JPH02265497A - Anti-human apolipoprotein c-ii antibody and determination of human apolipoprotein c-ii using thereof - Google Patents

Abstract

PURPOSE:To easily and accurately determine human apolipoprotein C-II by using one or more kinds of monoclonal antibodies specifically recognizing human apolipoprotein C-II. CONSTITUTION:An animal such as BALB/c mouse is immunized with purified human apolipoprotein C-II (A) to obtain an antibody-producing cell (B) such as mouse spleen cell. The cell B is fused with (C) a mouse myeloma cell FO by polyethylene glycol method, etc., and the obtained fused cell is cloned by enzyme immunoassay, etc., to obtain a hybridoma (D) capable of producing anti-apo C-II monoclonal antibody. The component D is cultured in a desired medium and the supernatant of the cultured product is separated and purified by ammonium sulfate fractionation, affinity chromatography, etc., to obtain an anti-human apolipoprotein C-II antibody. Human apolipoprotein C-II is determined as necessary with the antibody using enzyme immunoassay, immunological nephelometry, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an anti-human apolipoprotein C-II monoclonal antibody and a method for measuring human apolipoprotein C-II using the same. More specifically, human apolipoprotein C-
(2) An anti-human apolipoprotein C-n monoclonal antibody that specifically recognizes human apolipoprotein C-II, which is produced by a fusion cell of a mammalian antibody-producing cell immunized with a mammalian myeloma cell, and the anti-human apolipoprotein C-n monoclonal antibody that specifically recognizes human apolipoprotein C-II; The present invention relates to a method for measuring human apolipoprotein C-II, which is characterized by using one or more C-n monoclonal antibodies.

Problems to be Solved by the Prior Art and Inventions As the food situation becomes more nutritious and the aging society progresses, diseases such as hyperlipidemia and arteriosclerosis caused by abnormal lipid metabolism are becoming increasingly common.
In recent years, the trend of increase has become stronger. In diagnosing these diseases, it is widely practiced in the clinical field to measure blood lipids, particularly lipoproteins and their protein components, apolipoproteins.

Human apolipoprotein c-n (hereinafter referred to as "apoC-■") is mainly present in chylomicrons and very low density lipoproteins (hereinafter referred to as rVLDLJ) among lipoproteins in human blood, and is responsible for the production of lipoprotein lipase. Apolipoproteins are attracting particular attention because they are powerful activators. Measuring its blood concentration provides an important guideline for understanding lipid metabolism.

Currently, apoC-II is measured by various immunoassay methods using antiserum obtained by immunizing animals with apoC-■. However, immunoassay methods that use antisera obtained from animals utilize polyclonal antibodies contained in the antisera, and these antibodies may vary depending on the degree of purification of the antigen used for immunization and the individual nature of the immunized animal. Differences in specificity and strength between lofts occur due to differences in lofts, making it difficult to obtain consistent measurement values. Furthermore, when applied as a reagent kit, it was difficult to produce a product with consistent performance.

Means for Solving the Problems As a result of intensive research aimed at improving the above-mentioned drawbacks of polyclonal antibodies, the present inventors succeeded in producing a monoclonal antibody that specifically reacts with apoC-■. The present inventors have discovered that monoclonal antibodies are useful for measuring apoC-II, and have completed the present invention.

That is, the present invention provides a monoclonal antibody that specifically reacts with apoC-H, and a method for measuring apoC-H using the monoclonal antibody.

The monoclonal antibody against apoC-■ of the present invention is produced by a fusion cell (hereinafter referred to as "hybridoma") of a mammalian antibody-producing cell immunized with apoC-■ and a mammalian myeloma cell. The thus obtained anti-apoC-■ monoclonal antibody of the present invention (hereinafter referred to as the "antibody of the present invention") can be used as a specific antibody in various immunoassay methods, and can be used as a simple, rapid, and highly accurate antibody. Enables measurement of apoC-II.

Next, a method for producing a hybridoma producing the antibody of the present invention, a method for producing the antibody of the present invention, and a method for measuring human apoC-II using the antibody of the present invention will be explained in more detail.

A hybridoma producing the antibody of the present invention can be produced by the following steps: (i) mammalian immunization, (ii) cell fusion, and (iii) cloning.

