KR20000002227A - Novel monoclonic antibodies for human plasma apolipoprotein and hybridoma strains which produce them - Google Patents

Abstract

PURPOSE: The monoclonal antibody mAbB55 and HmAbB128 which are produced by hybridoma strains H-mAbB55 and mAbB128 showing a superior binding specificity to human apolipoprotein B and can be used for diagnosing blood circulatory organ disease. CONSTITUTION: The human apolipoprotein B-100 antibodies are injected into a mouse for immunization of the pancreas and coalesced with myeloma cell for producing hybridoma strains. The binding specificity to apolipoprotein B-100 of monoclonal antibodies, which are produced by hybridoma strains, is analyzed by Western Blatting Method and enzyme immunoassay. The potency of monoclonal antibodies and the concentration of apolipoprotein B-100, which is bound to monoclonal antibodies, are measured by enzyme immune-assay.

Description

Novel Monoclonal Antibodies to Human Plasma Apolipoprotein Ｂ-100 and Hybridoma Cell Lines Producing the Same

The present invention relates to novel monoclonal antibodies against human plasma apolipoprotein B-100, hybridoma cell lines and the use of the novel monoclonal antibodies to produce them. More specifically, the present invention provides a novel monoclonal antibody against apolipoprotein B-100, which is a protein part of low-density lipoprotein (LDL) that carries cholesterol and triglycerides in the plasma to the tissues required by the body. And a method for measuring the concentration of apolipoprotein B-100 in human blood using the hybridoma cell line producing the same and the novel monoclonal antibody.

Fats in the blood (triglycerides, cholesterol and cholesterol esters, phosphate fats) are transported together with apolipoproteins in a complex called lipoproteins. Lipoproteins have triglycerides (triacyl glycerol, TG) and cholesterol esters, which are nonpolar lipids, in the center of them, and the surface (around) is surrounded by polar fats such as phosphate and cholesterol. There is a protein coating, which makes the particles water-soluble as a whole. Apolipoprotein is a protein part obtained by treating lipoproteins from plasma with organic solvents to remove fats. There are about 10 types of apolipoproteins that differ in immunological and chemical characteristics. It plays an important key role in lipid metabolism, such as activating factors of enzymes involved, maintaining the water-soluble structure of lipoproteins, and acting as a target protein for lipoprotein receptors.

Many immunological methods for measuring blood apolipoproteins in the art include, for example, radial immunodiffusion, electroimmunoassay, radioimmunoassay, and enzyme-linked immunosorbent assays. ELISA), immunoturbidimetry, and the like (Labeur C., Shepherd J., and Rosseneu,

M., Clin. Chem. 36, 591-597 (1990)). Among several apolipoproteins in plasma lipoproteins, especially low-density lipoprotein (LDL), apolipoprotein B-100 is more recently compared to conventional cholesterol analysis (low-density lipoprotein-cholesterol) in the diagnosis of circulatory diseases such as atherosclerosis and coronary heart disease. It is known as a more reliable indicator of positive (Brustolin D. et al., Clin. Chem. 37, 742-747 (1991); Dona V. et al., Giorn. It. Chim. Clin. 12,205- 214 (1987). Therefore, by measuring blood levels of apolipoprotein B-100, circulatory disease can be diagnosed primarily and reliably. For this purpose, it is essential to develop monoclonal antibodies specific for apolipoprotein B-100.

Accordingly, an object of the present invention is to provide a monoclonal antibody that specifically reacts against apolipoprotein B-100 and a hybridoma cell line producing the same. Another object of the present invention is to measure the concentration of apolipoprotein B-100 using the monoclonal antibody that specifically reacts against apolipoprotein B-100, thereby diagnosing atherosclerosis or coronary artery disease. To provide a diagnostic method.

The object of the present invention is to inject a human apolipoprotein B-100 antigen into a mouse to immunize a mouse with apolipoprotein B-100 and to produce a hybridoma cell line by fusion of pancreatic cells of the mouse with myeloma cells. Among hybridoma cell lines, stable hybridoma cell lines were selected while producing monoclonal antibodies against apolipoprotein B-100. The specificity of binding of the monoclonal antibody produced by the selected hybridoma cell line to apolipoprotein B-100 was analyzed by Western blotting method, and the isotype of the monoclonal antibody was analyzed by enzyme-immunoassay using specific secondary antibodies. The titer of the monoclonal antibody and the concentration of apolipoprotein B-100 bound to the monoclonal antibody were then also achieved by measuring the enzyme immunoassay.

