JP2759365B2 - Immunological assay method and reagents - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a measurement method and a measurement reagent utilizing an immunological agglutination reaction. In particular, the present invention relates to a method for immunological measurement of low molecular compounds and a reagent for measurement.

[Related Art] In recent years, in the medical field, it is important to measure trace components present in a biological fluid sample such as blood or urine quickly, simply, and with high accuracy for observing the progress of treatment and diagnosing disease. Is an important issue. For such measurement, a method utilizing aggregation by an antigen-antibody reaction is widely used. For example, a particulate insoluble carrier is sensitized with an immunologically active substance such as an antigen or an antibody, and the sensitized carrier is brought into contact with a liquid sample containing the test substance, and the antigen-antibody reaction between the sensitized carrier and the test substance Aggregation caused by the test substance can be visually or optically confirmed, and the presence (qualitative) and / or concentration (quantitative) of the test substance can be analyzed.

Here, the test target substances to be clinically tested include, specifically, a wide range of compounds ranging from high molecular compounds such as proteins and polypeptides to low molecular compounds such as drugs, peptides, saccharides or nucleic acids. Exists.

However, especially when a low molecular weight compound is analyzed by an agglutination method derived from an immune reaction, there are difficult problems unique to the low molecular weight compound.

That is, in the case of a low molecular weight compound, the number of antigenic determinants contained therein is small, so that an analysis method for a high molecular weight compound cannot be used as it is. For example, when an insoluble carrier sensitized with an antibody is brought into contact with a high molecular compound containing a large number of antigenic determinants, an agglutination reaction occurs, whereas a low molecular weight compound containing only one antigenic determinant is brought into contact with the polymer. No agglutination reaction naturally occurs. Even if the low-molecular compound has about two or three antigenic determinants, the molecule itself is small, so that steric hindrance does not cause aggregation or becomes extremely difficult.

JP-A-60-20149 describes a technique using two types of carriers. That is, a carrier sensitized with a hapten (a low-molecular compound) as a test substance, a carrier sensitized with an antibody against the hapten, and a test liquid sample are brought into contact with each other, and the agglutination reaction of the two types of carriers is measured by the sample. A technique for analyzing the presence or concentration of a test substance by measuring the degree of inhibition of the test substance therein is described. However, the technique described in this patent publication also has a problem that the sensitivity is low.

[Problems to be Solved by the Invention] The present inventor has found that the above-mentioned problems in the prior art can be solved by means completely different from the viewpoint of the prior art. That is, the test target compound (a) in which it is impossible or difficult to directly cause an agglutination reaction
An antigen-antibody reaction complex [(a) + (b)] of the antibody (b) against the (particularly low-molecular compound) and the test compound (a) itself is prepared, and then the complex [(a ) + (B)] and found that the above problem can be solved by using the second antibody (c). The present invention is based on this finding.

[Means for Solving the Problems] Accordingly, the present invention provides: (i) a test compound (a); and a primary antibody (b) against the test compound (a).
An insoluble carrier (X) carrying a secondary antibody (c) for the antigen-antibody reaction complex of (a) and (b), an insoluble carrier carrying the primary antibody (b) or the primary antibody (b) Alternatively, the soluble carrier (Y) and (iii) the test sample (d) are brought into contact with each other, and then the test compound (a) in the sample (d), the insoluble carrier (X), and the primary antibody (b) ) Or an agglutination reaction with the carrier (Y).

Further, the present invention provides: (i) a compound to be tested (a); and a primary antibody (b) against the compound to be tested (a).
An insoluble carrier (X) carrying a secondary antibody (c) against the antigen-antibody reaction complex of and, and (ii) the primary antibody (b) or the primary antibody (b) The present invention also relates to a reagent for immunological measurement, which comprises an insoluble or soluble carrier (Y).

 Hereinafter, the configuration of the present invention will be described in detail.

