JPH11239479A - Hybridoma, monoclonal antibody, assay method and immunoassay reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a monoclonal antibody capable of specifically recognizing apo B-48, thereby accurately and easily measure the amount of a risk factor for arteriosclerosis, and diagnose and treat it. Provide a way. SOLUTION: A hybridoma obtained by fusing lymphocytes obtained by immunizing a mammal with a peptide derived from Apo B-48 and myeloma cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody which specifically recognizes apoprotein ApoB-48 (ApoB-48), a hybridoma producing the same, and an apoB-48 utilizing the same. And / or Apo B-4
8 relates to measurement of lipoprotein. Apo B-48 is a constituent protein of lipoprotein which is closely related to hyperlipidemia in human blood, and its measurement is useful for diagnosis and treatment of hyperlipidemia and arteriosclerosis. is there.

[0002]

2. Description of the Related Art As one of the typical modern diseases of arteriosclerosis, establishment of effective diagnostic methods and therapeutic methods is expected. As one of the causes of arteriosclerosis, among lipoproteins present in the blood circulating in the body, various lipoproteins that promote the deposition of cholesterol on the inner wall of blood vessels increase,
Alternatively, it is described that various lipoproteins that prevent the deposition of the cholesterol are reduced. Therefore, it is desired to measure the blood levels of these lipoproteins and use them for diagnosis and treatment of hyperlipidemia and arteriosclerosis.

[0003] Representative lipoproteins involved in hyperlipidemia include chylomicron (CM), chylomicron remnant (CM remnant), an intermediate metabolite of CM in blood, and ultra-low density lipoprotein (VLDL). ), VLDL intermediate metabolites in blood, ultra-low density lipoprotein remnant (VLDL remnant), low density lipoprotein (LDL),
High-density lipoprotein (HDL) and the like are known.

In terms of lipid metabolism, for example, CM remnants and VLDL remnants [these are remnant-like lipoproteins (remnant-like particles)]
s; RLP)], and LDL and the like are lipoproteins that carry cholesterol to the blood vessel wall, and reducing their blood levels is directly linked to the treatment of arteriosclerosis. Also, for example, HDL has a function of extracting cholesterol from atherosclerotic lesions, so that increasing its blood concentration will be useful for treating arteriosclerosis.

[0005] Among them, it has been pointed out that RLP is involved in the development of arteriosclerotic lesions of postprandial hyperlipidemia, and it has begun to be considered to be important as one of the risk factors for arteriosclerosis. Apo C, Apo E, Apo A-I, Apo B-100, Apo B-
48 and the like are known.

As one of the methods for measuring the blood concentration of RLP,
Currently, RLP-C (remnantlike part)
The method of measuring the cholesterol (oles cholesterol) is known. In this RLP-C method, a mixed gel is prepared by immobilizing an anti-apoA-I monoclonal antibody and an anti-apoB-100 monoclonal antibody, and the mixed gel is reacted with a sample.
The lipoprotein bound to these antibodies is removed by centrifugation, and the amount of unbound lipoprotein present in the supernatant is measured as the amount of cholesterol.

ApoA-I exists as a major apoprotein of CM and HDL, and apoB-100 is VLDL and VL
DL remnant is a major apoprotein of LDL. Therefore, in theory, CM and HDL bind to the A-I monoclonal antibody, and anti-apoB-1
Since the VLDL, VLDL remnant and LDL bind to the 00 monoclonal antibody, the RLP-C
According to the assay, these lipoproteins are bound and removed.

[0008] Anti-Apo B- used in RLP-C assay
The 100 monoclonal antibody is 229 of Apo B-100.
It specifically recognizes the amino acid region from position 1 to position 2318 and does not recognize apo B-48 consisting of the amino acid sequence up to position 2152 of apo B-100, and therefore contains apo B-48 as an apoprotein. Are lipoproteins that do not contain apoB-100 as an apoprotein are subject to the RLP-C assay.

[0009] However, the RLP-C measurement method requires an adsorption removal operation, which is complicated, requires complete adsorption, and causes variations in measured values depending on measurement conditions. was there.

Apo B-48 is contained as apoprotein in CM and CM remnants. CM is immediately converted to CM remnant by lipoprotein lipase after eating,
M remnant will be taken into the liver by the remnant receptor present in the liver. Therefore, CM does not exist in the blood on an empty stomach or the like. Therefore, if a monoclonal antibody capable of specifically recognizing apoB-48 can be obtained, the blood at such a time can be used as a sample. By doing so, it is possible to directly measure the amount of only the CM remnant. This makes it possible to more reliably diagnose hyperlipidemia, and thus can be useful for diagnosis and treatment of arteriosclerosis.

