JPH0264457A - Determination of serum apo a-i - Google Patents

Abstract

PURPOSE:To perform measurement within a short time and to obtain the concentration of serum apo A-1 with good reproducibility by performing the antigen- antibody reaction with serum using two kinds of anti-human apo A-1 monoclonal antibodies having different antigenic determinants to be recognized and spectroscopically measuring the turbidity due to the generated antigen-antibody composite. CONSTITUTION:Two kinds of anti-human apo A-1 monoclonal antibodies having different antigenic determinants to be recognized are used in a method for the determination of serum apo A-1 due to immune nephelometry not requiring the dilution of serum. By the use of these anti-human apo A-1 monoclonal antibodies, the antigen-antibody reaction with serum is performed and the turbidity due to the generated antigen-antibody composite is spectroscopically measured and serum apo A-1 is measured within a short time with good reproducibility.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for quantifying serum apoA-I, more specifically,
A method for quantifying serum apoA-1, which is characterized by causing an antigen-antibody reaction with human serum using two types of monoclonal antibodies against serum apoAI that recognize different antigenic determinants, and spectroscopically measuring the resulting turbidity. Regarding.

Conventional technology ApoA-4 in the blood is a high-density lipoprotein (HDL), a lipoprotein that plays an important role in lipid metabolism and transport.
In recent years, apolipoproteins have attracted attention in relation to hyperlipidemia, especially as a preventive factor for arteriosclerosis.

Therefore, measurement of blood apoA-1 has become important as an index for diagnosis and treatment of hyperlipidemia.

Methods for measuring apoA-[in the blood include radioimmunoassay (RIA) using anti-apoA-1 polyclonal antibodies from animals such as rabbits and goats, enzyme immunoassay (EIA), Expansion ffi method (S RI
D), electroimmunoassay, immunoturbidimetry (T I
A) is known, and 5RID plate reagents and TIA reagents that can be measured with automatic analyzers are already commercially available. However, since these reagents use polyclonal antibodies obtained by immunizing animals, it is difficult to obtain consistent measured values due to differences in titer and specificity resulting from individual differences in immunized animals.

In addition, in this measurement, since serum apoA and I contents are high, it is necessary to dilute the serum in order to measure with appropriate sensitivity.
In RIA and RIA, it is necessary to use samples diluted from 100 times to to or even 00 times, which causes complicated operations and variations.

In recent years, monoclonal antibodies with high specificity and consistent measurement values have been developed to improve the drawbacks of polyclonal antibodies.
A, rgrvy [JP-A-60-237363; Ja
p,J,CNn,Chem,, Vol, 14.185
-191 (1985)] and C-reactive protein (CRP)
(Japanese Unexamined Patent Publication No. 62-192661) uses a TIA method using a monoclonal antibody. In the case of these test substances, since one antigen has multiple identical antigenic determinants, turbidity is likely to occur due to the antigen-antibody reaction, and even one type of monoclonal antibody will cause turbidity, so it is necessary to adjust the type of monoclonal antibody. By doing so, you can obtain appropriate sensitivity. However, in general, the antigen to be measured often has only one specific antigenic determinant (and if only a monoclonal antibody against that specific antigenic determinant is used, it will cause turbidity due to the antigen-antibody reaction). degree is unlikely to occur.

Therefore, in order to obtain appropriate turbidity, it is necessary to use a combination of multiple monoclonal antibodies that each recognize different types of antigenic determinants, but it is difficult to prepare and select monoclonal antibodies that are suitable for the TIA method. there were.

Monoclonal antibodies have also been developed for apoA-1, and several have been reported [JP-A-60-2538
71. Special table Showa 62-502705, C11nical
Chemistry Vol, 322155-9 (19
86)]. However, this measurement method is not suitable for EIA or RIA, which requires complicated serum dilution, or for normal measurement.
This method is based on 5RID, which is time-consuming, and is problematic as a routine testing method.

