DE69822066T2 - Monoclonal antibody to Apo B-48, Hybridoma, and method of use - Google Patents

Description

 The present disclosure includes a monoclonal antibody that specifically binds to Apo-B-48 and a hybridoma that binds to it produced, and the invention relates to a method for measuring of Apo-B-48 using such a monoclonal antibody. The monoclonal antibodies is for the diagnosis and treatment of hyperlipemia and arteriosclerosis are suitable.

Atherosclerosis is one of the most typical Adult diseases, and the development of an effective diagnosis and therapy for this disease is desirable. The causes of Atherosclerosis include an increase in the levels of various Lipoproteins in the blood that circulates through the body, causing the deposition of cholesterol on the inner walls accelerated by blood vessels, and a decline various lipoproteins which cause such cholesterol deposition prevent.

Representative lipoproteins, from who are known to contribute to hyperlipemia include chylomicrons (CM), Chylomicron residues (CM residues), which is a metabolic intermediate of CM in the blood, extremely low density lipoproteins (VLDL), Lipoprotein residues extremely low Density (VLDL residues), which is a metabolic intermediate of VLDL in the blood are low density lipoproteins (LDL) and lipoproteins high density (HDL).

CM residues, VLDL residues and the like are called residual particles (RLP). The rest of the particles and LDL are lipoproteins that carry cholesterol to the blood vessel walls. A decrease in the level of these lipoproteins in the blood provides a direct therapy for Arteriosclerosis. Because, for example, a function of HDL in it consists of cholesterol from injury lead away by atherosclerosis, is an increase of HDL levels in the blood for the Treatment of atherosclerosis.

Of the above lipoproteins it has been reported that RLP is due to expression of atherosclerosis postprandial hyperlipemia contributes which is why it is considered an important risk factor for atherosclerosis becomes. Known apolipoproteins that represent RLP include Apo-C, Apo-E, Apo-A-I, Apo-B-100 and Apo-B-48.

One of the known methods for Measuring the RLP values in the blood is the RLP-C ("remnant-like particles" -, residual particle cholesterol) Method. With the RLP-C process will produce an anti-Apo-A-I monoclonal and an anti-Apo-B-100 monoclonal immobilized on a gel, and the gel is reacted with a test sample, after which the to these antibodies bound lipoproteins are removed by centrifugation. After that will be in the supernatant remaining amount of lipoprotein based on the amount of cholesterol measured.

Apo-A-I occurs as the major apolipoprotein of CM and HDL, and Apo-B-100 occurs as the major apolipoprotein of VLDL, VLDL residues and LDL.

Thus CM and HDL become theoretical on the monoclonal anti-Apo-A-I antibody and VLDL, VLDL residues and LDL bound to the anti-Apo-B-100 monoclonal antibody. So be these lipoproteins are removed in the RLP-C process.

The anti-Apo B-100 monoclonal antibody recognizes specifically the region from the 2291st to the 2318th amino acid, see above that he has Apo-B-48, which is from the 1st to the 2152nd amino acid of Apo-B-100 exists, does not recognize. Therefore, through the RLP-C process the lipoprotein measured the Apo-B-48 as apolipoprotein, but not Apo-B-100 as apolipoprotein.

The structure of the epitopes on the Apolipoproteins and the homogeneity of the epitopes in the plasma lipoprotein population are still unclear.

For the RLP-C process, this is above removal of the various lipoproteins by binding of lipoproteins on gel indispensable. However, this step is problematic and difficult to reproduce because of the degree of removal varies from run to run. Therefore, the measurements are not very reliable.

Apo-B-48 is in use as apolipoprotein CM and CM residues included. CM is rapidly removed by lipoprotein lipase after eating converted into a CM remainder, and the CM rest will through residual receptors in the liver absorbed into the liver so that CM in sober Condition not present in the blood. I.e. Chylomicrons are lipoproteins, the only are synthesized from the intestine to make fat from foods and fat-soluble vitamins to transport. Of the apolipoproteins found in chylomicrons, such as Apo-B-48, Apo-A-I, Apo-A-IV and Cs is only that Synthesis of Apo-B-48 for the assembly of chylomicrons required. Apo-B-48 is a 2152 amino acids long polypeptide found in the gut of adults of the same gene like Apo-B-100 translated by the action of a number of enzymes and is pooled. While as they circulate in the blood, chylomicrons are exposed to lipolysis and apolipoprotein exchange and converted into "chylomicron residues". Therefore, if a monoclonal Antibody, the Apo-B-48 specifically recognizes, is present, a CM residue alone can be measured directly by using the blood when sober as a test sample as a risk marker for postprandial lipemia and / or atherosclerosis is used. So it is possible to get hyperlipemia exactly diagnose what in turn for diagnosis and therapy of atherosclerosis useful is.