[0011]

SUMMARY OF THE INVENTION In view of the above situation, the present invention provides a monoclonal antibody capable of specifically recognizing apoB-48, thereby accurately and easily obtaining the risk of arteriosclerosis. An object of the present invention is to measure the amount of a factor and to provide a diagnostic and therapeutic method.

[0012]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that lymphocytes obtained by immunizing a mammal with a peptide derived from apoB-48 and myeloma cells are fused. By establishing a hybridoma and culturing the same, a monoclonal antibody capable of specifically recognizing apoB-48 was successfully obtained, thereby completing the present invention. Hereinafter, the present invention will be described in detail.

[0013] The present invention has been established for the first time by establishing a method for obtaining a monoclonal antibody capable of specifically recognizing apo B-48. This method was established when the present inventors first established a hybridoma producing an anti-apoB-48 monoclonal antibody.

Apo B-48 is a peptide having the same amino acid sequence as a part of the amino acid sequence of Apo B-100, and the amino acid sequence of Apo B-100 and the amino acid sequence of Apo B-48 are already known. Some [Nature
re) 323, 738. October 1986]. Also,
Anti-apoB-48 specific antisera is also already known [Journal of Biological Chemistry (J. Biol.
Chem.,), 265, No. 15, p. 8358, 1990.
Year. Journal of Biological Chemistry (J. Biol. Chem.,) 267, No. 2, p. 1175,
1992. Clinical Scien
ce), 85, 521, 1993].

However, there has been no report that a monoclonal antibody that specifically reacts with only apo B-48 has been obtained so far, and the literature (clinical examination, volume 40, 9).
No. (1996), p. 1025, left column, line 8 from the bottom left) states that it is theoretically difficult to produce an antibody that specifically reacts only with apo B-48. The anti-apoB-48 monoclonal antibody according to the present invention fundamentally reverses these conventional ideas.

The method for establishing a hybridoma according to the present invention will be described below. In the present invention, first, a peptide derived from apo B-48 is synthesized. The peptide derived from Apo B-48 is preferably a peptide containing a 20 amino acid sequence from the C-terminus of Apo B-48, more preferably a 10 amino acid sequence, and still more preferably a 6 amino acid sequence. In the present invention, for example, Cys Thr Tyr Met Ile in which cysteine is bonded to the N-terminal of a peptide corresponding to four amino acid sequences counted from the C-terminal in the amino acid sequence of already known apo B-48. (Hereinafter, this peptide is referred to as “C4” in the present specification). Such an amino acid sequence can be synthesized very easily by using a known peptide synthesizer.

Similarly, of the amino acid sequence of apo B-48, five amino acid sequences counted from the C-terminus and six amino acid sequences are synthesized ("C5" and "C6", respectively). ). These peptides can be purified according to a conventional method. Thereafter, complexes of these peptides with hemocyanin and the like are synthesized.

On the other hand, blood collected from a human who has passed 2 hours after a meal is pooled, and this human serum is centrifuged to obtain a VLDL fraction and an LDL fraction, which are used as antigens for screening and the like.

On the other hand, a hybridoma producing an anti-apoB-48 monoclonal antibody immunizes a mammal with the C4-KLH, C5-KLH and C6-KLH complex synthesized by the above-described method, and immunizes the lymphocyte and myeloma cell with the mammal. Can be established by fusing.

For example, a mammal such as a BALB / C mouse is immunized with an apoB-48-derived peptide of a C4-KLH, C5-KLH and C6-KLH complex emulsified with Freund's complete adjuvant, and the serum of the animal is immunized. Is used to confirm an antibody that reacts with native apo B-48 by Western blotting (WB).
The free complex is intravenously administered to the immunized animal in which the most intense band was confirmed by the WB method, and 3 to 4 days later, the spleen is removed from the animal to prepare spleen cells.

Separately, myeloma cells cultured in a medium and the above-mentioned splenocytes are mixed, and cell fusion is performed by a known method. The fused cells, for example, after floating in the medium,
Dispense into a culture plate and culture. For cultured cells, VLDL fraction was used as antigen and SDS-P
After performing AGE, etc., the migrated antigen is transferred to a nitrocellulose membrane, and after blocking the transfer membrane, cut into strips, and a reaction is performed by adding a culture solution pooled with a part of the culture plate as a group. Let it. Thereafter, after washing with shaking for 5 minutes in a washing buffer, a POD-labeled anti-mouse immunoglobulin antibody was added, and the mixture was reacted.
The band at the position corresponding to 48 is confirmed.

If a band is found, the wells producing the antibody are selected. Well cells producing the desired antibody are cloned by cloning by the limiting dilution method. The cells producing the anti-apoB-48 monoclonal antibody are cultured in large quantities and administered to the mouse peritoneal cavity, and ascites containing the anti-apoB-48 monoclonal antibody is collected. Further, the antibody is purified from the ascites fluid using Protein A-Sepharose or the like to obtain a monoclonal antibody.

