KR0141313B1 - Novel monoclonal antibody against human blood apolipoprotein a-1 and recombinat hybridoma cell line for thd production thereof - Google Patents

Abstract

The present invention provides a novel monoclonal antibody that recognizes human plasma apolipoprotein A-I, recombinant hybridoma cell lines producing them and a method for measuring the concentration of plasma apolipoprotein A-I using monoclonal antibodies.

Description

New Monoclonal Antibodies to Human Blood Apolipoprotein A-I and Recombinant Hybridoma Cell Lines for Their Production

Figure 1 a shows the results of the polyacrylamide gel electrophoresis of the plasma protein to analyze the specificity of the monoclonal antibody of the present invention,

B of FIG. 1 shows the result of Western blotting analysis of the protein of FIG. 1 using the monoclonal antibodies MabA12 to MabA34 of the present invention,

2 shows the results of isotyping the H-chains and L-chains of the monoclonal antibodies MabA12 and MabA34 of the present invention using an enzyme immunoassay,

3 shows the antibody titration curve of the non-competitive enzyme immunoassay (non-competitive ELISA) using the monoclonal antibodies MabA12 and MabA34 of the present invention,

4 shows a standard curve of noncompetitive enzyme immunoassay using the monoclonal antibodies MabA12 and MabA34 of the present invention,

5 shows the antibody titration curves of the competitive ELISA using the monoclonal antibodies MabA12 and MabA34 of the present invention,

6 shows the standard curve of the competitive enzyme immunoassay using the monoclonal antibodies MabA12 and MabA34 of the present invention.

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

More specifically, the present invention relates to apolipoprotein (HDL), which is one of the protein portions of high-density lipoprotein (HDL), which carries a function of transporting cholesterol in plasma lipoproteins involved in lipid metabolism from blood to peripheral liver. Novel monoclonal antibodies against apolipoprotein AI, hybridoma cell lines for producing them and their use for measuring the concentration of apolipoprotein AI in human blood.

In the blood, fats (triglycerides, cholesterol and cholesterol esters, phosphate fats) are carried along with apolipoproteins to form complexes called lipoproteins. Lipoproteins have triglycerides (triacyl glycerol, TG) and cholesterol esters, which are non-polar fats, in the center of them, and the surface (around) is surrounded by polar fats, phosphate fats, cholesterol, etc. There are coatings of several apolipoproteins to form water-soluble particles as a whole.

Apolipoproteins are proteins from which lipids are removed by treating lipoproteins obtained from plasma with an organic solvent, and up to now, about 10 species with different immunological and chemical characteristics are known. The in vivo function of apolipoproteins plays an important key role in lipid metabolism, including transport and redistribution of intercellular fat, activating factors of enzymes involved in lipid metabolism, maintaining the water-soluble structure of lipoproteins, and acting as target proteins for lipoprotein receptors. .

In the meantime, many immunological methods for the measurement of apolipoproteins in the blood, such as radioimmunoassay, electroimmunoassay, radioimmunoassay, and enzyme-linked immnosorbent assay: ELISA), immunoturbhidimetry, etc. (Labeur C., Shepherd J., and Rosseneu, M ,. Clin. Chem. 36 591-597 (1990)).

Among several apolipoproteins in plasma lipoproteins, apolipoprotein A-I transports cholesterol in the body from peripheral tissues to the liver to lower blood cholesterol levels, ie, reverse cholesterol transport.

It is a major protein that makes up about 65% of the plasma lipoprotein (HDL) that performs reverse cholesterol transport (HDL) function. It is composed of 243 amino acids and diagnoses circulatory diseases such as atherosclerosis and coronary artery disease. In parallel with the conventional high density lipoprotein-cholesterol assay, it is usefully used as a negative diagnostic indicator [Brustolin D. et al., Clin. Chem, 37, 742-747 (1991): Dona V, et al., Giorn. It. Chim. Clin. 12, 205-214 (1987)]

Therefore, in addition to measuring apolipoprotein B-100, which has a positive correlation with disease in proportion to the onset of circulatory disease, in order to easily and reliably diagnose circulatory disease, it is a negative indicator of apolipoprotein AI as a negative indicator. There is a need to consider the temperature together, it is necessary to produce specific monoclonal antibodies to measure the concentration of apolipoprotein AI in the blood.