(i) Administration of immunopurified mammalian apoC-II to a mammal as an antigen (
immunization). ApoC- used as antigen
■ After delipidating the chylomicron/VLDL fraction obtained by ultracentrifugation of human serum or plasma, Sephacryl S
It can be obtained by purification by column chromatography using -200 or the like. As the mammal to be immunized, mice, nude mice, rats, etc. can be used, but BALB/c mice are particularly preferred.

The immunization method is carried out by a conventional method, and the antigen is administered to the mammal at an appropriate site such as intraperitoneally, on the back, or subcutaneously. The dosage is determined appropriately depending on the type of mammal, the site of administration, etc., but when administering to mice, it is 10 μ9 to 1 liter per dose.
It is appropriate to set it to about my/mouse.

(ii) Cell fusion Cell fusion is carried out by a conventional method using the antibody-producing cells of the immunized mammal obtained in step (i) above and myeloma cells of the mammal. The most suitable antibody-producing cells are the BALB/c mouse lung cells described above, but other cells such as mouse lymph node cells, peripheral lymphocytes, and rat lymphocytes can also be used. As the myeloma cells, mouse myeloma cells FO are preferred, but other myeloma cells can also be used. Examples of cell fusion methods include polyethylene glycol method, HVJ method, electrofusion method, and the like.

The hybridoma obtained in this way was 1. OXlo-
Only hybridomas will proliferate when cultured in an appropriate medium such as Dulbecco's Modified Eagle's Medium (DMEM) containing 4M hypoxanthine, 4.0 x 10-'M aminopterin and 1.6 x 10-'M thymidine.

(iii) Cloning After screening for hybridomas producing the antibody of the present invention from the hybridomas proliferated in step (if) above, cloning is performed by a known method such as limiting dilution method. Screening for hybridomas involves testing hybridoma culture supernatants using enzyme-linked immunosorbent assay or Western blot method using purified apoC-■ or human chylomicron/VLDL fraction as antigens, and detecting antibodies of the present invention in culture supernatants. This can be done by checking the presence or absence of .

Then, the antibody of the present invention is produced from the above hybridoma.

That is, the antibody-producing hybridoma of the present invention obtained through the steps (i) to (iii) above is cultured in an appropriate medium and separated and purified from the culture supernatant (in vitro method);
Alternatively, the desired antibody of the present invention can be produced by intraperitoneally administering a hybridoma producing the antibody of the present invention to a compatible mammal, allowing it to proliferate, and separating and purifying it from the ascites of the animal (in vivo method). . A medium that can be used in the in vitro method is DMEM.

Examples include media such as RPM11640 medium and ASF medium 103. Separation and purification can be carried out by conventional methods such as ammonium sulfate fraction a<ammonium sulfate salting out), ion exchange chromatography using DEAE cellulose, and affinity chromatography.

The antibody of the present invention thus obtained can be used in an immunoassay for apoC-II, such as a conventional enzyme immunoassay (EIA).
It can be used for nephelometric immunoassay (TIA method), etc.

Application of the antibody of the present invention to enzyme immunoassay (EIA method) can be carried out as follows. That is, after reacting the insolubilized antibody of the invention (hereinafter referred to as "insolubilized antibody") with apoC-II, the antibody of the invention labeled with a labeling substance (hereinafter referred to as "labeled antibody") is reacted with apoC-II bound to the insolubilized antibody. (referred to as "antibody"). Next, after separating the labeled antibody bound to the insolubilized antibody/apoC-■ conjugate from the unbound labeled antibody, the labeling activity of either the bound labeled antibody or the unbound labeled antibody is measured to determine the apoC-■ conjugate in the sample. C-■ can be quantified.

Enzyme immunoassay (EIA method) generally uses the antibody of the present invention.
When measuring apoC-II, it is convenient to use two types of antibodies of the present invention that react with different antigenic determinants, one as an insolubilized antibody and the other as a labeled antibody, by the so-called Sand-Deutsch method. The insolubilized antibody is prepared by adding the antibody of the present invention to an insolubilized carrier (polystyrene beads,
It can be manufactured by chemically or physically bonding to glass beads, 96-well microplates, etc.). For example, when the antibody of the present invention is physically bound using a 96-well microplate (made of polystyrene) as an insolubilization carrier, the antibody concentration is 5 to 500 μg/s2.
, preferably 10 to 100 μg/laQ, with an appropriate buffer such as carbonate buffer [antibody concentration is calculated from the value measured with a mouse IgG or rgMRID plate (manufactured by Serotik)], and each well 50 to 1
It is sufficient to dispense 00 μQ each and keep warm at 37° C. for several hours.