As will be described in detail in the examples later, the present inventors succeeded in obtaining an anti-apoB-48 monoclonal antibody by the above-described method, and obtained the monoclonal antibody B4
It was named 8-151. In addition, cells producing the monoclonal antibody B48-151 were
-151. Hybridoma B48-151
Is the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology [Address: 1-3-3 Higashi, Tsukuba, Ibaraki, Japan (Postal code 305-
0046)], identification number B48-151, accession number F
ERM BP-6473 (Original deposit date; July 4, 1997)
Request for transfer to a deposit based on the Budapest Treaty;
August 26, 2000).

The reaction specificity of the obtained anti-apoB-48 monoclonal antibody is determined by, for example, CM, VLDL and LDL.
It can be confirmed by the WB method using the fraction as an antigen. An anti-apo B-48 monoclonal antibody obtained from a hybridoma producing an anti-apo B-48 monoclonal antibody can specifically recognize apo B-48,
And it does not react at all with Apo B-100,
Very well, Apo B-48 and / or Apo B in the sample
It can be used to detect -48 containing lipoproteins. The lipoprotein containing apoB-48 means a lipoprotein having the same amino acid sequence as that of apoB-48 in the amino acid sequence of the apoprotein. Like lipoprotein, lipoprotein can be specifically recognized by the anti-apoB-48 monoclonal antibody of the present invention, which can specifically recognize apoB-48.

Further, the reaction specificity of the above-mentioned anti-apoB-48 monoclonal antibody can also be confirmed by ELISA. Anti-apo B- obtained from a hybridoma producing anti-apo B-48 monoclonal antibody
As described in detail in the Examples below, the 48 monoclonal antibody was used for ApoB in ELISA using this as a solid phase.
It is clear that -48 can be specifically recognized, and the present invention can be applied not only to a measurement method using a membrane immobilization such as a WB method which has been conventionally used, but also to a solid-phase ELISA method.

The anti-apoB-48 monoclonal antibody of the present invention can be applied to a so-called sandwich assay. For example, in order to perform a sandwich ELISA, a diluted anti-apoB-48 monoclonal antibody is put in an ELISA plate, left to adsorb, masked, washed, and then appropriately prepared apoB-48 is put as an antigen. To react.

To confirm the reaction specificity, Apo B-10
0 is measured as an antigen in the same manner. After washing, a labeled anti-human apoB antibody is added and reacted, and then washed, a substrate is added and left to stand, and the amount of reaction is measured by measuring the absorption wavelength, etc., so that apoB-48 can be specifically reacted. It can be confirmed that it is recognized.

The anti-apoB-48 monoclonal antibody of the present invention can also be applied to an assay using serum as a direct sample. For example, apoB-48 can be measured by applying the above-described ELISA method to human serum as a sample.

Measurement of apo B-48 and / or lipoprotein containing apo B-48 using the monoclonal antibody of the present invention can be performed by applying the monoclonal antibody of the present invention to various human-derived specimens. . Examples of the sample include various body fluids derived from humans, such as serum and plasma. The measurement method for these samples is also one of the present invention.

The above-mentioned specimen is preferably subjected to a treatment for exposing the epitope of apo B-48. The epitope of the monoclonal antibody of the present invention is apo B-48 C
Although it is the terminal part, since this part is not exposed in the biological fluid, it is considered that the monoclonal antibody is difficult to bind when performing an immune reaction. Need to be easy to combine. The treatment for exposing the epitope may be performed before the measurement, or may be performed simultaneously with the measurement.

As a treatment for exposing the above epitope, for example, a method of treating a specimen with a surfactant,
A method of repeating freezing and thawing can be exemplified. As a method of treating a specimen with a surfactant, the abstracts of the 30th Annual Meeting of the Japanese Society of Atherosclerosis [June 11 and 12, 1998, Teikyo University Internal Medicine, Tsukuba Memorial Hospital Internal Medicine, Makoto Kinoshita et al.
P. 33, "Development of Apoprotein B48-Containing Lipoprotein Measurement Method"], a monoclonal antibody specifically recognizing apoB-48 was prepared, and an ELISA plate using the antibody was prepared using PBS containing 2% SDS. Add diluted serum,
Measuring Apo B-48 is described. However, in the present invention, apoB-48 in serum cannot be measured in 2% SDS, and SDS is not preferred as a surfactant.