Apo-B-48 has the same amino acid sequence on like part of the amino acid sequence from Apo-B-100. The amino acid sequences Apo-B-100 and Apo-B-48 are known (Nature 323, 738, October 1986). An anti-Apo B-48 antiserum is also known (Journal of Biological Chemistry 265 (15), 8358 (1990); Journal of Biological Chemistry 267 (2), 1176 (1992); and Clinical Science 85, 521 (1993)) as well as monoclonal Antibody, that bind to both Apo-B-100 and Apo-B-48 (JP61007299; Arteriosclerosis 8 (5), 635A, Sept. 1988).

From a monoclonal antibody that binds specifically to Apo-B-48 alone but has not been reported. Moreover has been described (Rinsho Kensa 40 (9), 1025 (left column, 8th line from below) (1996)) that it is theoretically difficult to produce an antibody that specifically reacts with Apo-B-48 alone.

The inventors of the present invention led intensive research to provide a monoclonal antibody which specifically reacts with Apo-B-48 alone to take such a monoclonal antibody Use of a conjugate of a very small peptide derived from the C-terminal region of Apo-B-48 and a carrier protein as an immunogen, to obtain.

I.e. the present disclosure comprises a monoclonal antibody, that specifically binds to Apo-B-48, but not specifically to Apo-B-100. The present disclosure also includes a hybridoma that monoclonal antibodies produced. The present invention provides a method as defined in the claims ready to measure Apo-B-48 in a test sample that is ready for use an antigen-antibody reaction between Apo-B-48 in the test sample and a monoclonal antibody which specifically binds to Apo-B-48 and essentially does not bind to Apo-B-100.

The present disclosure provides Moreover a reagent for prepared an immunoassay for measuring Apo-B-48 which is the monoclonal antibody includes. About that in addition, the present disclosure includes an immunoassay set for Measure Apo-B-48 comprising the monoclonal antibody.

A monoclonal antibody that binds specifically to Apo-B-48, but not specifically to Apo-B-100 disclosed. Since the monoclonal antibody is specific to Apo-B-48 binds but not specifically to Apo-B-100, Apo-B-48 can do so alone be measured. Thus, the monoclonal antibody is useful for diagnosis and therapy of arteriosclerosis.

SUMMARY THE PICTURES

1 Figure 3 is a schematic of the results of the Western blot showing the reactions between a B48-151 monoclonal antibody and various lipoprotein fractions, the results obtained in Example 5;

2 Fig. 3 is a schematic of the Western blot showing the reactions between a B48-151 monoclonal antibody and various lipoprotein fractions when the reactions were inhibited by a C6 peptide, the results obtained in Example 5;

3 shows the calibration curve obtained in an ELISA using an immobilized monoclonal antibody B48-151 obtained in Example 6;

4 shows the results of the ELISA using an immobilized monoclonal antibody B48-151, which was performed using sera from healthy people as samples, the results being obtained in Example 7;

5 shows the results of the ELISA using an immobilized monoclonal antibody B48-151, which was carried out in the presence or absence of various surfactants, the results being obtained in Example 8; and

6 shows the results of the ELISA using an immobilized peptide C6 in the presence or absence of various surfactants, the results being obtained in Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned above, it binds in the process according to the present Invention used monoclonal antibodies specific to Apo-B-48, but not specifically on Apo-B-100. The corresponding epitope of the monoclonal antibody is preferably in the C-terminal region of Apo-B-48. The The C-terminal region preferably comprises amino acids 1 to 20, more preferably 1 to 10, of the C-terminus of Apo-B-48. The corresponding Epitope preferably includes the C-terminus of Apo-B-48. How to continue explained in more detail below is the corresponding epitope of the preferred monoclonal Antibody, the actually is produced in the examples, which consists of only 6 amino acids C-terminal region of Apo-B-48 existing peptide.

The one in the process according to the present Monoclonal antibodies used in the invention can be obtained as follows become:

First, a peptide derived from Apo-B-48 is synthesized. The peptide preferably consists of Essentially from 5 to 20 amino acids, more preferably from 5 to 10 amino acids, from the C-terminus of Apo-B-48 (except for a cysteine that may be attached to the N-terminus of the peptide). For example, a peptide consisting of the 6 amino acids from the C-terminus of Apo-B-48 to which a cysteine is bound at the N-terminus thereof, ie a peptide composed of 7 amino acids with the sequence Cys Leu Gln Thr Tyr Met Ile (this peptide is hereinafter referred to as "C6") can be synthesized.