Labeling substances for antibodies include various enzymes such as β-galactosidase and peroxidase, and labeling may be performed according to conventional methods. Labeled antibodies have different measurement sensitivity, measurement range,
Alternatively, it may be used by diluting it with an appropriate buffer solution to an optimal concentration, taking into account compatibility with the insolubilized antibody.

The immune reaction is carried out at 4 to 37°C for several hours to about 24 hours.

When applying the antibody of the present invention to immunoturbidimetry (TIA method), the antibody of the present invention and apoC-II are reacted in an appropriate buffer to form an immune complex, and the turbidity generated at that time is reduced. Measure and do it. Generally, in the case of a test substance that has multiple identical antigenic determinants in one antigen, turbidity is likely to occur due to the antigen-antibody reaction, so even one type of monoclonal antibody is sufficient to generate turbidity. As in the case of C-II, when the antigen to be measured contains only one specific antigenic determinant, monoclonal antibodies against that specific antigenic determinant alone will not be sufficient to prevent the antigen-antibody reaction. 7. Less likely to cause turbidity. Therefore, in order to obtain appropriate turbidity in this case, it is necessary to use a combination of two or more monoclonal antibodies that each recognize different types of antigenic determinants, but the antibody of the present invention There are antibodies that produce turbidity alone, and antibodies that do not produce turbidity with one type of antibody but produce turbidity when two or more antibodies that react with different antigenic determinants are combined. One suitable for turbidimetry may be selected as appropriate.

The concentration of the antibody of the present invention used in immunoturbidimetry is 10 to 500 μg/m12 when one type of antibody is used, preferably 50 to 200 μg/m12, and 100 to 5.0 μg/m12 when two or more types of antibodies are used in combination. OOOμ9/IIQ, preferably 500 to 3,000μ9/x (l [antibody concentration is calculated from the value measured with a mouse IgG or IgMRID plate (manufactured by Serotik)], a reaction accelerator as a buffer Phosphate buffer, Tris-HCa buffer, Gud buffer, etc. prepared to have a buffering capacity around neutrality can be used.Specific examples of reaction accelerators include polyethylene glycol 6000, etc. The reaction is carried out at a temperature around 37° C. for several minutes to about 30 minutes.

Samples used in these immunoassays include human serum or plasma.

As mentioned above, by using the antibody of the present invention in enzyme immunoassay (EIA method), immunoturbidimetry (TIA method), etc., apoC-II in a sample can be measured easily and accurately. becomes possible.

Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

Example 1 (Preparation of hybridoma producing the antibody of the present invention) ■ Preparation of antigen Preparation of human chylomicron/VLDL fraction Normal human plasma was collected in a centrifuge tube (Beckman, Ultra Clear, 168
76m+*) and overlaid with physiological saline. This was carried out using a Hitachi 55P-2 ultracentrifuge (Hitachi RP40 rotor) for 40. OOOrpm.

Centrifugation was performed at 15°C for 24 hours. Chylomicron V for indoor shoes
The LDL fraction was collected.

Preparation of ApoC-■ Solution A 10-fold amount of ethanol:ether (3:1) was added to the chylomicron/VLDL fraction and defatted three times.

The precipitate was dissolved in 1O% SDS solution (containing 1% 2-mercaptoethanol) (7,5me). The resulting solution was mixed with 0.2% SDS, 1 sM EDTA.

10sM) Lisu HCg containing 0.9% sodium chloride
The mixture was applied to a Sephacryl S-200 (manufactured by Pharmacia) column equilibrated with a buffer solution (pH 7.2) and kept at 50° C., and eluted with the same buffer solution. Three peaks (F1% F2 and F3) were obtained, and when examined by SDS polyacrylamide gel electrophoresis, apoC-■ was observed in peak F3. Both fractions were collected and dialyzed twice against purified water (5,000+a). The dialysate was concentrated by ultrafiltration using a 7M5 membrane (manufactured by Amicon).

The total amount of liquid collected was 4.8-. When the total protein concentration was measured using the Lowry method, it was found to be 2.45 x 97 cm (1.
Stored at °C.