In the present invention, the surfactant is preferably a nonionic surfactant, and SDS is not preferred because it adversely inhibits an immune reaction. More preferably, Triton X-100, Triton X-114, Tween-2
0, NP-40. The surfactant alone,
Even if they are mixed, they can be used. In actual use, when a surfactant (Triton X-114 or the like) which has a high epitope exposure effect but is hardly soluble at room temperature is used, a surfactant (Tween-20 or the like) which does not inhibit the immune reaction and is easily soluble is used. It is preferable to use a mixture.

In the present invention, a surfactant is used for exposing an epitope. Generally, a surfactant inhibits an immune reaction. Therefore, a surfactant is used to balance the effect of exposing an epitope with the effect of inhibiting an immune reaction. The concentration used can be appropriately selected. The surfactant can also be made to coexist in a solution for performing an immune reaction between the specimen and the monoclonal antibody, and 0.01 to 2%, preferably,
Used at a concentration of 0.02-0.5%. The treatment solution and the treatment time of the surfactant are not particularly limited, and, for example, in a buffer used for performing an immune reaction usually,
The reaction can be performed at 4 to 40 ° C for 5 minutes to 48 hours.

In the present invention, it is also possible to destroy the lipoprotein structure and expose the epitope by repeatedly freezing and thawing the specimen. The above-mentioned freeze-thaw is 1
If it is performed more than once, the effect can be obtained.

By using the method for measuring apo B-48 and / or apo B-48-containing lipoprotein using the monoclonal antibody of the present invention, apo B-48 and / or apo B
An immunoassay reagent for measuring -48-containing lipoprotein can be produced, and such an immunoassay reagent is also one of the present invention. As such an immunoassay reagent, for example,
An agglutination immunoassay reagent containing gelatin particles or latex particles to which the monoclonal antibody of the present invention is bound,
It can be produced by an ordinary known method. Also,
Similarly, enzyme immunoassay (EIA) reagent, ELISA
A reagent and a radioimmunoassay (RIA) reagent can be used as a solid phase by using, for example, a polymer such as polystyrene, glass beads, magnetic particles, a microplate, a filter paper for immunochromatography, a glass filter, or the like. It can be manufactured by a method.

[0036]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 Synthesis of Peptide Derived from Apo B-48 A peptide in which cysteine was bonded to the N-terminus of a portion corresponding to the amino acid sequence of 4 residues from the C-terminus of Apo B-48 was synthesized (hereinafter, this specification). In the description, it is referred to as “C4”. Apo B-48
Was synthesized with a peptide having a cysteine bonded to the N-terminus at a portion corresponding to the amino acid sequence of 5 residues from the C-terminus (hereinafter referred to as “C5” in the present specification. Shown). Furthermore, a portion corresponding to the amino acid sequence of 6 residues from the C-terminal of apo B-48 has
A peptide having a cysteine bonded to the terminal was synthesized (hereinafter, referred to as "C6" in the present specification, which is shown in SEQ ID NO: 3 in the sequence listing).

For the synthesis, an automatic peptide synthesizer PSSM-8, a multi-item simultaneous solid-phase method, manufactured by Shimadzu Corporation was used. All amino acids use the L-form, the α-amino group is protected with a 9-fluorenylmethoxycarbonyl group (Fmoc group), the β-sulfhydryl group of cysteine and the γ-carboxamide group of glutamine are protected with a trityl group, Threonine β
-The hydroxyl group and the phenolic hydroxyl group of tyrosine were protected with a t-butyl group.

The solid phase carrier for initiating peptide synthesis has a C-terminal isoleucine of 0.65 mmol in advance.
30 mg of HMP isoleucine resin (manufactured by PerkinElmer) introduced at a ratio of 1 / g was used. For the condensation, 2- (1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, and N, N-diisopropylethylamine were used. For removal of the Fmoc group, a 30% piperidine / DMF solution was used, DMF was used as a solvent, and three kinds of synthesis were simultaneously performed using a standard program attached to PSSM-8.

After completion of the synthesis, the obtained peptide carrier containing a protecting group was washed with methylene chloride and dried. The yield is C4
Is 41.0 mg, C5 is 48.4 mg, and C6 is 52.6.
mg. Each of the above-mentioned peptide carriers is placed in a trifluoroacetic acid solution (trifluoroacetic acid 1 m) containing an additive.
l, water 50 µl, phenol 75 mg, thioanisole 50 µl, ethanedithiol 50 µl) at room temperature for 2 hours to remove free peptide chains. The peptide was precipitated with diethyl ether, collected by filtration, dissolved in water and lyophilized. The yield of the crude peptide thus obtained was 12.23 mg for C4, 16.17 mg for C5, and 17.6 mg for C6.
04 mg.

