 is total human plasma, 4 is low density lipoprotein (LDL), 5 is high density lipoprotein (HDL) and 6 is lipoprotein-deficient serum (LPDS).

B of FIG. 3 shows the result of Western blotting analysis of the gel of FIG. 3 A using the monoclonal antibody MabB9 of the present invention. 1 is inferior serum, 2 is low density lipoprotein (LDL), 3 is high density lipoprotein (HDL), 4 is lipoprotein-deficient serum (LPDS).

Figure C shows the results of Western blotting analysis of the gel of Figure 3 A using the monoclonal antibody MabB23 of the present invention. 1 is human serum, 2 is low density lipoprotein (LDL), 3 is high density lipoprotein (HDL), 4 is lipoprotein-deficient serum (LPDS). The multiple bands shown in Figure 2 are due to overloading high molecular weight apolipoprotein B-100 onto the electrophoretic gel or due to partial degradation of the apolipoprotein B-100 and the low band at 1 Corresponds to Apolipoprotein B-100 in lipoprotein.

Figure 4 shows the results of isotyping the H-chain and L-chain of the monoclonal antibodies MabB9 and MabB23 of the present invention using an enzyme immunoassay. Enzyme-immunoassays were antigen-dependent (horizontal lines A to D) and antigen-independent methods were either total lipoprotein complexes (horizontal lines A and B) or purely isolated apolipoprotein B-100 (horizontal lines C and D). Pretreatment coating was used. As a monoclonal antibody, culture medium of a hybridoma cell line producing MaBb9 (line A, C and E) or MaBb23 (line B, D and F) was used. Results are shown using rabbit antiserum specific to isotype. N, normal rabbit serum, G1 to K, each type of mouse antibody isolated from rabbit, ie IgG1 (G1), IgG2a (G2a), IgG2b (G2b), IgG3 (G3), IgM (M), IgA (A ), Lambda L-chain (λ) or kappa L-chain (K), vertical P is antiserum isolated from rabbits responding to all antibody types in mice (+) for control (horizontal lines E and F) ).

5 shows the titration curves of the monoclonal antibodies MabB9 and MabB23 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100. Monoclonal antibody-producing cultures were used diluted as indicated on the horizontal axis, and arrows indicate the titers of antibody use corresponding to 50% of the maximum binding.

Figure 6 shows the standard curve of the monoclonal antibodies MabB9 and MabB23 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples are illustrated for the purpose of illustrating the invention and are not intended to limit the scope of the invention.

Example 1 Preparation of Hybridoma Cell Line

Sequential centrifugation of apolipoprotein antigen to be used for immunization for the production of monoclonal antibody from human blood in a KBr solution of constant density (15 ° C., Method Enzym, 128, Section II III). 45,000 rpm, Beckman Ti70 (24 hours each) to separate low-density low-proteins with a density purchase of 1.03 <d <1.05, and to remove pure water under organic solvent (50%, isopropanol) and gel recovery by electrophoresis. It was. 20 μg of isolated apolipoprotein B-100 was mixed with Freund's complete adjuvant and dead Salonella cells intraperitoneally to mouse Balb / c females and injected with the same amount of apolipoprotein B-100 20 days later. It was. Subsequently, an additional injection of 20 μg of apolipoprotein B-100 dissolved in PBS buffer (10 mM sodium phosphate, 150 mM Nacl pH 7.2) was performed every 10 days until experimental serum from mice was positively tested for apolipoprotein B-100. It was. The final injection was administered intravenously, three days after the last injection, the rats were opened and the pancreas was taken out and comminuted. Crushed pancreatic cells were mouse myeloma (1/10 of the number of pancreatic cells in the presence of 50% polyethylene glycol (PEG) according to known methods (Galfre G. et al., Nature 266, 550-552 (1977)). After fusion reaction with cells (Sp2 / 0), the cells were cultured in HAT (hypoxanthin, aminopterine, thymidine) selection medium (Gibco 31062-037) for one week. Sample serum was collected from the eyes of the immunized mice prior to the fusion reaction and the antibody production in the serum was confirmed by enzyme immunoassay (Elsia). As shown in FIG. 1, the antibody against human plasma apolipoprotein B-100 was well expressed in the serum of the mouse. It was confirmed that it was generated. After the fusion reaction, cell growth was observed in all 270 culture holes of the untreated plate used, among which cells not in the fusion reaction were added to HAT selective medium to inhibit the growth (see FIG. 2A). The production of antibodies against apolipoprotein B-100 in the culture solution was analyzed by enzyme immunoassay, and the cells of the positively-reacted cultured cells were transferred to a new titration plate and cultivated by limiting dilution to obtain the same antibody as in B of FIG. Two stable hybridoma cell lines were obtained, of which two hybridoma cell lines H-MaBb9 and H-MaBb23, which produce IgG-type monoclonal antibodies MaBb9 and MaBb23, were selected and these cell lines were genetically engineered by the Korea Institute of Science and Technology. It was deposited with the Institute Gene Bank on March 23, 1994 under accession numbers KCTC 0104 BP and KCTC 0105 PB, respectively.

