JPH08168394A - Monoclonal antibody to 4-hydroxy-2-nonenal modified protein, its production, hybrid cell producing the same and immonoassay kit for determining 4-hydroxy-2-nonenal - Google Patents

Abstract

PURPOSE: To provide a method for evaluating deterioration of industrial fats and oils and foods or oxidative stress of organisms, by using 4-hydroxy-2- nonenal, a peroxide of a lipid occurring in industrial fats and oils, foods and organisms as an index and immunochemically determining a very small amount of 4-hydroxy-2-nonenal rapidly. CONSTITUTION: This hybrid cell produces an anti 4-hydroxy-2-nonenal (HNE) modified protein antibody comprising a mouse splenocyte immunized against haemocyanin of KAGIANAKASAGAI modified by NHE as an antigen and a mouse myeloma cell. This monoclonal antibody against an anti-HNE modified protein is produced by preparing the hybrid cell of the mouse splenocyte immunized against the haemocyanin of KAGIANAKASAGAI modified by 4-hydroxy-2- nonenal (NHE) as the antigen and the mouse myeloma cell and proliferating the hybrid cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention uses, as an index, 4-hydroxy-2-nonenal (hereinafter referred to as HNE), which is a lipid peroxide generated in industrial fats and oils, foods and living bodies. It aims at making it possible to evaluate the deterioration of industrial fats and oils or foods or the oxidative stress of living organisms by rapid and minute quantification, and it is used in a wide range of fields such as applied engineering, food chemistry, medicine and pharmacy. In the present invention, it relates to a monoclonal antibody against an anti-HNE modified protein, hybrid cells producing the same, and an immunoassay kit for quantifying HNE.

[0002]

[Conventional Technology] For the quantification of conventional lipid peroxides, standard preparations were used for separation and quantification by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry.

[0003]

In the qualitative / quantitative determination of natural products or biological samples, each of the above-mentioned methods is used for obstacles to coexisting similar substances, when the target substance is in a trace amount below the detection sensitivity of chromatography, and for separation by column. This requires a long time, requires a relatively expensive device, and requires a large maintenance cost for column replacement, which is not economical.

In addition, the first requirement is that the method is specific for the quantitative evaluation of low molecular weight trace substances existing naturally or in the living body. Although immunochemical methods are superior for that purpose, when considering coexisting interfering substances, it is advisable to use monoclonal antibodies to confirm their specificity. However, preparation of a monoclonal antibody has a problem that it requires low cross-reactivity with a similar substance.

[0005]

The present invention makes it possible to relatively economically and easily prepare a monoclonal antibody with spleen cells and mouse myeloma cells of a mouse immunized with a hemocyanin antigen of the limpet, P. aeruginosa, which has been modified by HNE. It succeeded in making it, and solved the said problem.

That is, it was possible to create stable hybrid cells (hereinafter referred to as hybridomas) that produce a monoclonal antibody with high specificity for the HNE modified protein. Further, by constructing a kit for quantifying HNE using this anti-HNE modified protein antibody, it was possible to put the present invention into practical use.

First, HNE was reacted with limpet hemocyanin (hereinafter referred to as KLH) to prepare a conjugate, which was used as an antigen to immunize mice, and a monoclonal antibody against the HNE-modified protein was prepared by a cell fusion method. As a result of the cell fusion, antibody-producing hybridomas were obtained, and antibody-producing clones were selected from these by the limiting dilution method twice. As a result, clones having extremely high specificity for the HNE-modified antiprotein were obtained. This clone could be inoculated into the abdominal cavity of nude mice to produce antibodies in ascites. We also succeeded in producing the same monoclonal antibody by cell culture of this clone.

That is, the invention of the cell is characterized in that it is composed of spleen cells of mouse immunized and mouse myeloma cells using hemocyanin of limpet limpet modified by 4-hydroxy-2-nonenal (HNE) as an antigen. It is a hybrid cell that produces an anti-HNE modified protein antibody. Still another invention is to prepare hybrid cells of spleen cells of immunized mice and mouse myeloma cells using hemocyanin of limpet limpets modified by 4-hydroxy-2-nonenal (HNE) as an antigen, It is a monoclonal antibody against an anti-HNE modified protein characterized by being produced by the growth of hybrid cells.

Next, the invention of the manufacturing method is 4-hydroxy-2.
Monoclonal antibody against anti-HNE modified protein by preparing hybrid cells of spleen cells of immunized mouse and syngeneic myeloma using hapten modified by nonenal (HNE) as an antigen and growing the hybridomas A method for producing a monoclonal antibody for a HNE-modified protein, which comprises separating a monoclonal antibody against the HNE-modified protein after increasing the amount.