In the present invention, the compound to be tested (a) means that the number of antigenic determinants is small or a steric hindrance that hinders an antigen-antibody reaction at multiple sites occurs. It is a compound that is impossible or difficult to cause an agglutination reaction. For example, in the case of a toxicant having a molecular weight of about several hundred, it is expected that there are several types of antibodies that recognize the basic skeleton from various directions. However, in an actual antigen-antibody reaction field, an agglutination reaction cannot generally be caused directly due to steric hindrance.
It is generally considered that a peptide antigen has one antigenic determinant per 2,000 to 3,000 molecular weight. However, a peptide having a molecular weight of about 5,000 or less has a high possibility of causing steric hindrance that hinders antigen-antibody reactions at multiple sites. Therefore, the test compound (a) of the present invention is a compound having a molecular weight of about several hundreds in the case of a toxicant, and a compound having a molecular weight of about 5,000 or less in the case of a peptide or protein.

Specific examples of these low molecular weight compounds include toxicants (eg, barbiturate hypnotics), peptides (eg, kinins, calcitonin), low molecular weight proteins (eg, enzymatic degradation products), saccharides (eg, glucose, Tumor-associated carbohydrate antigens), steroid haptens (e.g., estrogen, progestin, androgen, adrenocortical hormone,
Vitamin Ds, cholesterol, bile acids, inotropic steroids, saponins) and bioactive amines (eg, catecholamines).

The primary antibody (b) against the test compound (a) can be prepared by a known method. When the test compound (a) itself has immunogenicity,
By immunizing an animal (eg, mouse, rat, rabbit, goat, sheep, horse, pig) with the substance, preparing a polyclonal antibody from the antiserum of the immunized animal, or preferably, by cell fusion using the spleen cells, Monoclonal antibodies can be prepared.

When the test compound (a) itself does not have immunogenicity, it is combined with an immunogenic polymer carrier compound to impart immunogenicity, and then administered to an animal. Similarly, a primary antibody (a) is prepared.

The polymeric carrier compound having immunogenicity includes, for example, proteins (particularly bovine serum albumin, ovalbumin,
Bovine immunoglobulin, KLH).

For example, a glutaraldehyde method or a carbodiimide method can be used for binding the test target compound (a) and the polymer carrier compound.

The thus obtained primary antibody (b) can be supported on an insoluble or soluble carrier (Y), or can be used alone for measurement of the test compound (a) without being supported on a carrier. The primary antibody (b) is a multimer (mainly
If the primary antibody (b) is a monomer (mainly IgG), the reaction field of the secondary antibody is multivalent when the primary antibody (b) is a monomer (mainly IgG). It is preferable to use the primary antibody (b) supported on an insoluble or soluble carrier (Y) in order to provide the primary antibody (b).

As the insoluble carrier (Y), for example, latex particles or red blood cells can be used. The sensitization of the carrier (Y) is mainly performed by using a physical adsorption method based on hydrophobic bonding.
Alternatively, the method can be carried out using a method in which the carrier (Y) modified with a carboxyl group, amino group, or the like and the antibody (b) are chemically bonded with a cross-linking agent (eg, glutaraldehyde, carbodiimide).

As the soluble carrier (Y), for example, dextran can be used. The sensitization in this case can be carried out using a chemical bonding method using periodic acid.

The preparation of the antigen-antibody reaction complex [(a) + (b)] of the test compound (a) and the primary antibody (b) can be performed as follows.

That is, when the primary antibody (b) is a polyclonal antibody, after obtaining a primary antibody (b) that specifically reacts with the detection target compound (a) using an affinity chromatography technique, The test compound (a) is reacted with the primary antibody (b) in the presence of an excess amount of the test compound (a), the test compound (a) not involved in the reaction is removed by a gel filtration method, and the antigen-antibody reaction complex [(a) + (B)] can be obtained.

Further, when the primary antibody (b) is a monoclonal antibody, after purifying the antibody, the excess amount of the compound (a) to be tested is determined in the same manner as described in relation to the polyclonal antibody. It can be reacted with the primary antibody (b), if present, and treated by gel filtration to obtain an antigen-antibody reaction complex [(a) + (b)].

Furthermore, the secondary antibody (c) against the antigen-antibody reaction complex [(a) + (b)] is converted into an animal (eg, mouse, rat, rabbit, goat, Sheep,
Horse, pig) and prepare a polyclonal antibody from the antiserum of the immunized animal, or preferably, prepare a monoclonal antibody by cell fusion using the spleen cells.