Purpose of the Invention Under these circumstances, the present inventors attempted to develop a measurement method published by Serum Apo A that does not require dilution of serum, is accurate in a short time, and always provides constant measurement values. , we have completed the present invention.

That is, the present invention is a method for quantifying serum apoA-1 by immunoturbidimetry that does not require serum dilution, and uses two types of anti-human apoA-1 monoclonal antibodies that recognize different antigenic determinants. The present invention provides a method for quantifying serum apoA-1, which is characterized by causing an antigen-antibody reaction with serum and spectroscopically measuring the turbidity due to the resulting antigen-antibody complex.

Structure and effect of the invention In the method of the present invention, two different antigenic determinants are recognized.
A variety of anti-human apoA-1 monoclonal antibodies are used.

This monoclonal antibody can be produced as follows: a) ApoA-I isolated and purified from human serum or plasma
immunize an animal using the antigen as an antigen, fuse the antibody-producing cells of the animal with myeloma cells of the animal, select a hybridoma that produces a monoclonal antibody that specifically reacts with ApoA-r, and iii) The monoclonal antibody is recovered by culturing under appropriate conditions.

For the immunization of animals in step 1), HDL is fractionated from human serum or plasma by ultracentrifugation, defatted, purified by DEAE-Sepharose CL-6B chromatography, and purified ApoA-I is used as an antigen. This can be done by administering the antigen to an animal and, after a certain period of time, further treating the animal with the same antigen.

Usually, this apoA-[ is diluted with physiological saline, mixed with Freund's complete adjuvant, and administered to animals.

As the animal species to be immunized, animals such as mice and rats can be used, and among them, BALB/c mice are particularly preferred.

Cell fusion in step 11) can be performed by a conventional method using the antibody-producing cells of the animal immunized in step 1) and myeloma cells of the animal.

As the antibody-producing cells, the lung cells of the above-mentioned BALB/c mice are most preferable, but in addition to these, for example,
Mouse 1178 node cells, peripheral lymphocytes, and rat lymphocytes can also be used.

As myeloma cells, mouse myeloma cells FO
is preferred, but other cells can also be used.

Cell fusion methods include polyethylene glycol method, HV
J method, electric fusion method, etc. can be mentioned.

Moreover, the selection of the obtained hybrid 17 can be performed, for example, as follows. That is, hybridomas are cultured in an appropriate medium, the culture solution is added to a human HDL-sensitized microplate prepared in advance, and reacted. Hybridomas producing the desired antibody may be selected by reacting with the antibody and adding a coloring solution. The selected /''1 hybridomas are then cloned by limiting dilution.

The monoclonal antibody obtained in step 1) can be recovered, for example, by culturing the hybridoma clone obtained in step 11) in a suitable animal cell culture medium and recovering it from the culture solution in a conventional manner. Alternatively, the recovered antibody may be purified by ammonium sulfate salting out, ion exchange chromatography, affinity chromatography, or the like.

By operating as described above, in the production example described later, four types of anti-apoA-I monoclonal antibodies, MN
HA-[-1, 2, 3 and 4 were obtained. Of these, M
When NHA-I-4 was used in combination with any of the other three monoclonal antibodies, turbidity occurred due to the reaction with human serum ApoA-1. Therefore, this MNHA-r-4
It is thought that the other three monoclonal antibodies recognize different antigenic determinants, and in the method of the present invention, anti-human apoA-1 monoclonal antibodies MNHA-I4 and MNHA-I, 2 or 3 are used. Use one of the following. It is preferable to use a combination of MNHA-1-4 and MNHA-1-2.

The method of the present invention may be carried out according to a conventional immunoturbidimetric method, and can be carried out, for example, as follows. Anti-apo A-I monoclonal antibody MNHA-I-4 and MN
Dilute any one of HA-[-1, 2 or 3 with an appropriate buffer to give a total antibody concentration of 0.5 to 10 rg9/y (1
, preferably 1 to 5 m9/m (preferably, the test antibody solution is prepared so that the ratio of both antibodies is 1 to 3:3 to 1, preferably about 1:1), and the test antibody solution and human serum are mixed. The turbidity generated by the reaction is measured with a spectrophotometer, and the serum apoA-4 concentration can be obtained from a calibration curve prepared in advance.

