JPS6358262A - Analysis of biocomponent by using specific monoclonal antibody - Google Patents

Abstract

PURPOSE:To improve sensitivity by optically measuring the extent (degree) at which a free antigen prohibits the agglutination to be generated by the reaction of the monoclonal antibody sensitized carrier reacting specifically with the antigen and the same antigen sensitized carrier as the antigen. CONSTITUTION:The antigen sensitized carrier prepd. by using the same antigen as the antigen to be measured (for example, human hemoglobin sensitized carrier) is added into the liquid of the specific monoclonal antibody sensitized carrier (for example, anti-human hemoglobin monoclonal antibody sensitized carrier) and the free antigen to be measured (for example, occult blood of feces). The extent (degree) at which the free antigen to be measured prohibits the agglutination to be generated by the reaction of the monoclonal antibody sensitized carrier and the antigen sensitized carrier can be thereby visually or optically measured. The objective material in the vital sample contg. other kinds of material is, therefore, measurable with high selectivity and high sensitivity. A slight amt. of the occult blood in the human feces in particular is thus easily measured with the high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for analyzing biological components using a specific monoclonal antibody.

<Prior art> As a method for analyzing the image components of a living body using an antigen-antibody reaction, a specific polyclonal antibody-sensitized carrier and a free antigen are mixed in a liquid, and the agglutination reaction that occurs is visually observed on a slide agglutination plate. Methods of measuring the temperature visually or optically are known.

This method is generally widely used because it can highly selectively measure target biological components in samples containing biological components. There is a method of measuring human hemoglobin in feces by reacting antibody-sensitized latex particles, which are made by bonding antibody-sensitized latex particles to latex particles, with human hemoglobin in feces (see JP-A-59-125064).
.

<Problems to be Solved by the Invention> However, since the conventional analysis methods described above use polyclonal antibodies, they have the following disadvantages specific to polyclonal antibodies.

(1) Polyclonal antibodies are heterogeneous antibodies that contain many types of antibodies against various antigenic determinants (epitopes), so when measuring antigens with similar structures, immunological cross-reactions occur and the measured value is affected and becomes inaccurate.

(2) Antigens used for antibody production must be highly purified by producing 'N4.

(3) In order to collect large amounts of antibodies, large animals such as horses must be immunized in large quantities each time.

(4) Since immunization is carried out for each of the 171 substances, the properties of the antibodies obtained vary from animal to animal and are not the same, which is inconvenient.

Therefore, the present inventors attempted to use monoclonal antibodies instead of polyclonal antibodies.

The advantages of replacing polyclonal antibodies with monoclonal antibodies are as follows.

(1) Because the chemical and immunological properties are uniform, it has high specificity for one type of antigenic determinant, and the target antibody can be obtained by culturing antibody-producing cloned cells. Therefore, antibodies with the same properties and no lot differences can be obtained stably over a long period of time.

(2) It is not affected even if the purity of the antigen used for antibody production is considerably lower than that of polyclonal antibodies.

(3) Only a small amount of antigen is required (a small amount is sufficient since small animals such as mice are used).

The above (2) and (3) are particularly advantageous compared to polyclonal antibodies for antigens that are difficult to purify or rare antigens that are difficult to obtain in quantity.

However, when a monoclonal antibody is used instead of a polyclonal antibody, monoclonal antibodies have high specificity for one type of antigenic determinant. It was found that even when mixed with and in a liquid, the agglutination reaction did not occur in most cases, and free antigen could not be measured as it was.

Therefore, the present inventors investigated various methods of measuring free antigen by agglutination reaction using a carrier sensitized with a specific monoclonal antibody. The present invention was completed by developing a new method of producing an antigen-sensitized carrier and introducing (adding) it to the reaction system.

<Means for solving the problem> In other words, the present invention provides a monoclonal antibody-sensitized carrier that specifically reacts with the antigen to be measured, and an antigen-sensitized carrier that is the same as the antigen to be measured. By visually or optically measuring the degree to which the free antigen to be measured blocks the agglutination phenomenon that occurs,
It was developed with the aim of immunologically measuring the free antigen to be measured with high specificity and sensitivity. The analysis method according to the present invention is particularly effective when the specific monoclonal antibody-sensitized carrier and the free antigen to be measured do not aggregate even if they react with each other and cannot be measured visually or optically.

Next, the principle of the method for analyzing biological components using a specific monoclonal antibody according to the present invention will be described in more detail.