Hereinafter will be described the detailed configuration and operation of the present invention.

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

Figure 2a is a photograph of the observation of the microscope after culturing the recombinant hybridoma cell line in HAT selective medium for one day.

Figure 2b is a micrograph of colonies formed after culturing recombinant hybridoma cell lines in HAT selective medium.

Figure 3a shows the results of polyacrylamide gel electrophoresis of plasma proteins to analyze the specificity of the monoclonal antibody mAbB55 of the present invention.

Figure 3b shows the results of Western blotting analysis of the protein of Figure 3a using the monoclonal antibody mAbB55.

Figure 4a shows the results of polyacrylamide gel electrophoresis of the plasma protein to analyze the specificity of the monoclonal antibody mAbB128 of the present invention.

FIG. 4B shows the result of Western blotting analysis of the protein of FIG. 4A using the monoclonal antibody mAbB128. FIG.

Figure 5 shows the titration curves of the monoclonal antibodies mAbB55 and mAbB128 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100.

Figure 6 shows the standard curve of the monoclonal antibodies mAbB55 and mAbB128 antibodies of the present invention for analyzing the concentration of apolipoprotein B-100.

The present invention comprises the steps of immunizing a mouse with apolipoprotein B-100 antigen by injecting a human with apolipoprotein B-100 antigen to be used as a vaccine, and then injected into the mouse with a supplement. Pancreatic cells of the immunized mice were fused with myeloma cells in the presence of polyethylene glycol and cultured to obtain hybridoma cell lines. Screening; Assaying the antigen-binding specificity of the monoclonal antibodies mAbB55 and mAbB128 by Western blotting method; Analyzing the isotype of each of the monoclonal antibodies by specific antigen-dependent enzymatic immunoassay using secondary antibodies specific for the monoclonal antibodies mAbB55 and mAbB128, respectively; Reacting the low density lipoprotein (LDL) -apolipoprotein B standard with the monoclonal antibodies mAbB55 and mAbB128, binding the secondary antibodies, and measuring the titers of the monoclonal antibodies mAbB55 and mAbB128 using an Eliza reader; It consists of reacting different concentrations of LDL-apolipoprotein B standard with the monoclonal antibodies mAbB55 and mAbB128, binding the secondary antibody, and measuring the concentration of apolipoprotein B-100 using an Eliza reader.

Hereinafter, the specific method of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

Example 1: Hybridoma Cell Line Preparation

Experimental Example 1: Immunization of Mice

Sequential centrifugation of apolipoprotein antigens for use in immuno-injection for the production of monoclonal antibodies of the present invention from human blood in a KBr solution of constant density according to conventional methods (References: Methods Enzym. 128, Section II & III) (15oC, 45,000 rpm, Beckman Ti70, 24 hours each) to separate low-density lipoproteins by density gradient (1.03 <d <1.05), to remove fats under organic solvent (50% isopropanol), and gel recovery by electrophoresis. Pure separation with water. 20 µg of the isolated antigenic apolipoprotein B-100 was mixed with Freund's complete adjuvant and dead Salmonella cells intraperitoneally to mouse Balb / c females. After 20 days, the same amount of apolipoprotein B-100 was supplemented. Apolipoise dissolved in PBS buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.2) until mixed with Freund's incomplete adjuvant and experimental serum from mice was tested positive for apolipoprotein B-100. 20 μg of protein B-100 was injected at 10 day intervals and the last injection was given intravenously.

Experimental Example 2: Cell Fusion

In Experimental Example 1, the rat was opened three days after the last intravenous injection, and the pancreas was taken out.