The peptide derived from Apo-B-48, such as the above-mentioned C6, is then mixed with a carrier protein, such as hemocyanin, conjugated. The C6 can attach to the carrier protein through the N-terminal cysteine be bound. This can be done by using the examples below procedures described in more detail can be achieved.

Using the thus obtained The monoclonal antibody can be conjugated as an immunogen the well-known conventional Processes are made. An example of the method is in the Examples explained in more detail below. Briefly summarized becomes a mammal immunized with the conjugate, and antibody-producing cells, such as lymphocytes and spleen cells, are immunized from the mammal taken. The antibody producing Cells are then fused to myeloma cells and hybridomas selected by selective cultures. The hybridomas thus obtained produced monoclonal antibodies are then by Western Blot (WB) method or by ELISA using Apo-B-48 or Apo-B-100 as an antigen to them specificities tested, and the hybridomas, the monoclonal antibodies that bind to Apo-B-48 and not to Apo-B-100, produce, are selected, whereby the monoclonal antibody is obtained. As an antigen not only the free apolipoproteins, but also lipoproteins, which contain the apolipoproteins.

Through this procedure, which in the Examples will be explained in more detail below, a hybridoma named B48-151, which produces the monoclonal antibody. The hybridoma B48-151 was developed at the National Institute for Life Sciences and Human Technology from the Agency for Industrial Science and Technology, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, Japan, under the Accession number No. FERM BP-6473 deposited.

The one produced by the hybridoma B-48-151 monoclonal antibodies is a preferred monoclonal antibody for use in the method according to the present Invention that is strong and specific with Apo-B-48 and with Apo-B-100 at all not responding specifically. Monoclonal antibodies produced by variants of the Hybridomas B-48-151 are also preferred. The term "variant" stands for hybridomas, which are from the hybridoma B-48-151 and which are the same or modified have physiological and / or morphological features like that Hybridoma B-48-151 and produce a monoclonal antibody that is specific binds to Apo-B-48, but not specifically to Apo-B-100.

The monoclonal antibody can for measuring Apo-B-48 in a test sample by an immunoassay be used. Since not only free Apo-B-48, but also in Apo-B-48 containing lipoproteins can be measured monoclonal antibodies for immunoassays can be used to measure the lipoproteins containing Apo-B-48.

In the present description and the claims the term "measurement" includes both quantification as well as detection.

The test sample can be a body fluid, such as blood, a serum or blood plasma and a dilution thereof, although the test sample is not limited to this.

Different immunoassay methods are known in the art and the monoclonal antibody can can be used in each of these processes. I.e. known immunoassay methods include if they are classified according to the reaction mode, sandwich method, Immunoblotting, competitive, agglutination etc. If they are classified according to the marking used, include known immunoassays, enzyme immunoassays, radioimmunoassays, Fluorescence immunoassays, biotin immunoassays, etc.

These include sandwich ELISA and western blot to prefer. These methods are in the field of the invention generally known per se, and detailed procedures are described in the Examples explained in more detail below.

In short, a sandwich ELISA for example as follows. The monoclonal antibody is on a solid support, such as the walls of bowls a microtiter plate or microspheres, immobilized. To immobilization becomes the solid support with proteins such as Skimmed milk, casein, gelatin, bovine serum albumin or the like, blocked to prevent non-specific binding of the antigen. A test sample is applied to the solid support and an antigen-antibody reaction takes place. After washing the carrier, an enzyme-labeled second becomes antibody plotted against Apo-B-48, and an antigen-antibody reaction takes place. After washing, the second antibody, the to the carrier is based on the color reaction after the addition of a Substrate of the enzyme measured.

Western blot procedures can be carried out as follows, for example. A test sample is subjected to fractionation by gel electrophoresis such as SDS-PAGE and the electropho resemuster is transferred to a membrane, such as a nitrocellulose membrane. After the membrane is blocked, the monoclonal antibody is applied to the membrane and an antigen-antibody reaction takes place. After washing, a labeled second antibody against the monoclonal antibody is applied and an antigen-antibody reaction takes place. After washing, the second antibody bound to the membrane is measured by measuring the label.

In the process according to the present Invention is the test sample of a treatment with a nonionic Subjected to surfactant which exposes the epitope of Apo-B-48, wherein the epitope is recognized by the monoclonal antibody. That in cases in which the monoclonal antibody recognizes the C-terminate region of Apo-B-48 and in which the test sample a body fluid, which contains lipoprotein particles, or a dilution of which, the antigen-antibody response between Apo-B-48 and the monoclonal antibody may not progress well because the C-terminal region of Apo-B-48 may not be well exposed in lipoprotein particles is. Therefore, in such a case, the C-terminal region of Apo-B-48 exposed.