As shown in the examples below, the method of the present invention does not require dilution of serum, the antigen-antibody reaction reaches a plateau in about 15 minutes, so it can be measured in an extremely short time, and it is highly reproducible. This is a method for quantifying serum apoAd levels. According to this method, serum apoA of up to about 300 H/d (l)
-I concentration, an extremely linear calibration curve passing through the origin can be obtained, and therefore serum apoA-1 with high accuracy can be obtained.
Can measure 4 degrees.

The present invention will be explained in more detail with reference to production examples and examples below, but these are not intended to limit the present invention.

Production example: Preparation of anti-human apo A-1 monoclonal antibody ■ Immunization of animals Preparation of human HDL Potassium bromide was added to the plasma of a healthy person to reduce the specific gravity to 1.0.
63 and placed in a centrifugal tube (manufactured by Beckman, Ultra Clear, 16 x 76 mm), and overlaid with a potassium bromide solution having a specific gravity of 1.063. Using a Hitachi 55P-2 ultracentrifuge (Hitachi RP40Cf+-tar),
Centrifugation was performed at 0 rpm and 15°C for 24 hours. After removing the upper layer of chylomicron, VLDL, and LDL fractions, the lower layer was collected and potassium bromide was added to adjust the specific gravity to 1.21. A potassium bromide solution with a specific gravity of 1.21 was layered thereon, followed by centrifugation at 40.00 Orpm and 15°C for 48 hours, and the upper HDL fraction was collected.

Preparation of Apo A-1 solution Add to the amount of ethanol:ether (3:
2) was added and defatted three times. The precipitate was added to a 30 mM Tris-HC12 buffer (pH 8.0; hereinafter referred to as
It was dissolved in 151C (referred to as "urea-Tris buffer") and dialyzed for 2 hours against 500 ml of urea-Tris buffer. 3. O
After centrifuging for 10 minutes at OOrpm to remove insoluble materials, DEAE-Sepharose C equilibrated with urea-Tris buffer was added.
It was applied to an L-6B column. Urea Tris Buffer 300x &
After washing with water, elution was performed with a salt gradient urea tonos buffer containing 0 to 0.125 M NaCQ. Two peaks (F
1 and F2) were obtained, and immunoturbidimetry using anti-apoA-I polyclonal antibodies (ApoA-I auto "Daiichi",
When apo A-1 was confirmed using a method (manufactured by Daiichi Chemical Co., Ltd.), apo A-1 was observed in peak F2.

This fraction was collected and dialyzed three times against 3,000 mL of physiological saline. The total volume of collected fluid was 6.4 μm.

When the total protein concentration was measured by the Lowry method and the apoA-1 concentration was measured by the immunoturbidimetry described above, the total protein concentration was 9.1 my/rxQ, apoA-1i! AV is 6.45
It was u/xQ. The resulting ApoA-I solution was divided into portions and stored at -75°C.

The Ichthyosis ApoA-f solution was diluted with physiological saline so that the ApoA-1 concentration was 200 μg/waste a. This diluted apoA-I
Solution and Freund's Complete Adjuvant at 1:l
(liquid volume ratio) to prepare an antigen emulsion. 1 ml of this antigen emulsion was subcutaneously administered to the back of BALB/c mice (male, 6 weeks old) (first immunization).

One month after the first immunization, diluted ApoA-I solution 1ffl 0
, 251ρ was further administered intraperitoneally (booster immunization).

■ Preparation of cell fusion lung cell suspension Three days after the booster immunization, the lungs were removed and Tulbecco's modified Eagle's medium (Gibco, Glufus 4,500M9/ρ)
, penicillin 100 units/πQ, streptomycin 1
Contains 00μg/IIQ, no fetal bovine serum; below, 1
Lung cells were removed from the lungs in a medium (referred to as "basal medium").