■ Immunization of animals The apoC-■ solution obtained in the above step ■ was added to a protein concentration of 20μ
It was diluted with physiological saline to give g/sN.

This diluted ApoC-■ solution and Freund's complete adzinibant were mixed at a ratio of 1:1 (volume ratio) to prepare an antigen emulsion.

This antigen emulsion (1 mQ) was applied to BALB/c mice (male, 6
Initial immunization (initial immunization) to be administered subcutaneously to the back of children (age). One month after the first immunization, the above diluted ApoC-■ solution (0.25sN)
was further administered intraperitoneally (booster immunization).

■ Preparation of cell fusion lung cell suspension The lungs were removed from the mice 3 days after the booster immunization, and DMEM (
Manufactured by Gibco; Contains 4 glucose, 500319/12, 10 each of penicillin and streptomycin.
Lung cells were taken out from the lungs in a medium containing fetal bovine serum (hereinafter referred to as "basal medium"). The removed lung cells were suspended in a basal medium and stored at 4°C until used for cell fusion. 2.36×10 lung cells were obtained.

Preparation of myeloma cells FO was used as myeloma cells. 10% fetal bovine serum,
Basal medium containing laM sodium pyruvate (hereinafter referred to as “
After washing the FOs cultured in the basal medium twice with the basal medium, they were suspended in the basal medium. Cell density is 8XI
O” pieces/rs (l).

Cell fusion The above lung cell suspension is added to the above myeloma cell suspension (approximately 7.
After mixing 5 m and centrifuging at 1,000 rpm+ for 5 minutes, the supernatant was removed. A sterile 50% polyethylene glycol 4000 solution (ln+) kept at 37°C was added dropwise over 1 minute with stirring. After stirring the mixture for 1 minute, the basal medium (2d) was added over 2 minutes with stirring. Further, 8 mQ of the basal medium was added dropwise over 2 minutes while stirring. After centrifuging at 1.000 rpIl for 5 minutes and removing the supernatant, 1.0 x 10-'M hypoxanthin, 4.0 x 10-'M Aminopterin, 1.6X10
- Basal medium containing 5M thymidine (hereinafter referred to as rHAT medium)
) (50n+12) was added to prepare a hybridoma suspension.

■Culture of hybridomas and screening of hybridomas producing antibodies of the present invention 100 μQ of the above hybridoma suspension was added to 5 96-well culture microplates (manufactured by Coaster) in which 200 μσ of HAT medium had been dispensed in advance. The cells were cultured in an incubator at 5% carbon dioxide. Hybridomas producing the antibody of the present invention were screened by enzyme immunoassay (EIA) using human chylomicron/VLDL sensitized microplates.

Preparation of human chylomicron/VLDL sensitized microplate The human chylomicron/VLDL fraction obtained in the apoC-II purification process was mixed with 50 μM carbonate buffer (pi-19,6
) and dispensed 100 μQ per well into a 96-well microplate for EIA (manufactured by Coaster), followed by heating at 37° C. for 1 hour. Then, 25mM phosphate buffered saline (pH 7.3
, containing 1% Tween 20; hereinafter referred to as "PBS"), and then washed with a 3% bovine serum albumin solution (PBS).
(prepared in ) (200 μI2) was dispensed and heated at 37° C. for 1 hour. Human chylomicron/VLD obtained in this way
The L-sensitized microplates were stored at 4°C until use.

EIA method Culture supernatant of wells in which hybridomas are growing (10
0 μC) was dispensed into a human chylomicron/VLDL sensitized microplate and heated at 37° C. for 1 hour. After washing with PBS, anti-mouse IgG (H+L) goat antibody labeled with horseradish peroxyf-ase (manufactured by Bio-Rad, diluted 500 times with 3% bovine serum albumin solution).
(100 μm2) was dispensed and heated at 37°C for 1 hour.

Wash with PBS, and add a coloring solution (20 μg of 0-phenylenediamine dihydrochloride, 10 μg of 30% hydrogen peroxide, 525 mg of citric acid, 1°79 of disodium phosphate dodecahydrate).
After adding 100 μg of purified water (50 ei2) and allowing the enzyme reaction to occur at room temperature for about 20 minutes, the reaction was stopped by adding 2N sulfuric acid (50 μ).