Among the nonionic surfactants that are useful for exposing the C-terminal region of Apo-B-48, belong nonionic surfactants, such as polyoxyethylene p-t-octylphenyl ether, such as B. Triton X-100 (brand name), Triton X-114 (brand name) and Nonidet P-40 (brand name); and polyoxyethylene sorbitan alkyl esters such as z. B. Tween 20 (brand name). These surfactants can be used individually or in combinations be used. If a Triton series surfactant, such as Triton X-114 (brand name), which has a high epitope exposure ability but is not readily soluble at room temperature, is used, is preferably a mixture of the Triton series surfactant and a Tween series surfactant such as Tween 20 (brand name), that is readily soluble at room temperature is and the antigen-antibody reactions essentially does not inhibit used.

Although about using SDS for the exposure of an epitope from Apo-B-48 has been reported (Makoto Kinoshita et al., Abstract of the 30th Meeting of Japan Arterial Sclerosis Association, p. 133, June 11 and 12, 1998), becomes SDS not in the process according to the present Invention used because the antigen-antibody response is inhibited can.

Because surfactants tend to be immunological To inhibit reactions, the concentration of the surfactant is preferred chosen so that the surfactant essentially does not inhibit the immunological reactions. The preferred concentration of the surfactants is preferably 0.01 to 2 % By weight, more preferably 0.02 to 0.5% by weight. The treatment will preferably in a buffer at 4 to 40 ° C. for 5 minutes to 48 hours, the for the antibody-antigen response used. The surfactant can be in the reaction mixture of the antigen-antibody reaction to be available. Thus, the treatment can coexist with the antigen-antibody response or before the antigen-antibody reaction carried out become.

A key reagent for one Immunoassay for measuring Apo-B-48 can be in the form of a solid support, on which the monoclonal antibody is immobilized. The monoclonal antibody can be obtained by a conventional one Process immobilized on gelatin particles or latex particles become a reagent for to prepare an immunoassay by an agglutination method. Similar ones Way, the monoclonal antibody using a conventional Process for the preparation of a reagent for an enzyme immunoassay (EIA), ELISA, radioimmunoassay (RIA) or the like on a solid Carrier, such as the walls of bowls a microtiter plate, polymers such. B. polystyrene, glass beads, magnetic Particles, filter paper for Immunochromatography, glass filters or the like can be immobilized.

You can also use an immunoassay set for measuring Apo-B-48 in addition to the above reagent, including the to a solid support bound monoclonal antibody, at least one or more of a second labeled antibody, reagents for measuring the label of the second antibody, a blocking agent, include a buffer and the like.

EXAMPLES

The following is the present Invention explained in more detail using examples. The examples serve For illustrative purposes only, and are not meant to be limiting understand.

Example 1: Synthesis of a from peptide derived from Apo-B-48

A peptide made from the 4 amino acids of Apo-B-48 has a C-terminus with cysteine bound to its N-terminus was, d. H. a peptide consisting of 5 amino acids with the sequence Cys Thr Tyr Met Ile (this peptide is hereinafter referred to as "C4") using a commercially available peptide synthesizer synthesized.

Similarly, a peptide that from the 5 amino acids from the C-terminus of Apo-B-48, at the N-terminus of cysteine was bound, d. H. a peptide consisting of 6 amino acids with the sequence Cys Gln Thr Tyr Met Ile consists (this peptide is hereinafter referred to as referred to as "C5").

Similarly, a peptide that from the 6 amino acids from the C-terminus of Apo-B-48, at the N-terminus of cysteine was bound, d. H. a peptide consisting of 7 amino acids with the sequence Cys Leu Gln Thr Tyr Mel Ile (this peptide is used herein as above mentioned, hereinafter referred to as "C6").

The synthesis of the peptides was under Use of a PSSM-8 automatic peptide synthesizer developed by Shimazu Corporation, Kyoto, Japan, for simultaneous distribution Synthesis of a large number of species using a compact process carried out. All amino acids were L-amino acids. The α-amino group was by a 9-fluorenylmethoxycarbonyl group (Fmoc group), the β-sulfhydryl group of cysteine and the γ-carboxamide group of glutamine were replaced by a trityl group and the β-hydroxy group of threonine and the phenolic hydroxy group of tyrosine protected a t-butyl group.