The removed lung cells were suspended in basal medium and stored at 4°C until used for cell fusion. 1°92×lO8 lung cells were obtained.

Preparation of myeloma cell suspension Myeloma cells It, FO were used. Basal medium containing 10% fetal bovine serum, 1mM sodium pyruvate (
The FO cultured in the basal medium (hereinafter referred to as "basal medium") was washed twice with the basal medium and then suspended in the basal medium. The cell density was 8xlO' cells/me.

2. Mix the myeloma cell suspension of 6rtrQ with the lung cell suspension; 1. After centrifuging for 5 minutes at OOOrpm,
The supernatant was removed. Add to this 50% sterilization kept at 37°C.
Polyethylene glycol 4000 solution i&'rmQ was added dropwise over 1 minute while stirring. After stirring for 1 minute, basal medium 2+11f2 was added dropwise over 2 minutes while stirring. Furthermore, 8 volumes of basal medium were added dropwise over 2 minutes while stirring. Next, 1. After centrifuging for 5 min at OOOrpm and removing the supernatant, 1.0X 10-'M hypoxanthine, 4.0X 1
Basic medium (hereinafter referred to as rl (AT medium)) containing 0-7M aminopterin and 1.6 x 10-5M thymidine 50
1N (to prepare a fused cell suspension).

(2) Culturing of fused cells and screening of antibody-producing cells The above fused cell suspension was plated on five 96-well culture microplates (manufactured by Coaster) and cultured at 37°C in a 15% carbon dioxide incubator.

Antibody-producing cells were screened by EIA using human HDL-sensitized microplates.

Preparation of human HDL sensitized microplate Human HDL obtained in the purification process of apoΔ-■ was mixed with 5QmM carbonate buffer (p
) 19.6) diluted appropriately 50 to 200 times), EIA
1 in a 96-well microplate (manufactured by Coaster)
Dispense 100μQ per well and incubate at 37°C for 1 hour.
It was heated with Next, a 25mM'/phosphate buffered saline solution (pH 7.3; containing 0.1% Tween 20; hereinafter,
After washing with "phosphate buffer"), 200 μQ of 3% bovine serum albumin solution (prepared with phosphate buffer) was dispensed and heated at 37°C for 1 hour. The human HDL-sensitized microplate thus obtained was kept at 4°C until use.
I saved it.

EIA method: Dispense 100 μC of culture supernatant from wells in which cells are proliferating into a human HDL-sensitized microplate, and incubate at 37° for 1 hour.
It was heated at C. After washing with phosphate buffer, anti-mouse I
100 μQ of gG (H+L) goat antibody labeled with horseradish peroxidase (manufactured by Bio-Rad; diluted 500 times with 3% bovine serum albumin solution) was dispensed and heated at 37° C. for 1 hour. Wash with phosphate buffer, color developing solution (0-phenylenediamine 20 places, hydrogen peroxide 10μ)
1. Add 100 uQ of queic acid heptacid 525R9, 1.79 g of disodium phosphate dodecahydrate, purified water 50 + 12), allow enzymatic reaction at room temperature for about 20 minutes, and then add 2N sulfuric acid 50
The reaction was stopped by adding μe.

■ Cloning of antibody-producing cells and antibody specificity Cloning by limiting dilution The cells (hybridoma) in the antibody-positive wells were transferred to a 24-well microplate (manufactured by Coaster), and HAT
The cells were cultured in medium for 3 to 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 subjected to HAT so that the cell density was 10 to 25 cells/Iρ.
Diluted with culture medium. This cell dilution solution was prepared by dispensing 100 μQ of feeder cells (mouse thymocytes) in advance.
Plate 200μρ in each well microplate, 37°
C, cultured in a 5% carbon dioxide incubator.

This cloning was performed twice.