Accordingly, the present invention provides a monoclonal antibody MabA12 having a structure of H-chain IgG2b and L-chain κ, which specifically reacts against apolipoprotein AI in human blood, and a structure of H-chain IgG1 and L-chain κ. It provides a monoclonal antibody MabA34.

The present invention also provides hybridoma cell lines H-MabA12 (KCTC 0112 BP) and H-MabA34 (KTCT 0113 BP) for producing the monoclonal antibody.

The present invention also provides a method for producing the monoclonal antibody using the hybridoma cell lines H-MabA12 (KCTC 0112 BP) and H-MabA34 (KTCT 0113 BP).

The present invention also provides a method for measuring the concentration of lipoproteins comprising plasma apolipoprotein A-I using the monoclonal antibody.

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

Centrifugation of blood in a KBr density gradient, a conventional method (see Methods Enzym. 128, Section II III), for use in immunization for the production of monoclonal antibodies from human blood. Pure water is separated by removal and chromatographic methods. The isolated apolipoprotein AI is well mixed with Freund's complete adjuvant and dead Salmonella cells and intraperitoneally injected into mouse Bal / c females. After 20 days, the same apolipoprotein AI is mixed with the Freund's complete adjuvant. Additional injections. After that, the test serum obtained from the mice is injected with apolipoprotein A-I dissolved in PBS buffer (10 mM sodium phosphate, 150 mM NaCl, ph 7.2) at 10-day intervals until the test result shows excellent positive response to the apolipoprotein A-I. The final injection is given intravenously, and on the third day after the last injection, the rat is opened and the pancreas is taken out and ground. Massed 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)). ) Fusion to cells (Sp2 / 0) and HAT (hypo-

xanthine, aminopterine, thymidine) incubate for one week in selective medium. The resulting colonies are diluted and cultured in 96-co-titer 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 solution was passed through an antigen-immobilized column or Protein-A column to purify the monoclonal antibodies MabA12 and MabA34 against apolipoprotein AI. Use it.

Enzyme-linked immunoassays (ELISA) for assaying the concentration of apolipoprotein A-I in blood using monoclonal antibodies are performed by either non-competitive enzyme immunoassay or competitive enzyme immunoassay.

Noncompetitive enzyme immunoassays are as follows:

A blood sample or a sample containing apolipoprotein A-I is reacted with adsorption and coating overnight at 4 ° C. to a suitable temperature on a 96-microtiter plate or a suitable solid surface. Then, reacted by adding bovine serum aliphatic or skim milk powder to mask nonspecific binding, and to which monoclonal antibodies MabA12 and MabA34 (primary antibody) were added, reacted and washed for 3 hours at an appropriate temperature of room temperature to 37 ° C. According to the following enzyme immunoassay, the enzyme-bound secondary antibody and the substrate of the enzyme are added to the pumice plate and developed, and the absorbance is read with an ELISA reader to determine the concentration of apolipoprotein AI in the sample.

As the secondary antibody, an antibody combined with peroxidase or alkaline phosphatase is used. Instead of using a secondary antibody, the primary antibody is directly combined with the enzyme and used instead of the secondary antibody bound to the enzyme. The analysis step can be reduced.

Competitive enzyme immunoassays were as follows:

Place an appropriate amount of epitope portion of apolipoprotein AI, high density lipoprotein (HDL), or apolipoprotein molecule produced by chemical synthesis or genetic recombination techniques, or a combination thereof on a 96-coated microplate or a suitable solid surface, and then After reacting overnight at adsorption coating. Dilute the sample to be measured at about 200-fold to an appropriate ratio and place it in the assay plate together with the monoclonal antibody MabA12 or MabA34 (primary antibody), and react and wash overnight at an appropriate temperature of room temperature to 37 ° C. The remaining steps are performed in the same manner as in the noncompetitive enzyme immunoassay above.