As a solid support for initiation of the Peptide synthesis was 30 mg of HMP isoleucine resin (commercially available from Perkin Elmer), to which previously isoleucine (the C-terminus of the peptides) in an amount of 0.65 mmol / g was used. to Condensation was 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N, N-diisopropylethylamine used. To remove an Fmoc group, 30% piperidine / DMF- (dimethylformamide) solvent used. As a solvent for the DMF was used in the reaction. The three peptides were made simultaneously according to the standard working instructions of the instructions enclosed with the PSSM-8 synthesized.

After the synthesis, the carriers were on the peptides containing the protecting groups were bound washed with methylene chloride and dried. The yields at C4, C5 and C6 were 41.0 mg, 48.4 mg and 52.6 mg, respectively.

Each of the carriers to which the peptide is bound with a trifluoroacetic acid solution (1 ml trifluoroacetic acid, 50 μl water, 75 mg phenol, 50 ul Thioanisole and 50 μl Ethanediol) treated for 2 hours at room temperature to get a to get free peptide chain. The peptide was treated with diethyl ether precipitated and the precipitate was filtered off. The rainfall was in Water dissolved and freeze-dried. The yields of the crude peptides thus obtained C4, C5 and C6 were 12.23 mg, 16.17 mg and 17.04 mg, respectively.

The crude peptides thus obtained were purified by reverse phase high performance chromatography. The pillar was a Cosmosil 5C18-AR-300 (20 mm inner diameter × 150 mm Length, available in the stores by Nacalai Tesque), and elution was performed using 18 to 28% linear gradients an aqueous acetonitrile with 0.1% trifluoroacetic acid carried out. The yields of the purified peptides C4, C5 and C6 after the Freeze drying was 7.12 mg, 11.75 mg and 10.71 mg, respectively. aliquots these peptides have been tested and their sequences were confirmed using a protein sequencer (Procise 494, commercially available by Perkin Elmer).

Example 2: Synthesis of Peptide-KLH conjugates

In 400 μl 0.1 M phosphate buffer (pH 7.5) 5 mg keyhole limpet hemocyanin (KLH) (commercially available from Calbiochem) solved, and 1 mg GMBS (N-γ-maleimidobutyryloxysuccinimide ester, available in the stores from Dojindo Laboratories) was added to the solution, after which the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was on a PD-10 column (available in the stores from Pharmacia), with 0.1 M phosphate buffer (pH 7.0), the Contained 1 mM EDTA, equilibrated and eluted with the same buffer. The initial 2.5 ml was discarded and the subsequent 2.0 ml was collected.

The result was about 1 mg Added C4, which was synthesized in Example 1, in 1 ml of water, and the mixture was stirred at room temperature for 2 hours. The Reaction mixture was transferred to a dialysis tube and overnight dialyzed against phosphate buffer (PBS). The same procedure was followed performed with C5 and C6, um for each peptide about 5 ml of a solution of a KLH-peptide conjugate. The measured amounts of Proteins for C4-KLH, C5-KLH and C6-KLH were 768 μg / ml, 861 μg / ml and 1140 μg / ml, respectively.

Example 3: Production of lipoproteins from human serum

4 ml of pooled human serum, which was taken 2 hours after eating, was mixed with 4 ml of a solution prepared by dissolving 1.21 g of tris-hydroxymethylaminomethane, 9.0 g of sodium chloride and 0.372 g of disodium EDTA in 1 l of distilled water and subsequent adjustment of the pH to 7.4 was obtained (this solution is hereinafter referred to as "d = 1.006 solution") and the resultant was centrifuged for 1 hour at 26,000 x g in a Beckman ultracentrifuge. The upper phase was pooled as a CM fraction. The lower phase was overlaid with d = 1.006 solution and centrifuged for 20 hours at 114,000 × g. The upper phase was pooled as a VLDL fraction. The density of the lower phase was adjusted to 1.063 with a sodium bromide solution , and the resultant was centrifuged at 114,000 x g for 20 hours, the supernatant was pooled as an LDL fraction, and the pooled fractions were concentrated and used as antigens for the antigen-antibody reactions.

Example 4: Formation of anti-Apo B-48 monoclonal antibodies producing hybridomas and production of anti-Apo B-48 monoclonal antibodies

Hybridomas that produce anti-Apo B-48 monoclonal antibodies were by immunizing BALB / C mice with C4-KLH, or C5-KLH C6-KLH and fusion of splenic lymphocytes immunized by the mice were obtained, and myeloma cells were obtained.