When a conventionally used polyclonal antibody sensitized carrier is mixed with a free antigen, the polyclonal antibody can react with various antigenic determinants on the antigen molecule one after another, forming an aggregate. However, even if a monoclonal antibody-sensitized carrier is mixed with a free antigen, there is a limit to the number of antigenic determinants that can react with the monoclonal antibody.
Visually observable aggregation often does not occur.

Therefore, a separate antigen-sensitized carrier for the same antigen as the measurement target is prepared, and this antigen-sensitized carrier is added to the reaction system to ensure an agglutination reaction with the monouronal antibody-sensitized carrier.
The free antigen to be measured was made to function as a factor (component) that inhibits the Group 7 reaction. As a result, the degree of agglutination that occurs in the reaction is visually or optically measured (with and without addition of the free antigen to be measured). This made it possible to measure with high sensitivity.

Next, as a specific example of the present invention, an anti-human hemoglobin monoclonal antibody is used as the specific monoclonal antibody, and human hemoglobin is used as the antigen, and from these, an anti-human hemoglobin monoclonal antibody sensitized carrier and a human hemoglobin A method for producing a sensitized carrier and measuring fecal occult blood using the same will be described.

As the human hemoglobin (antigen) used in the production of the anti-human hemoglobin monoclonal antibody used in the present invention, commercially available products can be used as they are. The amount of contaminants contained therein can be ignored, unlike in the case of producing polyuronal antibodies.

The method for producing the anti-human hemoglobin monoclonal antibody of the present invention is sufficient to be carried out by methods described in ordinary literature and does not require any special methods. After immunization twice, spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. and,
Among these fused cells, those that produce the antibody are grown as monoclonal cells by cloning, and by growing them in the peritoneal cavity of a mouse, a single antibody, that is, an anti-human hemoglobin monoclonal antibody, is produced in large quantities with the same properties. can be manufactured.

The carrier particles used in the present invention may be those for indirect aggregation reactions, that is, polyethylene latex,
Various latexes, animal red blood cells, kaolin, carbon particles, etc. can be used.

The method for sensitizing a carrier with the anti-human hemoglobin monoclonal antibody or human hemoglobin antigen of the present invention may be any known method for sensitizing general antibodies6.For example, the most common method is physical adsorption. It is. There are also covalent bonding methods using carboxylated polystyrene latex, and methods using so-called coupling agents such as glucuraldehyde, tolylene diisocyanate, carbodiimides, and chromium chloride.

Next, the specific method will be described in more detail.

(a) The specific monoclonal antibody-sensitized carrier used in the practice of the present invention was prepared as follows.

(b) Preparation of immunized spleen cells: Human hemoglobin immunogen solution (A280nm=2.0
)%ffi of Freund's complete adjuvant until emulsified, and immunized mice by intraperitoneally administering 100 Uβ of the mixture (first immunization).
After recovery, the mice were intraperitoneally administered in the same manner as above (second immunization). 21 days have passed since the second immunization, human hemoglobin immunogen solution (A280nm=2
．． 0) with an equal volume of physiological saline, and the diluted solution is 100%
uff was administered intravenously to the mouse (RP immunization),
1&3 days after immunization, spleen cells were taken out from the mouse and used for cell fusion.

(b) Cell fusion: The spleen removed aseptically is placed in a petri dish containing 5 mI2 of DME medium containing 10-15% fetal bovine serum.Next, the spleen is mixed with 10-15% fetal bovine serum. D containing
The spleen cells were refluxed with 15 rrl of ME medium.
After draining the splenic cell suspension, the spleen cell suspension is passed through a nylon mesh.

Collect these splenic cells in a 50rnβ centrifuge tube and
Centrifuge at 0x9 for 10 minutes. The beret obtained in this way has 3
~5mu of hemolizing solution (155ml NH2
Cl, 10mM KHCO3, 1mM
Naz EDTA.

pH 7.0) and suspend. The red blood cells in the suspension are destroyed by incubation at 0°C for 10-20 minutes. Furthermore, 10
DME medium containing ~20 mβ of 10-15% fetal bovine serum is added and centrifuged. The cell pellet thus obtained is washed with DME medium by centrifugation, and the number of mature spleen cells is measured. . On the other hand, pre-cultured mouse myeloma cotton #2! (Myeloma cells) Approximately 2 x 10 914 cells and 1 x 10' of the above spleen cells were added to SP210-, mixed well with DME medium, and centrifuged (
500×9, 10 minutes), aspirate the supernatant, loosen the pellet well, drop 0.5 rrl of a solution of 40% polynylene glycol 400o kept at 38°C, and gently gently remove the centrifuge tube by hand for 1 minute. The polyethylene glycol solution and cell pellet were mixed by rotation. Next, 1 mJ2 of DME medium stored at 38° C. was added every 30 seconds and the tube was gently rotated. After repeating this operation 10 times, the distance is 10-20m.
Add DME medium containing 10-15% fetal bovine serum in I2 and centrifuge @ (500xq.