That is, when a polyclonal antibody is obtained, first, antiserum is applied to an affinity column obtained by immobilizing the antigen-antibody reaction complex [(a) + (b)] to prepare an adsorbed fraction. Then, the above-mentioned adsorbed fraction is passed through the column for immobilizing the compound to be tested (a) and the column for immobilizing the primary antibody (b), and the non-adsorbed fraction is collected to obtain the secondary antibody (c). be able to.

In the case of a monoclonal antibody, an antibody that does not react with the test compound (a) and the primary antibody (b) but reacts with the antigen-antibody reaction complex [(a) + (b)] by ELISA By selecting (2), the secondary antibody (c) can be obtained.

The secondary antibody (c) thus obtained is used to sensitize the insoluble carrier (X). The sensitization of the carrier (X) is carried out mainly by a physical adsorption method using a hydrophobic bond, or a crosslinking agent (for example, glutaraldehyde, eg, glutaraldehyde, a carrier (X) modified with a carboxyl group or an amino group) and the antibody (c). Carbodiimide). As the carrier (X), those similar to the insoluble carrier (Y) can be used. Thus, the secondary antibody-sensitized insoluble carrier (X) used in the present invention can be obtained.

The test sample (d) used in the present invention is a sample which may contain the test compound (a), for example, an aqueous liquid, particularly a biological sample (for example, whole blood, serum, urine).

The test sample (d) is placed in a liquid containing the secondary antibody (c), the sensitized insoluble carrier (X), and the primary antibody (b) or the carrier (Y) supporting the primary antibody (b). In addition, when the test compound (a) is present in the test sample (d), an agglutination reaction occurs. That is, as shown in FIG. 1, the secondary antibody 2 carried on the insoluble carrier 1 mutually binds via the complex 5 of the test substance 3 and the primary antibody 4,
This results in agglomeration. Here, even if the insoluble carrier 1 is sensitized with the primary antibody 4 and the carrier 1 is brought into contact with the test substance 3, even if the number of antigenic determinants of the test substance 3 is small, Due to the obstruction, little or no mutual binding occurs and no aggregation is observed.

Since the agglutination reaction depends on the amount of the test substance contained in the test sample, the degree of agglutination is measured visually (visually) or optically (using an optical measurement device) by a conventionally known method. As a result, the determination of the substance to be inspected can be performed batchwise or continuously.

The reagent of the present invention comprises a first component comprising an insoluble carrier (X) carrying a secondary antibody (c), and a primary carrier (b) or an insoluble or soluble carrier carrying the primary antibody (b). And the second component comprising the carrier (Y). The first component and the second component are suspensions or solutions, and can be stored at a low temperature (preferably 1 to 10 ° C.), preferably in the presence of an antibacterial agent (eg, sodium azide).

Measurement of the test compound (a) in the test sample (d)
The test sample (d), the first component and the second component are simultaneously mixed and reacted for an arbitrary time (for example, 1 minute or more).
It can be measured visually (visually) or optically (using optical measuring instruments). Further, after the test sample (d) and the second component are first mixed and allowed to react for an arbitrary time (for example, 1 minute or more), the first component is further added for an arbitrary time (for example, 1 minute or more). ) Reaction, and can be measured visually (visually) or optically (using an optical measuring instrument).

[Examples] Hereinafter, the present invention will be described more specifically with reference to Examples, but these do not limit the scope of the present invention.

Example 1 (1) Preparation of digoxin-BSA conjugate (complex) Carbodiimide method (BBA: BiochimicaetBiophysicaA)
In accordance with CTA 842, 90-99), digoxin (molecular weight 780) was covalently bound to bovine serum albumin (BSA) as a carrier protein. That is, 6 mg of digoxin, 3 mg of BSA and 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide were dissolved in 1 ml of 0.9% sodium chloride solution, kept at 20 ° C. for 16 hours, and dissolved in a 0.9% sodium chloride solution (4%). C). The obtained dialysate (1.0 mg / ml, 25m
l) is used as an immunizing antigen for preparing a monoclonal antibody against digoxin, and an ELISA for selecting hybridomas producing anti-digoxin monoclonal antibody.
Used as an antigen. Coupling efficiency was about 30%.