■ Cloning by limiting dilution method Hybridomas from antibody-positive wells were transferred to a 24-well microplate (manufactured by Coaster) and incubated with HAT medium for 3 to 3 hours.
It was cultured for 5 days. This culture solution was stirred to obtain a cell suspension, a portion of which was stained with trypan blue staining solution (manufactured by Gibco), and the number of cells was counted.

Next, the cell suspension was diluted with HAT medium so that the cell density was 10 to 25 cells/m (l).
100 feeder cells (mouse thymocytes) in advance
200μg into a 96-well microplate dispensed with μQ
and cultured in an incubator at 37° C. and 5% carbon dioxide. This cloning was performed twice.

As a result of this cloning, four types of hybridomas producing the antibodies of the present invention were obtained. These four types of hybridomas producing the antibody of the present invention were NHC-nl and NHC-n-4.
, named NHC-11-8 and NHc-n-io,
The antibodies produced by these hybridomas are each MN
HC-11-1, MNHC-■-4, MNHC-11
-8 and MNHc-n-to.

The above-mentioned four types of hybridomas producing antibodies of the present invention, NHC-1
1-1, NHC-II-4, NHC-II-8 and N
HC-n-10, respectively,
No. (FERM P-10625), No. 10626 (FERM P-10625), No.
FERM P-10626), No. 10627 (FERM
RM P-10627) and No. 10628 (FE
RM P-10628).

■ Cryopreservation of the hybridoma producing the antibody of the present invention After cloning was performed twice, the hybridoma producing the antibody of the present invention was
Transfer to a 4-well microplate and add 1.0Xlo-'M
The cells were cultured in a basal medium (hereinafter referred to as white (T medium)) containing hypoxanthin and 1.6×10 −'M thymidine.Then, the medium was gradually changed from the HT medium to the basal medium over about one week.

After culturing the cells in basal medium, the cells were collected and suspended in fetal calf serum containing 10% dimethyl sulfoxide (DMSO) so that the cell density was 10' to 108 cells/m (!). The contents were dispensed into serum tubes (manufactured by Corning) in units of I+12.The serum tubes were wrapped in absorbent cotton and left in a -75°C freezer overnight to freeze the contents, then transferred to a liquid nitrogen storage container and stored. did.

Example 2 (Specificity of the antibody of the present invention) Using the culture supernatants of the four types of antibody-producing hybridomas of the present invention obtained in Example 1, the specificity of the antibody of the present invention was determined using human chylomicron/VLDL fraction as an antigen. When confirmed by Western blotting, it was confirmed that all of them reacted only with apoC-H.

Furthermore, when the class of the monoclonal antibodies of the present invention was investigated using a mouse monoclonal antibody typing kit (manufactured by ICN), MNHC-n-1 and MNHC-n-4 were found to be I
It was found that MNHC-I[-8 belonged to the 1gM class, and NHC-Ir-10 belonged to the IgG+ subclass.

Example 3 (Purification of antibodies of the present invention) MNHC-II-1, MNHC-11-4 and MN
After pre-culturing NHC-n-1, NHC-n-4 and NHC-II-10, which are purified cryopreserved HC-Ir-10, in basic medium, AS
F medium 103 (serum-free medium for animal cell culture, manufactured by Ajinomoto Co., Ltd.) (1,000 m) was cultured for about 10 days. Ammonium sulfate was added to the culture supernatant to achieve 50% saturation, and salting out overnight at 4°C. Centrifuge at 7°000 rpm for 30 minutes,
The precipitate was dissolved in 20I containing 150IIM sodium chloride.
It was dissolved in IIM phosphate buffer (pH 7.2; hereinafter referred to as "basic buffer") and dialyzed against the same buffer. The crude antibody solution obtained in this way was mixed with a 1°5M glycy7-NaOH buffer containing 3M sodium chloride (pH 9,0;
Equal volumes of the mixture (hereinafter referred to as "binding buffer") were mixed and applied to an immobilized protein A (manufactured by Repligen Ir) column equilibrated with the binding buffer.

After washing the column with 15 times the bed volume of binding buffer, add O.1M citrate buffer (pH 3°0).
It was eluted. This eluate was immediately neutralized with 1M Tris-HC12 buffer (pH 9,0). After dialyzing against basic buffer, it was concentrated by ultrafiltration using Ultrafilter PO200 (manufactured by Atovanchik Toyo Co., Ltd.).