10 minutes). After removing the supernatant, the cell pellet was placed in HAT medium (DM) containing 10-15% fetal bovine serum.
Aminopterin 4X10-7M, thymidine 1.
6x 10-5M, hypoxanthin 1X10-'M) was purified using the same method by centrifugation.
40ml of the above HAT medium tT! ! Suspend in Pour this cell suspension into each well of a 96-well cell culture plate at 2°C.
Two portions of OuI were dispensed, and culture was started at 37° C. in a carbon dioxide gas incubator containing 5% carbon dioxide gas. During culture, remove approximately 100 um of the medium from each well at intervals of 2 to 3 days, and add freshly added HA from each well.
Hybridomas growing in HAT medium were selected by adding 8100 uj2 of T medium. 10 from around the 8th day
HT medium containing ~15% fetal bovine serum (1.6 x 10-'M thymidine, lXl hypoxanthine in DME medium)
At about 108th day, the human hemoglobin antibody M main hybridoma was screened by the following ELISA method while observing the proliferation of the hybridoma.

(c) Establishment of hybridoma The presence or absence of produced antibodies in the hybridoma culture supernatant is determined by ELIS.
Measured by method A. The purified human hemoglobin solution (A280nm=
0.05. Diluted with physiological saline. )! Dispense 50 uu each and store at 25°C at 2 o'clock.
After washing three times with een20-physiological saline, 50 uI of the culture supernatant was added to each well and reacted at 25°C for 1 hour.

Next, 50 uI of peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200 times in Tween 20-saline was added to each well.

To complete the reaction, add 0.05% Tween20-Physiological saline
Wash each well three times with 250 L of a solution containing 0.5 mM aminoanthidrine, 10 mM phenol, and 0.005% hydrogen peroxide. was added to each well, reacted at 25°C for 30 minutes, and the absorbance of each well at 490 nm was measured. As a result, antibody production was observed in 12 out of 192 wells.

Next, the above clone was cloned by the limiting dilution method. The limiting dilution method requires 5 hybridomas in the HTig site.
cells/m 12 (the diluted cell suspension was
100 LIβ was dispensed into each well of a 96-well plate in which 2×10′ peritoneal cells of a normal BALB/C mouse had been dispensed per well. After about 10 days, ELI
Hybridoma clones producing anti-human hemoglodon-specific antibodies were screened by the SA method. As a result, 20 to 40 antibody-producing clones were obtained for each hybridoma. Among these clones, a clone with good growth, high antibody secretion ability, and stability was selected and re-cloned in the same manner as above to obtain anti-human hemoglobin-specific antibody-producing hybridoma l-1-1. was established.

(b) Production of monoclonal antibodies (a) In vitro method Mouse hybridoma H-1v! : Each was cultured in a DME medium containing 15% fetal bovine serum at 37° C. in a 5% carbon dioxide atmosphere for 72 to 96 hours. Cultures were incubated far q, > min M (10
000xq, 10 minutes) Solid ammonium sulfate was gradually added to the supernatant to give a final concentration of 50%. The mixture was stirred for 30 minutes under water cooling, left to stand for 60 minutes, and then centrifuged!
(10,000 x 9.10 min), the resulting precipitate was dissolved in a small amount of 10mM') Mn buffer (pH 8,0) L, 1
It was dialyzed against 10mM phosphate buffer from 000baiya.

This was added to DE equilibrated with 10mM phosphate buffer.
It was packed into an AE-cellulose column. Elution of the monoclonal antibody was carried out using a gradient method using +OrnM phosphoM buffer (pH 8,0) and 10mM phosphoM solution (pH 8,0) containing 0.2M Na (12@). The monoclonal antibodies obtained were concentrated using an ultraviolet method and dialyzed against 0.11M Phosphorus MIS Equilibrium (pH 8.0).To remove bovine serum 9G, the dialysate was mixed with goat anti-bovine serum 19G-Sepharose 4B. The permeate was passed through a column of % O, IM phosphate buffer (pH 8, o
)' was loaded onto a protein A-Sepharose 4B column equilibrated with . Anti-human hemoglobin antibody antibody H-1 purified by eluting the column with buffer 1))-13,5
I got it.