More specifically, C4-KLH, C5-KLH and C6-KLH emulsified with complete Freund's adjuvant, and the Were mice with the emulsions in a dose of 25 to 100 μg of the conjugate immunized per mouse. The individual conjugates were made two to three weeks later emulsified with incomplete Freund's adjuvant and the mice were additionally with the emulsions in a dose of 25 to 100 μg of the conjugate immunized per mouse. Samples of sera from the mice were drawn and run tested the Western blot (WB) procedure for the presence of anti-Apo-B-48 antibody. The WB procedure was the same as that below described WB method performed to screen the desired monoclonal antibody carried out has been. The mouse immunized with C6-KLH had the WB method strongest Gang up. This mouse was given 25 to 100 μg of free C6-KLH intravenously administered. Three or four days later the mouse became the spleen and spleen cells were made from it.

Mouse myeloma cells (P3U1), which had been previously grown on RPMI-1640 medium, and the spleen cells obtained were mixed in a ratio of 1: 2 to 1: 5, and cell fusion was performed using PEG (from Boehringer) carried out. The fused cells were suspended in a HAT medium and then transferred to wells of a 96-well culture plate, after which the cells were grown in a CO ₂ incubator at 37 ° C.

The culture supernatants of the cells were assayed screened the WB procedure. I.e. the VLDL faction in example 3, SDS polyacrylamide gel electrophoresis (SDS-PAGE) subjected to a gradient of 3-15%, and the electrophoresis pattern was transferred to a nitrocellulose membrane to a blotting membrane manufacture. The blotting membrane was blocked with skim milk and then cut into strips. Each of these strip-shaped membranes was placed in a groove of an Accutran incubation tray (commercially available from S & S), and a mixture of a pooled culture medium from 6 wells of 96-well culture plate was added to one of the grooves, after which a 1 hour response followed at room temperature.

The blotting membranes were under stir three times for 5 minutes with PBS, the 0.05% Tween 20 (brand name) (this buffer is hereinafter referred to as "washing buffer"), washed, and a peroxidase (POD) labeled anti-mouse immunoglobulin antibody (from Daco) was added to each groove, followed by a 1 hour reaction at room temperature. The membranes were then 4 times on the same Washed as above with the wash buffer, and the substrate 4-chloronaphthol was added. After that, the presence of the Apo-B-48 became corresponding Gangs checked.

Using the individual culture media, which represented the mixture of the culture media of 6 wells that corresponded to the a groove expressing a positive band has been added, WB was carried out again as above, and the bowl, which the desired Antigen produced was selected. The cells in the well were through a limit dilution process cloned. The cells that produced the anti-Apo B-48 antibody were isolated in greater Amount administered intraperitoneally to a mouse, and ascites that affect the anti-Apo B-48 monoclonal antibody were removed from the mouse. The monoclonal antibody was cleaned of ascites using Protein A Sepharose (brand name) has been.

This anti-Apo B-48 monoclonal antibody was as a monoclonal antibody B48-151, and the hybridoma producing this monoclonal antibody B48-151 was Hybridoma called B48-151.

Example 5: Confirmation of reaction specificity of the monoclonal antibody B48-151

The reaction specificity of the monoclonal antibody B48-151 was made by a WB process examined using CM, VLDL and LDL fractions.

More specifically, each of the above-mentioned fractions prepared as antigen in Example 3 was subjected to SDS-PAGE with a gradient of 3 to 15%, and the electrophoresis pattern obtained was transferred to a nitrocellulose membrane. The blotting membrane obtained was blocked with skim milk and the resulting membrane was subjected to a reaction with the antibodies. The monoclonal antibody B48-151 was used as antibody and a monoclonal anti-Apo-B-100 antibody MAB014 (from Chemicon) and an anti-Apo-B goat serum (from Chemicon) were used as controls. In order to further confirm the reaction specificity, an inhibition test was carried out using free C6. Some the blotting membranes were subjected to CCB staining to determine the position of the proteins.

The antibody-antigen reactions were carried out as follows. The individual antibodies were dissolved in a phosphate buffer containing 1% bovine serum albumin (1% BSA-PBS) (pH 7.4) at a concentration of 1 µg / ml, and the resulting solution was stirred for 1 hour Allow room temperature to react with the individual antigens blotted on the WB membrane. After the membrane was washed with the washing buffer three times for five minutes, a POD-labeled anti-mouse immunoglobulin antibody (from Daco) was added to the membrane, and the whole was reacted at room temperature for 1 hour. After the membrane was washed four times in the same manner as above, a solution of the 4-chloronaphtol substrate was added and the bands were observed. The results are in 1 to see.

The inhibition test was carried out in the same manner as described above, except that C6 was present in a concentration of 5 μg / ml in the reaction between the WB membrane and the individual antibodies. The results are in 2 to see.