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

The accompanying drawings will be described in detail as follows.

A of FIG. 1 shows the results of electrophoresis of polyacrylamide gels on plasma proteins to analyze the specificity of monoclonal bodies.

1 is plain protein lacking serum (LPDS), 2 is purified apolipoprotein A-I, 3 is low density lipoprotein (LDL), 4 is total human plasma.

FIG. 1B shows the results of Western blotting analysis of the proteins of FIG. 1 using the monoclonal antibodies MabA12 and MabA34 of the present invention. 1 is lipoprotein-deficient serum (LPDS), 2 is stagnant apolipoprotein A-I, 3 is low density lipoprotein (LDL), and 4 is the result of analysis on total human plasma.

2 shows the results of isotyping the H-chains and L-chains of the monoclonal antibodies MabA12 and MabA34 of the present invention using an enzyme immunoassay. Enzyme immunoassay was carried out in parallel with the antigen-dependent method (horizontal lines 1 to 4) and the antigen-independent method (horizontal lines 5 and 6). Antigen-dependent methods were used by pretreatment coating of total lipoprotein complexes (horizontal lines 1 and 2) or purely isolated apolipoprotein A-I (horizontal lines 3 and 4) into an untitrated plate. As monoclonal antibodies, cultures of hybridoma cell lines producing MabA12 (lines 1, 3, and 5) or MabA34 (lines 2, 4, and 6) were used, and each letter in the vertical line is represented by each isotype of the mouse antibody as follows. It shows the result of using rabbit antiserum specific. : G1-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); N, regular rabbit serum. Column P is an antiserum isolated from rabbits responding to all antibody types in mice and is a positive control (for horizontal lines 5 and 6).

Figure 3 shows the antibody titration curve of the non-competitive enzyme immunoassay developed to use the monoclonal antibodies MabA12 and MabA34 of the present invention in the analysis of the concentration of apolipoprotein A-I in the blood. Monoclonal antibody-producing cultures were diluted as indicated on the horizontal axis and arrows indicate the appropriate concentration of antibody use corresponding to 50% of maximum binding.

Figure 4 shows the standard curve of the non-competitive enzyme immunoassay developed to use the monoclonal antibodies MabA12 and MabA34 of the present invention in the analysis of the concentration of lipoprotein A-I in the blood.

Figure 5 shows the antibody titration curves of the competitive enzyme immunoassay developed for the use of monoclonal antibodies MabA12 and MabA34 of the present invention in the analysis of the concentration of apolipoprotein A-I in blood.

Figure 6 shows the standard curve of a competitive enzyme immunoassay developed for the use of the monoclonal antibodies MabA12 and MabA34 of the present invention in the analysis of the concentration of apolipoprotein A-I in blood.

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 (15 ° C.) in a KBr solution of constant density according to a conventional method (Reference Methods: Enzym, 128 Section II III) for apolipoprotein antigen to be used for immunoinjection for the production of monoclonal antibodies from human blood. , 45000 rpm, Beckman T170, 24 hours each) 1.08d

The high density lipoprotein of 1.21 was separated, and pure water was separated by a method of removing fats from an organic solvent (ethanol: ether = 3: 2 by volume) and gel recovery by electrophoresis. 30 μg of isolated apolipoprotein AI was mixed with Freund's complete adjuvant and dead Salmonella cells intraperitoneally to mouse Balb / c females, and 20 days later, the same amount of apolipoprotein AI was supplemented (Freund's complete adjuvant). And additional injection in combination with. Subsequently, the apolipoprotein A-I dissolved in PBS buffer (10 mM sodium phosphate, 150 mMNaCl, pH 7.2) until the experimental serum from mice was positively tested for apolipoprotein A-I.