(2) Preparation of Immunized Splenocytes Digoxin-BSA conjugate immunogen solution (1.0 mg / m
l) was mixed with an equal amount of Freund's complete adjuvant until emulsified, and immunization was performed by intraperitoneally administering 200 μl of the mixture (first immunization). After 30 days, the mouse was intraperitoneally administered with 200 μl of the same mixture as described above (second immunization). Twenty-one days after the second immunization, the digoxin-BSA conjugate immunogen solution (1.0 mg / ml) was diluted with an equal volume of physiological saline, and the diluted solution was used.
200 μl was administered intravenously to the mice (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used for the next cell fusion step.

(3) Cell fusion step The spleen aseptically removed was placed in a Petri dish containing 5 L of DME medium containing 15% fetal bovine serum. Then spleen 1
The spleen cells were drained by refluxing with about 5 ml of DME medium containing 5% fetal bovine serum, and the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50 ml centrifuge tube and centrifuged at 500 × g for 10 minutes. The pellet obtained in this manner was subjected to a hemolyzing solution (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 1
4 ml of mM Na ₂ EDTA (pH 7.0) was added and suspended. It was left at 0 ° C. for 5 minutes to destroy red blood cells in the suspension. DME medium containing 10 ml of 15% fetal bovine serum was added, followed by centrifugation.
The cell pellet thus obtained was washed with DME medium by centrifugation, and the number of living spleen cells was measured.

On the other hand, 1 × 10 ^{8 of the} spleen cells were added to about 2 × 10 ⁷ mouse emiroma cells (myeloma cells) SP 2 / O-Ag14 (RIKEN Genebank Cell Bank), which had been cultured in advance.
Mix well in DME medium and centrifuge (500 × g,
10 minutes). The supernatant is aspirated, the pellet is thoroughly disentangled, 0.5 ml of a 40% polyethylene glycol 4000 solution (preserved at 38 ° C.) is added dropwise, and the polyethylene glycol solution is gently rotated by hand for 1 minute to remove the cell pellet from the polyethylene glycol solution. Mixed. Then keep it at 38 ° C
The tube was gently rotated by adding 1 ml of DME medium every 30 seconds. After repeating this operation 10 times, DME medium containing 20 ml of 15% fetal bovine serum was added, and centrifuged (500 × g, 10 ×
Min). After removing the supernatant, remove the cell pellet for 15 minutes.
% Hat fetal serum in a HAT medium (DME medium supplemented with 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine and 1 × 10 ⁻⁴ M hypoxanthine) by centrifugation. After washing twice, the cells were suspended in 40 ml of the HAT medium. Add this cell suspension to each well of a 96-well cell culture plate.
The mixture was dispensed in 0 ml portions, and culturing was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. Incubate the culture, remove about 100 μl of medium in each well at 2-3 day intervals, and add 100 μl of the HAT medium
By adding l, hybridomas that grew in the HAT medium injection were selected. From day 8 contains 15% fetal bovine serum
Replaced with HAT medium (DME medium added to give thymidine 1.6 × 10 ⁻⁵ M and hypoxanthine 1 × 10 ⁻⁴ M),
While observing the growth of the hybridoma, on day 10, the digoxin antibody-producing hybridoma was screened by the ELISA method described later.

(4) Establishment of hybridoma ELISA for the presence or absence of produced antibody in the culture supernatant of hybridoma
It was measured by the method. 96-well ELISA plate (Immulon
II, Nippon Dynatech Co., Ltd.) in each well of the digoxin-BSA conjugate immunogen solution (50 μg / m
l, diluted with physiological saline), dispensed 50 μl each,
Left for a time (Plate A).

Similarly, 50 μl of BSA (50 μg / ml) is dispensed into another plate (plate B). Next, after washing three times with 0.05% Tween 20-physiological saline, 50 μl of the culture supernatant was added to each well.
Was added and reacted at 25 ° C. for 1 hour.