As a result of this cloning, four types of hybridoma strains producing anti-human ApoA-T monoclonal antibodies were obtained. These four types of antibody-producing hybridoma strains were NHA-'
I-1, NHA-I-2, NHA1-3 and NHA
-1-4, and the antibodies produced by these strains were named MNHA-1-1, MNHAi-2, and MNHA-'I, respectively.
``3 and MNHA-14.

The above four types of antibody-producing hybridoma strains, NHAl-1,
NHA-I-2, NHA-■-3 and NWA-I-4
are respectively FERM P
-10238), No. 10239 (FERM P-10
239), No. 10240 (FERM P-10240
) and No. 10241 (FERMP-10241).

Specificity of the antibody When the specificity of the antibody was confirmed by Western blotting, it reacted only with apoAT. When the classes of antibodies produced by the four hybridoma strains were investigated using a mouse monoclonal antibody typing kit (manufactured by ICN), all antibodies were found to belong to the IgG class.

Cells in antibody-positive wells after cryopreservation cloning of antibody-producing cells twice were transferred to a 24-well microplate and incubated at 1.0xlO-'
The cells were cultured in a basal medium (hereinafter referred to as "rHT medium") containing M hypoxanthine and 1.6xlO-'M thymidine. The medium was then gradually converted from HT medium to basal medium over a period of approximately one week. After culturing the cells in basal medium, the cells were collected, suspended in fetal serum containing 10% dimethyl sulfoxide to a cell density of IO5 to 106 cells/m (l), and placed in a 2ttt (l serum tube). The serum tube was 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.

■ Purification of anti-apo A-1 monoclonal antibody After pre-culturing the cryopreserved antibody-producing cell line in basic medium, ASF medium 1
03 (serum-free medium for animal cell culture; manufactured by Ajinomoto Co., Inc.) 1. O
The cells were cultured in OM 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. The mixture was centrifuged at 7°,000 rpm for 30 minutes, and the precipitate was dissolved in 20 mM'J acid buffered saline (pH 7.2) and dialyzed against the same buffer. 3. The crudely purified antibody solution thus obtained; 1°5M glycine-NaOH buffer containing QM NaCQ (pH 9, o; hereinafter referred to as “
Immobilized r-protein A (Repl 1Ge) equilibrated with the binding buffer was mixed with equal amounts of
(manufactured by n company) column. After washing the column with a binding buffer solution 15 times the bed volume, it was eluted with a 0.1M citrate buffer (pH 3.0). Add this eluate to 1
It was immediately neutralized with M Tris-HC12 buffer (pH 9,0). After concentration by ultrafiltration, it was dialyzed against 20 mM phosphate buffered saline (pH 7.2). The rgG concentration was measured using a mouse IgGRrD plate (manufactured by Shichirochiku). Table 1 shows the amount of each monoclonal antibody recovered.

l Ependymal monoclonal antibody recovery amount 1 ['ll Immunoturbidimetry using anti-apoA-1 monoclonal antibody ■ Time course of turbidity reaction Using the above four types of monoclonal antibodies alone, or in combination of two or three types, When reacting with human serum, we investigated whether the reaction solution became turbid and the time course of the turbidity reaction. For all combinations when using 2 types of antibodies, MNHA-I-4 when using 3 types of antibodies
(Tested using three types of antibodies.

Stock solution of purified anti-apo A-1 monoclonal antibody obtained in step ① of Production Example, basic buffer (20mM IJ acid buffer, 150mM
mM sodium chloride, pH 7.2>, and 10% PE
G buffer (basic buffer containing 10% polyethylene glycol 6000) was mixed, and the total antibody concentration was 2 mg/zQ (
Test antibody solutions were prepared so that when two types of antibodies were used, each In/xf was 2, and when three types were used, each was 0.667 my/m, and the polyethylene glycol 6000 concentration was 5%.