Crushed. 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 the cells (Sp2 / 0), the cells were incubated for one week in a HAT (Hypoxanthine, Aminopterine, Thymidine) selection medium (Gibco, product number 31062-037). Sample serum was taken from the eyes of the immunized mice prior to the fusion reaction to confirm antibody production in serum by enzyme-linked immunoassay (ELISA). As a result, as shown in FIG. 1, it was confirmed that the antibody against human plasma apolipoprotein B-100 was well formed in the serum of the mouse, and after the fusion reaction, cell growth was observed in all 270 untreated plates. Relatively round lymphocyte cells were lymphocytes and dead cell residues that did not fuse with macrophage (FIG. 2A). Among them, cells that did not fusion reaction were inhibited by adding HAT (Hypoxanthine, Aminopterine, Thymidine) selection medium. The production of antibodies against the apolipoprotein B-100 antigen in the culture medium was analyzed by enzyme-immunoassay, and the cells of the positively-reacted cells were transferred to a new titration plate and cultivated by limiting dilution to 12 stable hybridomas. Cell lines were obtained (FIG. 2b), of which two hybridoma cell lines H-mAbB55 and H-mAbB128, which produce IgG-type monoclonal antibodies mAbB55 and mAbB128, were selected and these cell lines were attached to the Korea Institute of Science and Technology. On March 3, 1998, they deposited their gene banks with KCTC 0439BP and KCTC 0440BP.

Example 2: Specificity Analysis of Monoclonal Antibodies

Western blotting was performed to assay the antigen-binding specificity of the monoclonal antibodies mAbB55 and mAbB128 obtained in Example 1. That is, about 0.5 μg of plasma proteins

The separation was performed by electrophoresis on a polyacrylamide gel with a gradient of 4-15% in the presence of SDS according to the method of Laymmli, Nature 227, 680 (1970). 3a shows the result of electrophoresis of mAbB55 and FIG. 4a shows the result of electrophoresis of mAbB128. The protein isolated on the gel was placed on a nitrocellulose membrane (Millipore, pore size 0.45 ml) according to Tobin's method (Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1979)). Moved. The filter was reacted with a solution containing 0.5% bovine serum albumin (BSA) to mask nonspecific binding, followed by addition of monoclonal antibody mAbB55 or mAbB128 to PBS buffer containing 0.05% Tween 20 , Filter was washed twice with PBS buffer containing 0.05% Tween 20 for 15 minutes and reacted with a secondary antibody (horseradish peroxidase-labeled goat anti-mouse immunoglobulin G). The resultant was washed with the above PBS buffer, and finally, the filter was developed by adding hydrogen peroxide and o-phenylene diamine as a substrate. As shown in FIGS. 3B and 4B, the monoclonal antibodies mAbB55 and mAbB128 specifically bind to apolipoprotein B in human plasma proteins and to other proteins in high density lipoprotein (HDL) or lipoprotein-deficient serum (LPDS). Did not show excellent specificity.

Example 3: Isotype Analysis of Monoclonal Antibodies

In order to examine the H-chain and L-chain isotypes among the antibody molecules of the monoclonal antibodies mAbB55 and mAbB128, enzyme immunoassay was carried out in a specific antigen-dependent method using secondary antibodies specific to each type. . In other words, a monoclonal antibody mAbB55 or mAbB128 was added to an untreated plate coated with 1 µg of low-density lipoprotein (LDL), and one of the antisera specific to each isotype of mouse antibody isolated from rabbits was added to the enzyme immunoassay method (ELISA). As a result, an analysis was performed using anti-rabbit immunoglobulin isolated from goats as a secondary antibody conjugated with horseradish peroxidase, and the degree of color development was expressed as absorbance measurement at 650 nm. Antisera specific for this isotype are normal rabbit serum (N), anti-mouse IgG1 (G1), IgG2a (G2a), IgG2b (G2b), IgG3 (G3), IgM (M), IgA ( A), antiserum against kappa L-chain (K) or lambda L-chain (L) was used. Experimental results showed that the monoclonal antibody mAbB55 was of the H-chain IgG2a type and L-chain. Type, and the monoclonal antibody mAbB128 is H-chain IgG2b type, L-chain It turns out that he has a brother. This is summarized in Table 1.