Further, it is an immunoassay kit for HNE quantification constructed by using a monoclonal antibody against an anti-HNE modified protein.

[0011]

Example 1 10 mg / ml of KLH solubilized with a phosphate buffer having a pH of 7.2 was treated with 80 mM HNE at 37 ° C. for 2 times.
An antigen for immunization was prepared by reacting for a time.

[0012]

Example 2 Preparation of hybridoma producing anti-HNE modified protein antibody will be described.

Using HNE / KLH as an immunizing antigen, intraperitoneally in a mouse (BALB / c, female, 7 weeks old),
Subcutaneously and intramuscularly (1/2, 1/4, 1/4 respectively)
Amount) A single dose of 100 μg / mouse was administered with Freund's complete adjuvant (DIFCO). Part 4
Weeks later, the same amount of antigen was administered in the same manner with Freund's incomplete adjuvant (DIFCO). One week after the administration, serum of the mouse was collected, and HNE / BSA was used by Western blotting and ELISA to determine (1 mg
/ Ml BSA was reacted with 1 mM HNE at 37 ° C. for 2 hours, and an increase in antibody titer was confirmed using BSA as a control. After a further 3 weeks, the same amount of antigen alone was intravenously administered, and 3 days later, the spleen of the mouse was excised and cell fusion was performed.

Mouse 653 cells (P3X63-Ag8,653: CRL-1580) were used as myeloma cells, 10% FCS (fetal calf serum), 0.0015%.
RPM with azaguanine, (2.5 × 10 ^-7 ) M 2-mercaptoethanol, 0.03% L-glutamine
The culture was performed in the I-1640 medium.

Spleen cells of the immunized mouse and the above-mentioned 653 cells were subjected to cell fusion using polyethylene glycol in a conventional manner, and hybridomas were selectively grown in HAT medium. By this cell fusion, 320 colonies were obtained from 720 wells and the oxygen antibody method (ELISA
Method) for primary screening. As a result 28
Strongly positive colonies were detected. Among these, 5 is good for growth
Selected strains. Three of them (HNEJ-1, described below,
For 3 and 4), culture medium was used to maintain growth. Esclone cloning medium CM-B (Sanko Junyaku Co., Ltd.)
Changed to.

[0016]

Example 3 Next, an example of cloning antibody-producing cells and purifying a monoclonal antibody from mouse ascites will be described.

All 5 antibody-producing strains were cloned by the limiting dilution method twice. As a result, clones HNEJ-1, 2, 3, 4, 5 producing a monoclonal antibody
Cells were obtained. These 5 clones were stored in liquid nitrogen. HNEJ-1, from ascites obtained by transplanting these 5 types of cells (10 ⁷ cells / ml / mouse) into nude mice (BALB / c, nu / nu, female 8 weeks old),
The 2, 3, 4, and 5 antibodies were purified respectively.

Prior to purification, the mouse 1gG subclass assay kit (Amersham) was used to determine the subclass of each antibody. All were IgG ₁ and K (kappa). Purification was carried out by salting out with ammonium sulfate in the usual manner. The obtained 5 kinds of antibodies were stored at -80 ° C.

[0019]

Example 4 An example for confirming antibody cross-reactivity and reaction specificity will be described.

To confirm the specificity of the 5 types of monoclonal antibodies obtained in the above-mentioned examples, the Western blotting method was performed first. 25 μg of each of BSA and HNE / BSA was subjected to SDS polyacrylamide gel electrophoresis, and transferred to a PVDF membrane (Millipore) in the usual manner. 200 μg / of each monoclonal antibody
When the enzyme-antibody method was carried out at a concentration of m1, it reacted strongly with HNE / BSA, but hardly reacted with BSA. H
Strong reactivity was exhibited in the order of NEJ- (2> 1 to 3 = 4> 5) (FIG. 1).

Next, using the HNEJ-2 antibody, an ELISA
The specificity of the antibody was examined in more detail by Method A. The antibody showed almost no reactivity with modified proteins by 2-nonenal, 2-hexenal, 1-hexenol, and 4-hydroxy-2-hexenal (Fig. 2). Moreover, the modified protein with high concentration of glutanol aldehyde or formaldehyde showed almost no reactivity. This is an advantage when applying the monoclonal antibody of the present invention to tissues fixed by glutaraldehyde or formaldehyde.

[0022]

Test Example 1 An ELISA method for measuring free HNE was constructed.

In order to select a protein suitable for measurement of free HNE, the reactivity of the modified protein produced by the reaction with HNE with various antibodies was examined with the monoclonal antibody HNEJ-2. Since glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as GAPDH) showed the highest reactivity (FIG. 3), it was decided to use this protein in the ELISA method for measuring free HNE.