(2) Purification of MNHC-11-8 NHC-U-8, which is a cryopreserved product, was precultured in a basic medium and then cultured in ASF medium 103 (1, OOO+++12) for about 10 days. Ammonium sulfate was added to the culture supernatant to achieve 50% saturation, and salting out was carried out at 4°C overnight. 7.
After centrifugation at OOOrpm for 30 minutes, the precipitate was dissolved in basic buffer and dialyzed against the same buffer. The crudely purified antibody solution thus obtained was applied to a Sepharose 6B (manufactured by Pharmacia) column equilibrated with a basic buffer and eluted with the same buffer. Ultrafilter the eluate with PO20
0 (manufactured by Atobanchiku Toyo Co., Ltd.) by ultrafiltration. This concentrated solution was again applied to a Sepharose 6B (manufactured by Pharmacia) column equilibrated with the basic buffer and eluted with the same buffer. Ultrafilter the eluate with PO2
00 (manufactured by Atobanchiku Toyo Co., Ltd.) by ultrafiltration.

■ Recovery amount of monoclonal antibodies The antibody concentration of each monoclonal antibody purified in the above steps
and mouse IgG for MNHC-■-10.
MNHC-11 with RID plate (manufactured by Serotik)
-3 was measured using a mouse IgMRID plate (manufactured by Shichirochiku Co., Ltd.). Table 1 shows the amount of each monoclonal antibody recovered.

↓Considerations Recovery amount of monoclonal antibodies Example 4 (Immune turbidimetry (TIA method)) ■ Preparation of test antibody solution Stock solution of each monoclonal antibody obtained in Example 3, basic buffer and 10% PEG buffer (basic buffer containing 10% polyethylene glycol 6000) to adjust the antibody concentration to MNHC-11-1, MNHCn-4 and MNHC-
1100a/mQ for II-10, MNHC-11
In the case of -8, test antibody solutions were prepared so that the concentration was 85 μg/mQ and the concentration of polyethylene glycol 6000 was 5%. In addition, MNHC-n-4
and MNHC-n-10 were mixed in an amount of 1,000 u9/l each and prepared in the same manner.

■ Time course of turbidity reaction Place 7A of the test antibody solution prepared in step (■) above into a cuvette.
．． The solution was dispensed in 5ml/1J portions, set in a spectrophotometer (Shimadzu UV-160) equipped with a 37°C constant temperature device, and incubated for about 10 minutes. Then human serum (50μa)
Add it, mix by inverting, and measure the absorbance at 340 nm at 37°.
Measurements were taken every 2 minutes at C for up to 30 minutes. The blank is 5% PEG buffer (polyethylene glycol 6000
The measurement was performed using a basic buffer solution containing 5% of Results first
As shown in the figure.

(2) Relationship between apoC-nm and absorbance Using the MNHC-I[-8 test antibody solution prepared in step (2) above, the relationship between apoC-nm and absorbance was investigated. Human serum [-ApoC-■ concentration based on original immunodiffusion method (SRID method) as measured with ApoC-■ plate "Daiichi" (manufactured by Daiichi Chemical Co., Ltd.) is 2.8yt9/d (1) test tube i: 12.5.25.5o, 100 and 150
Sample each μa and add 1.5 μa of the test antibody solution to this sample.
d at a wavelength, and reacted for 25 minutes at 37°C.
The absorbance at 0r+m was measured. Note that the blank was measured using a 5% PEG11 solution. The results are shown in Figure 2. As is clear from the results shown in FIG. 2, the absorbance is directly proportional to the serum volume and therefore to the apo c-m.

(2) Measurement of apoC-H in human serum ApoC-II in human serum was measured using the MNHC-11-8 test antibody solution prepared in step (2) above. Human serum and standard solutions (ApoC-IN yield is 3 by SRID method)
．． 65 Shoulder 9/dd human pool serum) was sampled in an amount of 50 μQ each into a test tube, 1.5 μQ each of the test antibody solution was added thereto, reacted at 37° C. for 25 minutes, and the absorbance at a wavelength of 340 nrQ was measured. Blank is 5
% PEG buffer. The measurement results of this method are 5
The results are shown in Table 2 together with the results of the RID method. From the results in Table 2, it can be seen that the measured values by both methods are almost equivalent.