(b) Ishi-Dopo method 14-20 days after intraperitoneal administration of 0.5 mA of Bristan (2,6,10,14-tetramethylhentadecane) to 10-12 week old 84LB/C mice. Paipridoma H-1j grown in vitro in the peritoneal cavity of mice! 2 x 106 cells per mouse were inoculated.

Approximately 10-15 from one mouse for each vibridoma
mA of ascites was obtained. The antibody concentration is 2-10m9
/mβ. Purification of monoclonal antibodies in ascites (
(However, except for the operation of passing the goat anti-bovine serum 19G-Sepharose 4B through the column) was carried out in the same manner as the in vitro purification method described above.

(C) Identification of immunoglobulin class of monoclonal antibody Anti-human hemoglobin-specific monoclonal antibody H-1 Identification of immunoglobulin class and specificity was carried out by the offteloimmunodiffusion method and enzyme assay, respectively.

The results were as shown in Tables 1 and 2 below.

Table 1 Classes of immunoglobulins Table 2 Reaction, specificity (d) Preparation method of anti-Hb monoclonal antibody sensitized carrier (latex) The bond between the purified anti-Hb monoclonal antibody and the carrier (latex particles) is a physical bond, Examples of bonding methods that can be carried out by general chemical bonding are shown below.

(a) Physical method A suspension of polystyrene La-Ntex particles (manufactured by Japan Synthetic Rubber ■, 10.221 um) was added to a suspension of 0.25 μm containing 0.15 M sodium chloride. 1M Tris-HCl buffer (p)-1
8.0) and centrifuged and washed three times with this buffer. This precipitate is made into a 1% suspension with the above buffer. This was supplemented with anti-Hb monoclonal antibody (0,6rnq
/mβ) and stirred at room temperature for 1 hour, excess antibody that does not bind to the latex is washed away by centrifugation. A 0.15M sodium chloride-containing Tris-HCl buffer (pH 8.0) containing 0.01% bovine serum albumin is added to prepare a 1% latex suspension.

(b) Chemical method A 10% suspension of polystyrene latex particles (manufactured by Japan Synthetic Rubber, diameter 0.264 um) into which carboxyl groups have been introduced is prepared using IM Tris-HCl buffer (pH 8.0).
Dilute twice. An anti-Hb monoclonal antibody (0.6 mg/mβ) and a coupling agent are sequentially added to this diluted solution and pressed to cause covalent bonding. Excess antibody that does not bind to the latex is washed away by centrifugation. To this was added 0.15M sodium chloride-containing Tris-HCl buffer (pH 8.0)'J8 containing 0.01% bovine serum albumin,
Prepare a 1% latex suspension.

(e) Preparation method of Hb-sensitized carrier (latex particles) The bond between Ht) and the carrier (latex particles) is physically,
Alternatively, it can be carried out by general chemical bonding.

The binding method can be carried out in the same manner as the method for preparing the anti-Hb monoclonal antibody sensitized carrier (latex) (d) above, and the HbJ degree at that time is adjusted to (0.6 mq/mβ).

Example 1 Detection of occult blood in stool by slide agglutination plate method Stool 19 of a healthy person
suspended in 0.1M Tris-Salt M buffer 10rr17,
Centrifuge and collect supernatant. Next, purified human Hb
was added to the supernatant, and 700u9/rnI! ,50u
9/mI2.25uq/m! 2.10uq/mu, 5u
Samples with concentrations of q/mI2°2.5 uq/mβ and Ouq/mβ are prepared, respectively. 4 of the samples at each of these concentrations
0uI! were dropped onto the slide aggregation plate, and then 20 uf 2. and add and drop the +b antigen sensitized lux TOuI2 prepared in (e) above,
Observe while stirring gently for 2 minutes.

As a result, the agglomeration image was clearly generated due to Ouq/mj2
．． At 2.5 uq/mu, the agglutination image became weak at 5 IJ 9/mj2, and at 10 u q/mβ or higher, the agglutination image hardly became 1.

From this result, the sensitivity is 5u9/mJ7 (50L19
/9 (feces)), and the sensitivity (
It can be seen that this method has very good sensitivity compared to approximately 600 OL1 to 1/9), has high specificity because it uses an anti-Hb monoclonal antibody, and is not affected by coexisting substances.

Example 2 Detection of occult blood in stool using a spectrophotometer (reaction) 0, IMl
-Lis-hydrochloric acid buffer solution 1000uI2 sample used in Example 1 v! After adding 1oouβ, 40 μβ of the anti-Hb monoclonal antibody sensitized latex prepared in (a) of (d) above, and 4° LIρ of the Hb antigen sensitized latex prepared in (e) above, and mixing quickly. , placed in a cell with an optical path length of 0.5 mm, using a spectrophotometer (Hitachi Model 320) 7a, and opening at a wavelength of 700 nm, starting the reaction at 0.5
The absorbance was measured between minutes and 1.5 minutes.