Example 2 Specificity Analysis of Monoclonal Antibodies

Western blotting was performed to determine the antigen-binding specificity of the monoclonal antibodies MaBb9 and MaBb23. That is, about 0.5 μg of plasma proteins were electrophoresed in polyacrylamido gels with a gradient of 4 to 5% in the presence of SDS according to the method of Laemmli, Nature 227, 680 (1970), isolated on gels. Was transferred to a nitrocellulose membrane (Millipore, pore size 0.45 μm) according to Tobin's method (Towbin et al., Proc, Natl, Acad, Sci, USA 76, 4350-4354 (1979)). The filter was added to PBS buffer containing 0.5% Tween 20 and allowed to react, and then the filter was washed twice with 15 minutes PBS buffer containing 0.05% Tween 20. A secondary antibody (goat's anti-mouse immunoglobulin G labeled with horseradish peroxidase) was added to the filter and reacted with the PBS buffer. Finally, the filter was developed by adding hydrogen peroxide and ο-phenylenediamine as a substrate. As a result, the monoclonal antibodies MaBb9 and MaBb23 specifically bind to apolipoprotein B-100 in human plasma proteins as shown in FIGS. 3B and C and other proteins in high density lipoprotein (HDL) or lipoprotein-deficient serum (LPDS). It showed excellent specificity that did not bind to proteins.

Example 3 Isotype Analysis of Monoclonal Antibodies

Antigen-dependent methods using secondary antibodies specific to each type to investigate H-chain and L-chain isotypes among antibody molecules of the monoclonal antibodies MaBb9 and MaBb23 (Figure 4 and Table 1). Or enzymatic immunoassays in an antigen-independent manner (FIG. 4 and Table 2). As a result, it was found that monoclonal antibody MaBb9 has IgG2b type as H-chain, κ as L-chain, and monoclonal antibody MaBb23 has IgG2b type as H-chain and λ as L-chain.

Table 1 Results of isotype analysis of monoclonal antibodies using antigen-dependent enzyme immunoassay

Put the monoclonal antibody MaBb9 or MaBb23 into an unqualified plate of total lipoprotein complex (0.5 μg / 100 μl) and add one of the antisera specific to each isotype of mouse antibody isolated from rabbits according to the enzyme immunoassay (ELISA). Analysis was carried out using anti-rabbit immunoglobulins isolated from goats, with horseradish peroxidase bound, and their color development was expressed as absorbance measurements at 650 nm. 1 normal rabbit serum, anti-mouse IgG1 (2), IgG2a (3), IgG2b (4), IgG3 (5), IgM (6), IgA (7), lambda L-chains isolated from 2-9 rabbits (8) or antiserum against kappa L-chain (9).

Table 2 Results of isotype analysis of monoclonal antibodies using antigen-dependent enzyme immunoassay

In goats coated with horseradish peroxidase in accordance with an enzyme immunoassay (ELISA), which was coated with an unanticipated plate of one of the antisera specific for isotypes of mouse antibodies isolated from rabbits and which contained the monoclonal antibody MaBb9 or MaBb23. Analysis was performed using the isolated anti-rabbit immunoglobulin and the degree of development was expressed as absorbance measurements at 650 nm. 1 normal rabbit serum, anti-mouse IgG1 (2), IgG2a (3), IgG2b (4), IgG3 (5), IgM (6), IgA (7), lambda L-chains isolated from 2-9 rabbits (8) or antiserum against kappa L-chain (9), 10 (+) control serum.

Example 4 Determination of Blood Apolipoprotein B-100 Concentration

The following competitive enzyme immunoassay method was used to use the monoclonal antibody MaBb9 or MaBb23 for the measurement of blood apolipoprotein B-100 concentration. 100 μg of PBS buffer containing 0.5 μl of LDL-apolipoprotein B standard was adsorbed overnight at 4 ° C., followed by PBS containing 0.05% bovine serum albumin (BSA) and 0.05% Tween 20. The lipoproteins that were not adsorbed with the buffer were washed away. Then, in order to prevent nonspecific binding, PBS buffer containing 1% bovine serum albumin and 2% (Carnation Co.) was gently shaken at room temperature for 1 hour, and then washed again with the PBS buffer. Here, 50 µl of a solution containing various concentrations of apolipoprotein B-100 was added together with 50 µl of monoclonal antibody culture (primary antibody) diluted in PBS buffer and reacted gently at 4 ° C. overnight. Secondary antibody (goat anti-rabbit IgG) conjugated with horseradish peroxidase after washing out the remaining lipoproteins that were not adsorbed with PBS buffer containing 0.05% bovine serum albumin (BSA) and 0.05% Tween 20 Was developed using the above antibody or bound to these enzymes as the primary antibody, and the absorbance was measured at 450 nm with an ELISA reader.

The monoclonal antibody MaBb9 or MaBb23 against apolipoprotein B-100 was suitably diluted to about 600-fold as can be seen in FIG.

In addition, in order to determine the concentration range of the apolipoprotein B-100 suitable for the immunoassay, the standard curves of absorbance at 450 nm and the concentration function of the apolipoprotein B-100 were drawn. As shown in FIG. Was found to be 0.125-80 μl / ml, demonstrating that the monoclonal antibodies MaBb9 and MaBb23 of the present invention are clinically useful for measuring the concentration of Apolipoprotein B-100.

Claims (6)

Monoclonal antibodies specifically reactive against apolipoprotein B-100 in human blood and produced by the hybridoma cell line H-MabB9 (KCTC 0104 BP), wherein the H-chain is IgG2b and the L-chain is κ MaBb9. Monoclonal antibody with specific response to human blood apolipoprotein B-100, produced by hybridoma cell line H-MabB23 (KCTC 0105 BP), H-chain IgG2b, L-chain λ MaBb23. Hybridoma cell line H-MabB9 (KCTC 0104 BP), which specifically responds to human blood apolipoprotein B-100 and produces monoclonal antibodies. Hybridoma cell line H-MabB23 (KCTC 0105 BP) that specifically responds to human blood apolipoprotein B-100 and produces monoclonal antibodies. A method for producing a monoclonal antibody that specifically reacts against human blood apolipoprotein B-100, comprising culturing the hybridoma cell line according to claim 3 or 4 in a suitable medium or animal body. Method for measuring the concentration of plasma apolipoprotein B-100 using the monoclonal antibody of claim 1