Note that while the measurement using the 5RID method required 48 hours, the time required for the measurement using this method was only 25 minutes.

MNHC-11-1 and β-
An enzyme-labeled antibody was produced using galactosidase (for EIA, manufactured by Boehringer Mannheim). The stock solution of MNHC-II-1 obtained in step ① of Example 3 (1.32 m of basic buffer (1.18 m) of N-(ε-maleimidocaproyloxy) succinimide solution (2.24 x 9/
(dissolved in N,N-dimethylformamide to make 1) (125μ) was mixed and heated at 30°C for 30 minutes. This was mixed with 100mM phosphate buffer (pH 6,0
) and eluted with the same buffer solution. The collected antibody fraction was diluted with 100mM phosphate buffer (pH 6.0) for 5 minutes.
Add β-galactosidase solution (2 m) prepared to give 1.37% N-ethylmaleimide-treated bovine serum albumin solution (42 µ) and 100 mM magnesium chloride (84 µQ) at 30 °C for 2 hours. Then, 100+ nM 2-
Add mercaptoethylamine hydrochloride (640μ),
It was heated at 0°C for 20 minutes. This was mixed with 0.1% bovine serum albumin, 0.02% magnesium chloride hexahydrate,
Sepharose 6B (
The mixture was applied to a column (manufactured by Pharmacia) and eluted with the same buffer. Enzyme-labeled antibody fractions were collected.

■Preparation of antibody-sensitized microplate MNHC-n-4 obtained in step (2) of Example 3 and MNHC-11-8 obtained in step (2) of Example 3 were mixed with 50mM carbonate buffer (p149, 6) to produce an antibody. Concentration is 25μg/1rr
(100 μa/well was dispensed into a 96-well microplate for EIA (manufactured by Coaster) and heated at 37°C for 2 hours. Then, the solution was washed with PBS. After that, 3% bovine serum albumin solution (prepared with 50ffiM carbonate buffer) (200 μg
) was dispensed and heated at 37°C for 1 hour. The antibody-sensitized microplate thus obtained was stored at 4°C until use.

■Enzyme immunoassay (EIA method) Human serum (ApoC-■ concentration of 2.95 m9/d by SRID method) was added to the antibody-sensitized microplate obtained in the above step (■).
diluted 100, 200 and 400 times with 1% bovine serum albumin solution (prepared with basic buffer) and dispensed in 50 μa portions (1% bovine serum albumin solution was used as a blank), 37°C. The mixture was heated for 1 hour. Next, after washing this with PBS, 50 μg of the enzyme-labeled antibody obtained in step ① above diluted 100 times with 1% bovine serum albumin solution was dispensed into each well, and incubated at 37°C.
Warmed for hours. Next, after washing this with PBS, a substrate solution (3.01 g of o-nitrophenyl-β-D-galactopyranoside, sodium phosphate/2 aqueous phase 4.
6g, disodium phosphate dodecahydrate 7°36g, sodium chloride 4.38g, magnesium chloride hexahydrate 0.4g, sodium azide 0.2g, bovine serum albumin 0.5g, ethylene glycol 60g, Make the total volume 1.0001 with purified water) and add 50 μe each, and heat at 37°C.
The mixture was heated for 25 minutes. Next, 100 μg each of 1% sodium carbonate solution was added to stop the enzyme reaction, and then the absorbance at a wavelength of 414 ru++ was measured. 3rd result
As shown in the figure.

[Brief explanation of drawings]

Figure 1 is a graph showing the time course of an immunoturbid reaction with serum apoC-■ using the antibody of the present invention, and Figure 2 is a graph showing the time course of the immunoturbid reaction with serum apoC-■ using the antibody of the present invention.
ApoC-11f by immunoturbidimetry using NHC-U-8
Graph showing the relationship between fi and absorbance. FIG. 3 is a graph showing the relationship between apoC-II ffi and absorbance in enzyme immunoassay. 12.5 25 Fig. 2 50 bits 00150

Claims (2)

[Claims] (1) Monoclonal antibody that specifically recognizes human apolipoprotein C-II. (2) A method for measuring human apolipoprotein C-II in a sample, which comprises using one or more of the monoclonal antibodies according to claim 1.