Next, the crude peptide was purified by reversed-phase high performance liquid chromatography. The column is Cosmosil 5C18
-AR-300 20mmI. D. x150mmL.
(Nacalai Tesque, Inc.) and eluted with an 18-28% linear concentration gradient of acetonitrile aqueous solution containing 0.1% trifluoroacetic acid. The yield of the purified peptide obtained after lyophilization was 7.12 mg for C4, 11.75 mg for C5,
C6 was 10.71 mg. These peptides are
A part thereof was subjected to a protein sequencer (Procise494, manufactured by PerkinElmer) to confirm its structure.

Example 2 Synthesis of Peptide-KLH Complex 5 mg of hemocyanin (KLH) (manufactured by Calbiochem) was dissolved in 400 μl of a 0.1 M phosphate buffer (pH 7.5), and GMBS (Dojindo Chemical) dissolved in 100 μl of DMF 1 mg (manufactured by Laboratories) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was added to a 0.1 M phosphate buffer containing 1 mM EDTA (p
H7.0), and applied to a PD-10 column (Pharmacia) equilibrated with the same buffer. First 2.5
The ml was discarded and the next 2.0 ml was collected.

In addition, about 1 mg of C4 synthesized in Example 1
Was dissolved in 1 ml of water, and the mixture was stirred at room temperature for 2 hours. The reaction solution was transferred to a dialysis tube and dialyzed against phosphate buffer (PBS) overnight. The same operation was performed for C5 and C6 to obtain about 5 ml of a KLH complex solution. The protein quantification value was determined using the C4 KLH complex (C4-KL
H) is 768 μg / ml and the KLH complex of C5 (C5-
KLH) is 861 μg / ml, KLH complex of C6 (C
6-KLH) was 1140 μg / ml.

Example 3 Preparation of lipoprotein from human serum 1.21 g of trishydroxymethylaminomethane, sodium chloride 9.
0 g, 0.372 g of disodium EDTA, 1 liter of distilled water
And adjusted to pH 7.4 (hereinafter referred to as “d =
1.006 solution)) and centrifuged at 26,000 xg for 1 hour in a Beckman ultrahigh-speed centrifuge. The upper layer was pooled as a chylomicron (CM) fraction, and the lower layer was further overlaid with a d = 1.006 solution.
After centrifugation at 14000 × g for 20 hours, the upper layer was VLD
Pooled as L fraction. Further, the lower layer portion was adjusted to d = 1.0063 with a sodium bromide solution, and 1140
The mixture was centrifuged at 00 × g for 20 hours, and the supernatant was pooled as an LDL fraction. Each pool fraction was concentrated and used as an antigen for an immune reaction.

Example 4 Establishment of Hybridoma Producing Anti-Apo B-48 Monoclonal Antibody and Anti-Apo B-4
8. Preparation of Monoclonal Antibody Hybridomas producing anti-apoB-48 monoclonal antibody were synthesized using C4-KLH and C5-K synthesized in Example 2.
Immunizing BALB / C mice with LH and C6-KLH,
It was established by fusing the spleen lymphocytes with myeloma cells.

That is, C4-KL emulsified with Freund's complete adjuvant was added to BALB / C mice.
H, C5-KLH and C6-KLH were 25 to 1 respectively.
The same complex 25 immunized with 00 μg / mouse and emulsified with incomplete Freund's adjuvant 2-3 weeks later
Booster immunizations were performed at １００100 μg / mouse. Using a part of the mouse serum, an antibody that reacts with native apo B-48 was confirmed by Western blotting (WB). The WB was measured in the same manner as the screening method described later. Free C6-KLH25 was added to the C6-KLH-immunized mice in which the most intense band was confirmed by the WB method.
-100 μg was intravenously administered, and three to four days later, the spleen was removed from the mouse to prepare splenocytes.

Mouse myeloma cells (P3U1) previously cultured in RPMI-1640 medium and the above spleen cells were mixed at a ratio of 1: 2 to 1: 5, and cell fusion was performed using PEG (Boehringer). went. The fused cells were suspended in a HAT medium, dispensed into a 96-well culture plate, and cultured in a 37 ° C. carbon dioxide incubator.

The culture supernatant of the cultured cells was screened by the WB method. That is, the VLD prepared in Example 3
Using the L fraction as an antigen, SDS polyacrylamide gel electrophoresis (SDS-PAGE) with a 3 to 15% gradient
After performing E), the migrated antigen was transferred to a nitrocellulose membrane to prepare a transfer membrane for WB. After blocking the transfer membrane with skim milk, it was cut into strips. Accutran Incubation Tra
y) Into each groove (manufactured by S & S), a culture solution obtained by pooling a strip-shaped transfer membrane and 6 wells of a 96-well culture plate as a group was added and shaken at room temperature for 1 hour to carry out a reaction.