Dispense the test antibody solution into cuvettes in 1.5πQ portions,
It was sectioned into a spectrophotometer (Shimadzu UV-160) equipped with a °C constant temperature device, and incubated for about 10 minutes. Next, 10 µe of human serum (apoA-1, concentration 106 y/d as measured by the SRI D method) was added, mixed by inversion, and the absorbance at a wavelength of 340 nm was measured at 37°C.
Measurements were taken every minute up to 30 minutes. In addition, sample blank is 5%
PEG buffer (5% polyethylene glycol 6000)
The measurement was performed using a basic buffer solution containing

The results are shown in Figure 1. When MNHA-T-4 was used alone, almost no turbidity reaction was observed, but when MNHA-14 was combined with any of the other three antibodies, a strong turbidity reaction was observed. was observed, and the reaction reached a plateau in about 15 minutes. In addition, when an antibody other than MNHA-1-4 was used alone, when two types of antibodies other than the above combination were used, and when three types of antibodies other than MNHA-1-4 were used in combination, almost no turbidity was observed. No response was observed (data not shown).

(2) Relationship between apoA-4ffi and absorbance Using a combination of two monoclonal antibodies, MNHA-1-2 and MNHA-1-4, the relationship between apoA-1fi and absorbance was investigated.

Mix MNHA-i2 and MNHAi-4 antibody stock solutions, base buffer, and 10% PEG buffer to give a total antibody concentration of 2 rpy/mc (1 mg/m for each antibody), and polyethylene glycol 60
A test antibody solution was prepared so that the degree of 00/a was 5%.

2.5.5.10°2 human full serum (apoAI concentration measured by SRI D method: 95 mg/dN) in a test tube.
0 and 30 μg were sampled, 1.5 ia each of the test antibody solution was added thereto, and after reacting at 37° C. for 20 minutes, the absorbance at a wavelength of 340 nm was measured. The sample blank was measured using a 5% PEG buffer.

The results are shown in Figure 2.ApoA with high linearity passing through the origin
A relationship between -1fi and absorbance was obtained. from this result,
Using a measurement system in which the sample amount was 10 μρ and the test antibody solution was 1.51σ, it was possible to measure apoA-I concentrations up to about 300 m9/dQ.

(2) Simultaneous reproducibility Using three human serum samples and the test antibody solution prepared in (1) above, each serum was measured 10 times to examine reproducibility.

Human serum was sampled in an amount of 10μβ in a test tube, a test antibody solution was added in an amount of 152σ, reacted at 37°C for 20 minutes, and the absorbance at a wavelength of 340nm was measured.

The results are shown in Table 2. All coefficients of variation were less than 2%, indicating good reproducibility (measured values in the table are ○D
34G value).

! Simultaneous reproducibility for z people Correlation with the 5RID method Using this method and S RI D2 (ApoA-1 plate "Daiichi", manufactured by Daiichi Chemical Co., Ltd.), 44 human serum samples were measured. The correlation between the measured values by the method was investigated.

Measurements using this method were performed as follows. Human serum and a standard solution (human boule serum with apoAla level of 126.5zy/dρ measured by the S Rr D method) were each sampled from 10 animals in test tubes, and 1
Add 1.5 ml of the test antibody solution prepared at 37°C and incubate for 2
The reaction was carried out for 0 minutes, and the absorbance at a wavelength of 340 nm was measured.

The sample blank was measured using 5% PEG buffer.

The results are shown in Figure 3, and the correlation coefficient r = 0.9149 and the regression equation y = 0.986x-3,83 between both measurement methods.
A good correlation was shown with 7.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the time course when two types of monoclonal antibodies are used to react with serum apoA-1 in comparison with that when one type of monoclonal antibody is used. The figure shows that serum apoA-
FIG. 3 is a graph showing the relationship between apoA1ff1 and absorbance when measuring I, and FIG. 3 is a graph showing the correlation when serum apoA-1 was quantified using this method and the 5RID method.

Claims (1)

[Claims] 1. Two types of anti-apoA that recognize different antigenic determinants
A method for quantifying serum apoA-I by immunoturbidimetry, which is characterized by using a monoclonal antibody.