As in 1 As seen, use of the B48-151 monoclonal antibody resulted in only one position at a band corresponding to Apo-B-48 with a molecular weight of slightly more than 200,000 and one band at Apo-B-100 with a molecular weight of about 550,000, was not observed.

As in 2 the band corresponding to Apo-B-48 when monoclonal antibody B48-151 was used disappeared with the presence of C6.

These results clearly show that the monoclonal antibody B48-151 specifically recognizes Apo-B-48, but not specifically Apo-B-100.

If a VLDL fraction is used the gang that matched Apo-B-48 was the thickest, probably, because a CM residue is in the VLDL fraction. I.e. there the Fractions that have differences in particle size and weight, could have been separated the VLDL fractions were contaminated with chylomicron residues, about immunoreactivity to show against Apo-B-48.

Example 6: Measurement of Apo-B-48 by ELISA using an immobilized monoclonal antibody B48-151

A sandwich ELISA was used Apo-B-48 as an antigen extracted from the SDS-PAGE gel carried out.

More specifically was said by the same Procedure as in Example 2 carried out an SDS-PAGE. The percentage of gel that using the VLDL fraction gave the Apo B-48 band cut out, and the antigen was removed using an electroeluter (available in the stores by Bio Rad) eluted from the gel. On the other hand, everyone became Well of an ELISA plate (Maxisorb, commercially available from Nunc) 75 ul of the monoclonal antibody B48-151 in PBS (pH 7.4) added at a concentration of 10 μg / ml, and the plate was over Night at 4 ° C left standing so that the antibodies are adsorbed on the wells could. 150 μl of 1% BSA-PBS (pH 7.4) were added to each well, and the plate was incubated at 37 ° C for 5 hours, causing masking carried out has been. After washing the plates three times with the wash buffer 75 μl / well were duplicated in each well in succession (2 '') diluted, cleaned Apo-B-48 in 1% BSA-PBS at a concentration of 5 μg / ml, and the whole was allowed to react at 37 ° C for 1 hour.

After three times with the Wash buffer had been washed, a POD-labeled anti-human Apo-B antibody (from Chemicon) in an amount of 75 μl / well was added and the whole was reacted at 37 ° C for 1 hour calmly.

The record then went well with that Wash buffer was washed, and the substrate ABTS was in an amount of 75 ul / well to the bowls added, then stand at room temperature for 15 minutes was left followed by a measurement of absorbency at 405 nm.

To confirm the reaction specificity, was as a control Apo-B-100 (from Chemicon) successively to the same Way as above, but starting from a concentration of 40 μg / ml, diluted and used as antigens in the ELISA described above.

The results are in 3 to see. As in 3 Apo-B-48 can be measured by ELISA using an immobilized monoclonal antibody B48-151, while Apo-B-100 was not measured by this ELISA.

Example 7: Measurement of Apo-B-48 in sera by ELISA using an immobilized monoclonal antibody B48-151

Apo-B-48 was measured in the same manner as in Example 6, except that the samples were sera diluted 20-fold with 1% BSA-PBS (pH 7.4). The sample sera were pooled frozen sera from 8 healthy subjects (samples Nos. 1 to 8) that were fasting or 1 hour after eating were removed. The results are in 4 to see.

As in 4 It can be seen that the measured Apo-B-48 values 1 hour after eating were higher than those in the fasted state for all samples. This was in line with the behavior of CM.

Example 8: Treatment of serums with surfactants

Using the reaction in the ELISA of the immobilized monoclonal antibody B48-151 when reacting of the immobilized antibody and various surfactants were added to the reaction mixture in the sera was examined. The surfactants used were anionic Surfactants, d. H. SDS (from Nacalai Tesque) and sodium deoxycholate (from Wako Pure Chemicals), and nonionic surfactants, i.e. H. Triton X-100 (by Nacalai Tesque), Triton X-114 (by Nacalai Tesque), Tween-20 (by Nacalai Tesque), Tween-80 (from Nacalai Tesque), NP-40 (from Sigma), Mega-8 (from Dojindo Laboratories) and Brij-35 (from Sigma).