[0024]

[Test Example 2] Iron nitrilotriacetic acid (15 mg iron / k) was used as a system for inducing lipid peroxidation in vivo.
g) was intraperitoneally administered to rats, and free HNE was measured over time by the above-mentioned ELISA system in the cytosolic fraction of renal tissue, and the results shown in FIG. 4 were obtained.

[0025]

[Test Example 3] The degree of oxidation of edible oil was measured in vitro (in v
The results measured by the amount of HNE produced in vitro are as shown in FIG.

[0026]

[Test Example 4] Human serum was subjected to ultracentrifugation with lipoproteins (VLDL, LDL, HDL) in vitro and each lipoprotein (0.5 mg / ml) with copper ion (10 μm).
M) was reacted with phosphate buffer (pH 7.2) for 24 hours at 37 ° C., and TBA method (MDA measurement) and ELISA method (HNE measurement) were performed, and FIGS. 6 (a) (b) (c) were obtained. Got the result.

[0027]

[Test Example 5] The HNE measurement protocol by the ELISA method is described. 100 μl of a 1 mg / ml GAPDH solution is dispensed into each well of the plate and incubated at 4 ° C. for 24 hours. After that, TTBS (Tween
containing Tris buffered
Wash 3 times with saline) to remove uncoated protein.

A HNE standard sample or a sample containing HNE is dispensed at 100 μl each and incubated at 37 ° C. for 2 hours to generate HNE-modified protein in the wells. Wash as before.

Block Ace 2 of 0.01 g / ml
Dispense 00 μl and incubate at 37 ° C. for 30 minutes. Wash as before.

Monoclonal antibody against HNE modified protein (HNEJ-2 10 μg / ml in
TTBS) (100 μl) is dispensed and incubated at 37 ° C. for 3 hours or 4 ° C. for 24 hours, and then washed as in the same manner.

Secondary antibody against primary antibody (Pero
xidase-linked IgG of Mouse
e) was diluted 4000 times with TTBS, and 100 μl
Aliquot and incubate at 37 ° C for 1 hour. Wash as before.

100 μl of O-phenylenediamine (pH 5) containing 0.003% H ₂ O ₂ was dispensed in 100 μl aliquots and left at room temperature for 15 minutes for color development.

The reaction was stopped with 2N sulfuric acid and measured at 492 nm with a plate reader.

[0034]

EFFECTS OF THE INVENTION The present invention has the effects that, due to the above construction, not only expensive equipment is not required, but also a large maintenance cost is not required for column replacement.

[Brief description of drawings]

FIG. 1 EL of monoclonal antibody of HNE of the present invention
The graph which shows the test result of ISA.

FIG. 2 is a graph showing the cross-activity of HNEJ-2 monoclonal antibody.

FIG. 3 is a graph showing the reactivity of protein with HNE.

FIG. 4 is also iron-nitrilotriacetic acid (15 mg iron / k
g) A graph showing the time course of free HNE in the rat kidney cytosolic fraction produced by intraperitoneal administration.

FIG. 5 is a graph showing the quantification of free HNE produced when linoleic acid was heated at 60 ° C.

FIG. 6 (a) is a graph showing the oxidation of very low density lipoprotein. (B) A graph showing the oxidation of low density lipoprotein.
(C) Graph showing oxidation of high density lipoprotein

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 15/02 G01N 33/02 33/53 K 33/577 B // (C12P 21/08 C12R 1 : 91)

Claims (4)

[Claims] 1. 4-hydroxy-2-nonenal (HN
A hybrid cell producing an anti-HNE modified protein antibody, characterized by comprising spleen cells of an immunized mouse and mouse myeloma cells, using the limpet hemocyanin modified by E) as an antigen. 2. 4-hydroxy-2-nonenal (HN
Anti-HNE characterized by producing hybrid cells of spleen cells of immunized mice and mouse myeloma cells using hemocyanin of limpet limpet modified by E) as an antigen and proliferating the hybrid cells A monoclonal antibody against the modified protein. 3. An immunoassay kit for HNE quantification constructed using the monoclonal antibody against the anti-HNE modified protein according to claim 2. 4. 4-hydroxy-2-nonenal (HN
The hapten modified by E) was used as an antigen to prepare hybrid cells of immunized mouse spleen cells and syngeneic myeloma cells, and the hybridomas were grown to determine the amount of monoclonal antibody to the anti-HNE modified protein. A method for producing a monoclonal antibody against a HNE-modified protein, which comprises isolating the monoclonal antibody against the HNE-modified protein after the increase.