After washing with shaking for 5 minutes in PBS containing 0.05% Tween 20 (hereinafter referred to as “washing buffer”),
After performing the test twice, a POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was placed in each groove, and the mixture was further reacted at room temperature for 1 hour. Similarly, after washing four times with a washing buffer, a substrate 4-chloronaphthol solution was added, and a band at a position corresponding to apo B-48 was confirmed.

If a band is seen, add 6 wells to 1 more
Wells producing the desired antibody were selected in the same manner by dividing the wells. The cells of the well producing the desired antibody were cloned by cloning by the limiting dilution method. The cells producing the anti-apoB-48 monoclonal antibody were cultured in large quantities and administered to the mouse peritoneal cavity, and ascites fluid containing the anti-apoB-48 monoclonal antibody was collected.
Further, the antibody was purified from ascites using protein A-Sepharose to obtain a monoclonal antibody. This anti-apo B-4
8 monoclonal antibody to monoclonal antibody B48-1
The monoclonal antibody B48-151
Was designated as hybridoma B48-151.

Example 5 Monoclonal Antibody B48-1
Confirmation of the reaction specificity of the monoclonal antibody B48-151
WB using M, VLDL and LDL fractions as antigens
Confirmed by law. That is, after performing 3 to 15% gradient SDS-PAGE using each fraction prepared in Example 3 as an antigen, the migrated antigen was transferred to a nitrocellulose membrane to prepare a transfer membrane for WB. After blocking the transfer membrane with skim milk, the reaction with the antibody was performed. For the reaction, monoclonal antibody B48-151, anti-apo B-100 monoclonal antibody MAB014 (Chemicon) and anti-Apo B goat antiserum (Chemicon) were used as controls. Further, a suppression test with a free immunopeptide C6 was also performed to confirm the reaction specificity. A portion of the WB transfer film was confirmed for protein position by CCB staining.

The reactions between the antibody and various antigens were as follows. Each antibody was added to 1% bovine serum albumin (B
SA) containing phosphate buffer (1% BSA-PBS) (pH
The concentration was adjusted to 1 µg / ml using 7.4), and the mixture was shaken with the antigen-transferred WB membrane at room temperature for 1 hour to perform a reaction. After washing with a washing buffer for 3 times with shaking for 5 minutes, a POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was added, and the mixture was further reacted at room temperature for 1 hour. Similarly, after washing four times with the washing buffer, the substrate 4-chloronaphthol solution was added, and the band was confirmed. The results are shown in FIG. The suppression test is WB
The reaction was performed in the presence of 5 μg / ml C6 during the reaction between the membrane and the antibody. The results are shown in FIG.

As shown in FIG. 1, the monoclonal antibody B4
8-151 forms a band only at a position corresponding to apo B-48 having a molecular weight of slightly more than 200,000, and apo B-1 having a molecular weight of about 550,000.
No band was found at the position corresponding to 00. FIG.
As shown in the figure, the band at the position corresponding to apo B-48 of the monoclonal antibody B48-151 disappeared in the presence of C6. From these results, it was found that monoclonal antibody B48-1
It became clear that 51 is a monoclonal antibody that specifically recognizes apo B-48. The band corresponding to apo B-48 in the VLDL fraction appears most densely because the VLDL fraction contains CM remnants.

Example 6 Monoclonal antibody B48-1
51 Measurement of Apo B-48 by Solid Phase ELISA Apo B-48 extracted and prepared from SDS-PAGE gel was used as an antigen for sandwich ELISA. That is, in the same manner as in Example 5, the apo B-48 band portion of the VLDL fraction electrophoresed on an SDS-PAGE gel was cut out, and extracted from the gel using an electroeluter (manufactured by Bio-Rad) to purify the antigen. On the other hand, Nunc ELISA plate (Maxisorb)
Then, 75 μl of the monoclonal antibody B48-151 diluted to a concentration of 10 μg / ml with PBS (pH 7.4) was added to each well in an amount of 75 μl, allowed to stand at 4 ° C. overnight, and adsorbed.
% BSA-PBS (pH 7.4) was added at 150 μl / well and left at 37 ° C. for 5 hours to perform masking. After washing the antibody adsorption plate three times with the washing buffer, 1
Add 75 μl / well of purified apo B-48 diluted from 5 μg / ml to 2 n with% BSA-PBS at 37 ° C.
Allowed to react for hours.

After washing three times with a washing buffer, 75 μl / well of a POD-labeled anti-human apoB antibody (manufactured by Chemicon) was added and reacted at 37 ° C. for 1 hour. After thoroughly washing with a washing buffer, the substrate ABTS was added at 75 μl / well and left at room temperature for 15 minutes, and the wavelength of 405 nm was measured. In order to confirm the reaction specificity, Apo B-100 (manufactured by Chemicon) was measured as an antigen from 40 μg / ml by the same dilution method. The results are shown in FIG. As shown in FIG. 3, ELISA using monoclonal antibody B48-151 as a solid phase
It was confirmed that A could specifically measure Apo B-48 in A.