Isotype analysis of monoclonal antibodies mAbB55 and mAbB128 of the present invention N G1 G2a G2b G3 A M K L mAbB55 0.08 0.08 1.1 0.1 0.1 0.08 0.09 0.8 0. mAbB128 0.08 0.08 0.08 1.1 0.1 0.08 0.09 0.8 0.2

Example 4: Titer Determination of Monoclonal Antibodies

Enzyme immunoassay was performed to determine titers of monoclonal antibodies mAbB55 and mAbB128. In other words, 100 μl of PBS buffer containing 1 μg of LDL-Apolipoprotein B standard was added to a 96-coated titrant plate and adsorbed at 4 ° C. overnight, containing 0.05% bovine serum albumin (BSA) and 0.05% Tween 20. After washing the remaining lipoproteins that were not adsorbed with PBS buffer, PBS buffer containing 3% bovine serum albumin was added to prevent nonspecific binding. After shaking gently for 1 hour at room temperature, the solution was washed again with PBS buffer. The monoclonal antibody mAbB55 or mAbB128 culture solution was diluted annually, and then reacted at 37 ° C. for 3 hours, followed by washing, followed by addition of a horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG). After reacting at 37 ° C. for 1 hour, absorbance was measured at 450 nm using an ELISA reader for the color development by adding a substrate of peroxidase. As a result, the determined titers (T60%) of monoclonal antibodies mAbB55 and mAbB128 against apolipoprotein B-100 were found to be 1000 and 1250 fold dilutions, respectively, as shown in FIG. 5.

Example 5: Determination of Apolipoprotein B-100 Concentration Using Monoclonal Antibody

Enzyme immunoassay was performed to use monoclonal antibodies mAbB55 or mAbB128 for the measurement of blood apolipoprotein B-100 concentration. That is, 100 μl of PBS buffer containing various concentrations of low density lipoprotein (LDL) -apolipoprotein B standards diluted serially in 96-coated titriate was adsorbed overnight at 4 ° C., and 0.05% bovine serum albumin ( BSA) and PBS buffer containing 0.05% Tween 20 and the remaining lipoproteins were not adsorbed, and then PBS buffer containing 3% bovine serum albumin was added to prevent non-specific binding. Again washed with PBS buffer. A monoclonal antibody mAbB55 or mAbB128 solution was added thereto and reacted at 37 ° C. for 3 hours, followed by washing. Secondary horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG) was added for 1 hour at 37 ° C. After the reaction, the absorbance was measured at 450 nm using an ELISA reader. As shown in FIG. 6, excellent correlation coefficients of 0.95 and 0.97 between the log concentration and measured absorbance of Apo B-100 in the concentration range of 18-300 ng of Apo B-100 were measured, respectively. The relationship between the monoclonal antibodies mAbB55 and mAbB128 is clinically useful for enzymatic immunoassay for the determination of apolipoprotein B-100 in human blood.

The present invention is an IgG-type monoclonal antibody mAbB55 of hybridoma cell lines prepared by fusion of pancreatic cells of mice immunized with human apolipoprotein B-100 antigen with myeloma cells as described in the above Examples and Experimental Examples. The hybridoma cell lines H-mAbB55 and H-mAbB128, which produce mAbB128 and mAbB128, were selected. The monoclonal antibodies mAbB55 and mAbB128 produced by the hybridoma cell lines were H-chain IgG2a and L-chain, respectively. Phosphorus structure and H-chain is IgG2b and L-chain It has a phosphorus structure and excellent specificity that specifically binds to human apolipoprotein B. It has a very high titer, which is useful for measuring the concentration of apolipoprotein B-100 in human blood. It is a useful invention.

Claims (8)

Monoclonal antibody mAbB55 or mAbB128, which specifically responds to apolipoprotein B-100 in human blood. According to claim 1, wherein the structure of the monoclonal antibody mAbB55 is H-chain IgG2a, L-chain Monoclonal antibody mAbB55 characterized in that. The method of claim 1, wherein the structure of the monoclonal antibody mAbB128 is H-chain IgG2b, L-chain Monoclonal antibody mAbB128. The hybridoma cell line H-mAbB55 (KCTC 0439BP), which produces the monoclonal antibody mAbB55 of claim 1. The hybridoma cell line H-mAbB128 (KCTC 0440BP), which produces the monoclonal antibody mAbB128 of claim 1. A method of producing hybridoma cell lines H-mAbB55 (KCTC 0439BP) or H-mAbB128 (KCTC 0440BP) by fusing the somatic cells of mice immunized with human apolipoprotein B-100 antigen with myeloma cells. A method of producing monoclonal antibodies mAbB55 or mAbB128 by fusing the somatic cells of mice immunized with human apolipoprotein B-100 antigen with myeloma cells to produce hybridoma cell lines. A method for measuring the concentration of human plasma apolipoprotein B-100 using the monoclonal antibody according to claim 1.