20 μg was further injected at 10 day intervals. 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 subjected to myeloma by 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 cultured in HAT (hypoxanthine, aminopterine, thymidine) selection medium (Giboc 31062-037) for one week. Sample serum was taken from the eyes of the immunized mice prior to the fusion reaction and the antibody immunoassay (ELSIA) confirmed the production of antibodies in serum. As shown in Fig. 1, the antibodies of human plasma apolipoprotein AI were well formed in the serum of mice. It was. After the fusion reaction, cell growth was observed in all 270 culture holes of the unqualified plate used, among which the cells that did not fusion reaction inhibited the growth by adding HAT selection medium. The production of antibodies against apolipoprotein AI 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 obtain eight stable hybridoma cell lines. Obtained, of which IgG-type monoclonal antibody

Two hybridoma cell lines, H-MabA12 and H-MabA34, producing MabA12 and MabA34 were selected and these cell lines were assigned to the Genetic Bank of Korea Institute of Science and Technology, Genetic Bank, dated June 2, 1994. KTCT 0112 BP and KTCT Each deposited at 0113 BP.

Example 2. Specificity Analysis of Monoclonal Antibodies

Monoclonal antibody MabA12 Why Western blotting was performed as follows to assay the antigen-binding specificity of MabA34. That is, about 0.5 μg of plasma proteins were electrophoresed on polyacrylamide gels in a gradient of 4-15% in the presence of SDS in accordance with the method of Laemmli, Nature 227, 680 (1970) and 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)). This filter was reacted with a solution containing 0.5% bovine serum albumin (BSA) to mask nonspecific binding. Thereafter, the monoclonal antibodies MabA12 and MabA34 were added to the PBS buffer containing 0.5% Tween 20 and allowed to react, and then the filter was washed twice with PBS buffer containing 0.05% Tween 20 for 15 minutes each. A secondary antibody (anti-mouse immunoglobulin G of goats labeled with horseradish peroxidase) was added to the filter and reacted with the PBS buffer solution. Finally, the filter was developed by adding hydrogen peroxide and ο-phenylenediamine as a substrate. As a result, monoclonal antibodies MabA12 and MabA34 specifically bind to apolipoprotein AI in human plasma proteins and other proteins in low density lipoprotein (LDL) or lipoprotein-deficient hemoglobin (LPDS) as shown in FIGS. It showed excellent specificity that did not bind to them.

Example 3. Isotype Analysis of Monoclonal Antibodies

In order to examine the H-chain and L-chain isotypes of the antibody molecules of the monoclonal antibody MabA12 or MabA34, secondary enzyme immunoassays were performed in FIG. 2 using the secondary antibodies specific to each type. Enzyme-immunoassays were performed in two ways: antigen-dependent methods (horizontal lines 1 to 4) and antigen-independent methods (horizontal lines 5 and 6). Antigen-dependent methods were used by pretreatment coating of 0.5 μg of total lipoprotein complex (horizontal lines 1 and 2) or purely isolated apolipoprotein A-I (horizontal lines 3 and 4) in 100 μl volume on an untitrated plate. As monoclonal antibodies, cultures of hybridoma cell lines producing MabA12 (lines 1, 3 and 5) or MabA34 (lines 2, 4, and 6) were used, with each letter in the vertical line specific for each isotype of the mouse antibody. Rabbit antiserum is used. As a result, in the analysis by both methods, as can be seen in FIG. 2, the monoclonal antibody MabA12 has IgG2b type as H-chain and κ as L-chain, and monoclonal antibody MabA34 is IgG1 as H-chain. It was found to have a type, κ as the L-chain.