Example 7 Monoclonal antibody B48-1
51 Measurement of Serum Apo B-48 by Solid-Phase ELISA Apo B-48 was obtained in the same manner as in Example 6 by using serum diluted 20-fold with 1% BSA-PBS (pH 7.4) as a sample.
Was measured. Samples included healthy subjects on an empty stomach and after meals.
Eight cases (specimen numbers 1 to 8) of the cryopreserved sera for each time were used. FIG. 4 shows the results. As shown in FIG. 4, it was confirmed that apoB-48 became high one hour after the meal in all the samples, which was consistent with the behavior of CM.

Example 8 Treatment of Fresh Serum with Surfactant Various surfactants were added to the buffer solution during the reaction between the solid phase antibody and fresh serum, and the monoclonal antibody B48-151 solid phase E
The reactivity in LISA was examined. Surfactants are SDS (manufactured by Nacalai Tesque) as an anionic surfactant,
Sodium deoxycholate (manufactured by Wako Pure Chemical Industries), Triton X-100 (manufactured by Nakarai Tesque), Triton X-114 (manufactured by Nakarai Tesque), and Tween-20 (manufactured by Nakarai Tesque) as nonionic surfactants , Tween-80 (manufactured by Nakarai Tesque), NP-40 (manufactured by Sigma), MEGA-8 (manufactured by Dojindo Laboratories), Bri
j-35 (manufactured by Sigma) was used.

A monoclonal antibody B48-151 diluted with PBS (pH 7.4) to a concentration of 10 μg / ml was added to an ELISA plate (Maxisorb) manufactured by Nunc Co., Ltd.
5 μl / well was put in each well and allowed to stand at 4 ° C. overnight for adsorption.
Next, 150% of 1% BSA-PBS (pH 7.4) was added.
Each well was placed and left at 37 ° C. for 5 hours to perform masking. After washing the antibody adsorption plate three times with the washing buffer,
Using PBS (pH 7.4) containing various surfactants at concentrations of 2%, 0.4%, 0.08%, and 0.016%, respectively, add 20 μl of diluted serum at 75 μl / well,
The reaction was performed at 37 ° C. for 1 hour. After washing three times with a washing buffer, an alkaline phosphatase (hereinafter also referred to as Alp) -labeled anti-human apo B-100 monoclonal antibody (B100-228; homemade) labeled with Yoshitake et al.
Each μl / well was added and reacted at 37 ° C. for 1 hour. After sufficiently washing with a washing buffer, the substrate 4-nitrophenol phosphate (hereinafter, also referred to as pNPP) was added at 75 μl / well, left at 37 ° C. for 30 minutes, and the wavelength of 405 nm was measured. High fat serum and fasting serum were used as samples. The results are shown in FIGS.

As shown in FIG. 5, when the serum was diluted with PBS (pH 7.4) containing no surfactant, no color development was observed, and it was judged that the apoB48C-terminal epitope was not exposed. PBS containing a surfactant (pH 7.
When 4) is used, around 0.1% of Triton X-1
Strong color development was observed with nonionic surfactants such as 00, Tween 20, and NP-40. In these surfactant treatments, it was determined that the apoB48C terminal epitope was exposed. In contrast, MEGA-8, Brij-35, SD
With a surfactant such as S, coloring was weak or hardly observed. Considering the results of Example 9, MEGA
-8 has a weak ApoB48C end epitope exposure effect,
S, especially SDS of 0.1% or more inhibits the immune response,
rij-35 could be involved in both.

Example 9 Influence of Surfactant on Immune Reaction Immobilized peptide C6 and monoclonal antibody B48-15
At the time of reaction with 1, the same various surfactants as in Example 8 were added, and the reactivity was examined. 1 μg / m with Nunc ELISA plate (Maxisorb) with PBS (pH 7.4)
The peptide C6 diluted to a concentration of 1 l was added in an amount of 75 μl / well, and allowed to stand at 4 ° C. overnight for adsorption. Next, 1% BSA-
Add PBS (pH 7.4) 150 μl / well,
It was left at 37 ° C. for 5 hours to perform masking. After washing three times with a washing buffer, 2%, 0.4%, 0.08
%, PBS containing various surfactants at a concentration of 0.016%
(PH 7.4), 75 μl / well of monoclonal antibody B48-151 diluted to 2.5 μg / ml was added thereto, and reacted at 37 ° C. for 1 hour. 3 in wash buffer
After washing twice, add a POD-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) in an amount of 75 μl / well at 37 ° C.
Allowed to react for hours. Wash well with washing buffer and wash substrate AB
After putting TS at 75 μl / well and leaving at room temperature for 15 minutes,
The wavelength of 405 nm was measured. FIG. 7 shows the results.