Monoclonal antibodies B48-151 dissolved in a concentration of 10 μg / ml in PBS (pH 7.4) were in an amount of 75 μl / well in wells of an ELISA plate (Maxisorb, commercially available from Nunc) was given and the plate was left overnight at 4 ° C, whereby the antibodies could be adsorbed on the wells. Then 1% BSA-PBS (pH 7.4) was added to the wells in an amount of 150 µl / well and the plate was incubated for 5 hours at 37 ° C, whereby masking was carried out. After washing the plate three times with the washing buffer, sera diluted 20-fold with PBS (pH 7.4) were added, which was one of the surfactants in a concentration of 2% by weight, 0.4% by weight, 0.08 % Or 0.016% by weight was added to the wells in an amount of 75 µl / well, and the reaction was allowed to proceed at 37 ° C for 1 hour. After the plate was washed three times with the washing buffer, an anti-human apo B-100 monoclonal antibody (B100-228), dissolved in PBS (pH 7.4), which was labeled with alkaline phosphatase and by which The method of Yoshitake et al. 75 µl / well was added to the wells and the reaction was allowed to proceed at 37 ° C for 1 hour. The plate was then washed sufficiently with the wash buffer and the substrate 4-nitrophenol phosphate was added to the wells in an amount of 75 ul / well. After the plate was incubated at 37 ° C for 30 minutes, the absorbance was measured at 405 nm. Fresh sera from healthy subjects taken 1 hour after eating and fresh sera from healthy subjects taken when fasted were used as samples. The results are in 5 to see. In 5 "Tx-100" stands for Triton X-100, "Tx-114" for Triton X-114, "Tw-20" for Tween 20, "TW-80" for Tween 80, "Deoxy-ch." for sodium deoxycholate and PBS for PBS that does not contain surfactants.

As in 5 no staining was observed in cases where the serum was diluted with PBS (pH 7.4) containing no surfactant, so that these were evaluated so that the C-terminal epitope of Apo-B- 48 was not exposed. In cases where the PBS (pH 7.4) was used with a surfactant, strong staining occurred when a nonionic surfactant such as Triton X-100, Tween 20 or NP-40 at a concentration of about 0, 1% was used. Thus, these were evaluated so that the C-terminal epitope of Apo-B-48 was exposed by the treatments with these surfactants. On the other hand, in cases where MEGA-8, Brij-35 or SDS was used, the coloring was weak or almost absent. Inclusion of the results of Example 9 below led to the conclusion that MEGA-8 has little ability to expose the C-terminal epitope of Apo-B-48; that SDS, especially at a concentration of not less than 0.1%, inhibits the immunological response; and that Brij-35 has poor ability to expose the Apo-B-48 terminal epitope and inhibits the immunological response.

Example 9: Influence of Surfactants on the immunological response

The reaction between an immobilized peptide C6 and the monoclonal antibody B48-151 when the same surfactants as in Example 8 were added to the reaction mixture was examined. A peptide C6 dissolved in PBS (pH 7.4) at a concentration of 1 μg / ml was put in an amount of 75 μl / well in wells of an ELISA plate (Maxisorb, commercially available from Nunc), and the plate was left overnight at 4 ° C, whereby the peptide C6 was adsorbed on the walls of the wells. Then 1% BSA-PBS (pH 7.4) was added to the wells in an amount of 150 µl / well and the plate was incubated for 5 hours at 37 ° C, whereby masking was carried out. After the plate had been washed three times with the washing buffer, a monoclonal antibody B48-151 dissolved in PBS (pH 7.4) at a concentration of 2.5 μg / ml, the PBS being one of the surfactants at a concentration of 2 Wt.%, 0.4 wt.%, 0.08 wt.% Or 0.016 wt.% Was added to the wells in an amount of 75 ul / well and the reaction was carried out at 37 for 1 hour ° C allowed to proceed. After the plate was washed three times with the wash buffer, a POD-labeled mouse immunoglobulin antibody per (from Daco) was added to the wells in an amount of 75 µl / well and the reaction was allowed to proceed at 37 ° C for 1 hour. The plate was then washed sufficiently with the wash buffer and the substrate ABTS was added to the wells in an amount of 75 ul / well. After the plate was incubated at 37 ° C for 15 minutes, the absorbance was measured at 405 nm. The results are in 6 to see.

As in 6 no influence of a nonionic surfactant, such as Triton X-100, Tween 20 or NP-40, on the immunological reaction was observed. However, Brij-35 or SDS had an impact on the immunological response. SDS in particular completely inhibited the immunological response at a concentration of not less than 0.1% by weight.

(informal) sequence listing

SEQUENCE LISTING

Claims (3)

Method of measuring apo-B-48 in a test sample, comprehensively the use of antigen-antibody reaction between apo-B-48 in the test sample and a monoclonal antibody specific to apo-B-48 binds and which does not essentially bind to apo-B-100, wherein the apo-B-48 in the test sample before or simultaneously with the antigen-antibody reaction is treated with a nonionic surfactant. The method of claim 1, wherein the monoclonal antibody on a solid support is immobilized by the monoclonal antibody the carrier immobilized apo-B-48 after the antigen-antibody reaction is measured. Method according to claim 2, which is carried out by ELISA.