Example 4 Determination of Blood Apolipoprotein A-I Concentration (Noncompetitive Enzyme Immunoassay)

100 μl of a blood sample or various concentrations of standard apolipoprotein AI solution on a 96-coated titration plate or a suitable solid surface were reacted and coated overnight at 4 ° C. to moderate temperature, followed by 0.05% bovine serum albumin (BSA) and 0 .05 The remaining lipoproteins were washed off with PBS buffer containing Twin 20. Then, in order to mask nonspecific binding, PBS buffer containing 1% cow serum albumin and 2% skim milk powder (Carnation Co.) was hung, and gently shaken for 1 hour at room temperature to react, followed by washing with the PBS buffer again. 100 μl of monoclonal antibodies MabA12 and MabA34 (primary antibody) diluted at various magnifications were added thereto, reacted and washed for 3 hours at an appropriate temperature of room temperature to 37 ° C., followed by enzyme immunoassay. The concentration of apolipoprotein AI in the sample was determined by adding the substrates of the secondary antibody and the enzyme to the assay plate in order to develop the color and reading the absorbance with an ELISA reader to compare with the standard curve using the standard apolipoprotein AI. As the secondary antibody, an anti-mouse antibody produced by another animal such as goat or the like and a peroxidase or an alkaline letter phosphate degrading enzyme are used. In this case, instead of the secondary antibody bound to the enzyme, the primary antibody is directly By using the enzyme in combination, the analysis step can be reduced without using a secondary antibody. As a result of the non-competitive enzyme immunoassay method, as shown in FIG. 3, the dilution ratio of monoclonal antibodies suitable for measuring the concentration of apolipoprotein AI was 600 times for MabA12 and 80 times for MabA34. The effective concentration range of apolipoprotein AI to be used for the measurement was 0.125 to 1.0 μg as shown in the standard curve of FIG.

Example 5 Determination of Blood Apolipoprotein A-I Concentration (Competitive Enzyme Immune Analysis)

100 μg of 0.5 μg epitope portion of apolipoprotein AI, high density lipoprotein (HDL), or apolipoprotein molecule produced by chemical synthesis or genetic recombination, or a combination thereof, is placed in a 96 μg microtiter or in a suitable solid surface. Adsorption coating was carried out by reaction overnight at < RTI ID = 0.0 > Then, the sample to be measured was diluted in an appropriate ratio and put into the assay plate together with the monoclonal antibodies MabA12 and MabA34, reacted and washed overnight at an appropriate temperature of room temperature to 37 ° C. The remaining series of steps were then performed in the same manner as in the noncompetitive enzyme immunoassay above. As a result, as shown in FIG. 5, the dilution ratios of monoclonal antibodies suitable for measuring the concentration of apolipoprotein AI by the competitive enzyme immunoassay were appropriately diluted 100-fold for both MabA12 and MabA34. The effective concentration range of apolipoprotein AI ranged from 0.625 to 80 μg as shown in the standard curve of FIG. From the results of the director, it was confirmed that the monoclonal antibodies MabA12 and MabA34 of the present invention are clinically useful for measuring the concentration of apolipoprotein A-I.

Claims (6)

Monoclonal antibody MabA12, which specifically responds to human blood apolipoprotein A-I and is produced by the hybridoma cell line H-MabA12 (KCTC 0112 BP), wherein the H-chain is IgG2b and the L-chain is κ. The monoclonal antibody MabA34, which specifically responds to human hyperemia apolipoprotein A-I and is produced by the hybridoma cell line H-MabA34 (KCTC 0113BP), wherein the H-chain is IgG1 and the L-chain is κ. Hybridoma cell line H-MabA12 (KCTC 0112BP), which produces monoclonal antibodies that specifically respond to human blood apolipoprotein A-I. Hybridoma cell line H-MabA34 (KCTC 0113BP), which produces monoclonal antibodies that specifically respond to human blood apolipoprotein A-I. A method for producing a monoclonal antibody that specifically reacts against human blood apolipoprotein A-I, comprising culturing the hybridoma cell line of claim 3 or 4 in a suitable medium or animal body. A method for measuring the concentration of plasma apolipoprotein A-I using the monoclonal antibody of claim 1.