As shown in FIG. 7, Triton X-10
Nonionic surfactants such as 0, Tween 20, and NP-40 had no effect on the immune response. In contrast, Brij-35 and SDS had an effect on the immune response. In particular, in SDS, the immune reaction was completely inhibited at 0.1% or more.

[0061]

According to the present invention, which has the above-mentioned structure, it has become possible to obtain an anti-apoB-48 monoclonal antibody. Therefore, by specifically recognizing apoB-48, simple and reliable Diagnosis and treatment of arteriosclerosis became possible.

[0062]

[Sequence List] <110> FUJIREBIO INC. <120> Hybridoma, monoclonal antibody, assay method and immunoassay reagent <130> FR24 <150> JP 1997299439 <151> 1997-10-15 <160> 3 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220><223> Sequence Listing Free Text ApoB-48 has a N-part corresponding to the amino acid sequence of the four residues from the C-terminus. Cysteine-bound peptide <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220><223> Sequence Listing Free Text A portion corresponding to the amino acid sequence of 5 residues from the C-terminus of apo B-48 has its N -A peptide having a cysteine at the end <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220><223> Sequence Listing Free Text A portion corresponding to the amino acid sequence of 6 residues from the C-terminus of apo B-48 has its N -A peptide having a cysteine at the end

[Brief description of the drawings]

FIG. 1 shows B48-151 according to the present invention performed in Example 5.
It is a figure which shows the reaction with respect to each lipoprotein fraction of a monoclonal antibody.

FIG. 2 shows B48-151 according to the present invention performed in Example 5.
It is a figure which shows the suppression by C6 peptide of the reaction with respect to each lipoprotein fraction of a monoclonal antibody.

FIG. 3 shows B48-151 according to the present invention performed in Example 6.
It is a figure which shows the test result by ELISA measurement method using a monoclonal antibody as a solid phase.

FIG. 4 shows B48-151 according to the present invention performed in Example 7.
It is a figure which shows the test result by ELISA measurement method using a monoclonal antibody as a solid phase.

FIG. 5 is a diagram showing the results of a test performed in Example 8 by an ELISA measurement method using a B48-151 monoclonal antibody according to the present invention as a solid phase, using a high-fat serum treated with a surfactant. is there. Tx-100 is Triton X-
100, Tx-114 is Triton X-114, Tw-
20 is Tween-20, Tw-80 is Tween-80,
Deoxy-ch. Represents sodium deoxycholate. PBS indicates that the test was performed without the addition of surfactant.

FIG. 6 is a diagram showing the results of a test performed by ELISA using the B48-151 monoclonal antibody according to the present invention as a solid phase, when fasting serum treated with a surfactant was used in Example 8. is there.

FIG. 7: B48-151 according to the present invention performed in Example 9
It is a figure which shows the test result of the influence of the surfactant on the ELISA measurement method using a monoclonal antibody as a solid phase.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/53 G01N 33/577 B 33/577 A61K 39/395 // A61K 39/395 C12P 21/08 C12P 21/08 G01N 33 / 50 T G01N 33/50 C12N 5/00 B (C12P 21/08 C12R 1:91)

Claims (9)

[Claims] 1. A hybridoma obtained by fusing lymphocytes obtained by immunizing a mammal with a peptide derived from Apo B-48 and myeloma cells. 2. The method according to claim 2, wherein the peptide derived from Apo B-48 is
The hybridoma according to claim 1, which is a peptide present at the C-terminal of 48. 3. The peptide present at the C-terminus of Apo B-48 is preferably at least 6 peptides from the C-terminus of Apo B-48.
3. The hybridoma according to claim 2, which is a peptide comprising amino acid sequences. 4. A method obtained by culturing the hybridoma according to claim 1, 2 or 3,
A monoclonal antibody that specifically recognizes apo B-48. 5. The monoclonal antibody according to claim 4, which does not substantially react with Apo B-100. 6. The monoclonal antibody according to claim 4, which is produced by hybridoma B48-151. 7. ApoB-48 in a sample, characterized by using the monoclonal antibody according to claim 4, 5 or 6.
And / or a method for measuring apoB-48-containing lipoprotein. 8. The method according to claim 7, wherein a treatment for exposing the epitope of apo B-48 is performed. 9. Apo B-48 and / or Apo B-48 using the measurement method according to claim 7 or 8.
An immunoassay reagent for containing lipoprotein.