The results were as shown in Table 3.

Table 3 Changes in fecal human Hb concentration and absorbance From the decrease in absorbance shown in Table 3, fecal human Hb concentration can be measured with higher sensitivity than the slide agglutination method of Example 1.

Next, examples other than anti-human hemoglobin monoclonal antibodies will be given.

<Example 3> Serum C-reactive protein (hereinafter referred to as 0-day P
Detection (1) Reagent ① Anti-CRP monoclonal antibody was prepared in the same manner as the anti-Hl) monoclonal antibody. The immunoglobulin class was 19G1.

(2) The anti-CRP monoclonal antibody-sensitized carrier was prepared by the method (a) of (d) above.

(3) The CRP antigen-sensitized carrier was prepared by the method described in (e) above.

(2) Operation Add CRP purified from ascites to CRP-free serum, and add 1000 nq/rrl and 500 n9/mc 25
0ng/mc I ○Or+q/mc 50 n
9/mβ, 25n9/mc Onq/muJ 1 degree samples are prepared respectively. After dropping 40 μβ of each of these J degree samples onto the aggregation plate, the above reagent ■anti-C
RP monoclonal antibody sensitized carrier (latex) 20u
β, and the above reagent ■C day P antigen sensitization carrier (latex)
Add 10 uβ dropwise and observe while stirring gently for 2 minutes.

As a result, it was found that the agglomerated image was clearly generated when On g/
mβ, 25n9/mβ, 50nq/mj2. At 100n9/mI2 or more, the agglutination image became weak, and at 100n9/mI2 or more, no agglutination image was generated.

From this result, the sensitivity is 50 ng/mβ in serum (
500 nq/9 (fee)).

Example 4 Detection of α-fetoprotein (hereinafter referred to as AFP) in serum using a slide agglutination plate (1) Reagent ■An anti-HbFP monoclonal antibody was prepared in the same manner as the anti-Hb monoclonal antibody. By the way, the immunoglobulin class was l9G1.

(2) The anti-AFP monoclonal antibody-sensitized carrier was prepared by the method (a) of (d) above.

■Preparation of AFP antigen sensitized carrier (latex) is as described above (
This was done using method e).

(2) AFP purified from yfR cord blood was added to the manipulated AFP-free serum.
, 1000 nq/mβ, 500n 9
, / mβ, 25Or+q/mI2.100nq/m
p, 50ng/mβ, 25r+q/mff.

Onq/mffJ degree samples are each prepared. After dropping 40 Llβ of each of these AIH samples onto the agglutination plate, add 20 uC of the above reagent (1) anti-AFP monoclonal antibody sensitized carrier (latex) and 10 UI 2 U of the above reagent (1) AFP antigen sensitized carrier (latex) to purify it. ,2
Observe while stirring gently for a minute.

As a result, it was found that the aggregation image was produced in Salmonmei on 9/mβ
, 25n9/mβ, 50n9/mβ, 1100n/m
! At 2, the aggregation image became weak, and at 250 ng/mA or more, no agglutination image was formed.

From this result, the sensitivity is 100n9/mρ (100
0n9/q (fee)).

<Effects of the Invention> As described above, according to the method for analyzing biological components using a specific monoclonal antibody according to the present invention, other than 1! It has the effect of being able to measure a target substance in a biological sample containing a substance with high selectivity and high sensitivity, and in particular, it is possible to measure occult blood in an image amount in human stool easily and with high sensitivity.

Claims (3)

[Claims] (1) Aggregation reaction that occurs by mixing solutions containing the following components (A) and (B), and the above-mentioned (A) and (B).
A method for analyzing biological components using a specific monoclonal antibody, which comprises adding and mixing a solution containing the following component (C) to the component, and comparing and measuring the degree of agglutination reaction between the two components. (A) A monoclonal antibody-sensitized carrier that specifically reacts with the antigen to be measured (B) An antigen-sensitized carrier that is the same as the antigen to be measured (C)
Antigen to be measured (2) A method for analyzing biological components using a specific monoclonal antibody according to claim 1, in which an anti-human hemoglobin monoclonal antibody is used as the specific monoclonal antibody, and human hemoglobin is used as the antigen. (3) A method for analyzing biological components using a specific monoclonal antibody according to claim 2, in which fecal occult blood is the antigen to be measured.