Next, Tween 20-a peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold with saline
μl was added to each well. After completion of the reaction, 0.05% Tween20
Washing each well three times with saline, adding 250 μl of a solution containing 0.5 mM aminoantipyrine, 10 mM phenol and 0.005% hydrogen peroxide to each well, reacting for 30 minutes at 25 ° C., and reacting each well at 490 nm The absorbance was measured (search for wells that reacted on plate A and did not react on plate B). As a result, anti-digoxin antibody production was observed in 4 of the 192 wells. The hybridoma in the well was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 4 to 5 days. Then again ELIS
The presence or absence of the production of an anti-digoxin antibody that does not react with the carrier protein (BSA) was confirmed by Method A, and then cloned by the limiting dilution method. In the limiting dilution method, a cell suspension obtained by diluting a hybridoma to 5 cells / ml in an HT medium is used in a 96 well in which peritoneal cells of a normal BALB / C mouse are previously contained in 2 × 10 ⁴ cells per well. 100 μl was dispensed into each well of the plate. Ten days later, hybridoma clones producing an anti-digoxin-specific antibody were screened by ELISA. As a result, for each hybridoma, out of 20 to 40 antibody-producing clones, a clone having good growth potential, high antibody secretion ability, and a stable clone was selected and recloned in the same manner as described above. , An anti-digoxin-specific antibody-producing hybridoma DIG-1 was established.

(5) Production of monoclonal antibody (a) In vitro method Mouse hybridoma DIG-1 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. Centrifuge the culture (10000x
g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. Mix the mixture under ice cooling for 30 minutes.
After stirring for 60 minutes, leave for 60 minutes and centrifuge (10000 ×
g, 10 minutes), the resulting precipitate was dissolved in a small amount of 10 mM phosphate buffer (pH 8.0), and dialyzed against a 1000-fold amount of 10 mM phosphate buffer. This was loaded onto a DEAE-cellulose column already equilibrated with 10 mM phosphate buffer. Elution of monoclonal antibody is performed with 10 mM phosphate buffer (pH 8.0) and 0.2 MN
The measurement was performed by a concentration gradient method between 10 mM phosphate buffer (pH 8.0) containing aCl. The eluted monoclonal antibody was concentrated by the marginal excess method, and dialyzed against 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG-Sepharose 4B to remove 1 gG of bovine serum. Next, the permeate was packed in a protein A-cephalol 4B column equilibrated with 0.1 M phosphate buffer (pH 8.0). The column was eluted with a buffer of pH 3.5 to obtain a solution of purified digoxin-specific antibody DIG-1.

(B) In vivo method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to BALB / c mice of 10 to 12 weeks of age, and after 14 to 20 days, Hybridoma DIG-1 grown in vitro was inoculated into the mouse abdominal cavity at 2 × 10 ⁶ cells / mouse.

About 10-15 ml from one mouse for each hybridoma
Of ascites was obtained. The antibody concentration was 2-10 mg / ml. Purification of the monoclonal antibody in the ascites fluid is carried out in the same manner as the above-mentioned in vitro purification method (except for goat anti-bovine serum).
Except for the operation of passing through a column of IgG-Sepharose 4B).

(6) Identification of Immunoglobulin Class and Specificity of Monoclonal Antibody Identification of the immunoglobulin class and specificity of DIG-1 of the anti-digoxin-specific monoclonal antibody was performed by the octeroney immunodiffusion method and the enzyme immunoassay method. The immunoglobulin class of -1 was found to be IgG1.

(7) Preparation of digoxin-DIG-1 (anti-digoxin monoclonal antibody) complex, preparation of immunized spleen cells,
Cell fusion and establishment of hybridoma 1 mg (1 mg / ml, 0.9% NaCl) of purified antibody DIG-1 was mixed with 1 mg (1 mg / ml, 0.9NaCl) of digoxin, and the mixture was incubated at 20 ° C. for 1 hour. This mixture was applied to a Sephadex G-25 gel column to remove free digoxin, thereby obtaining a digoxin-DIG-1 complex. This digoxin-DIG-1 complex (1.0 mg / ml, 0.9% NaCl) was mixed with an equivalent amount of Freund's complete adjuvant until emulsified, and 200 μl of the mixture was administered intraperitoneally to mice to immunize ( First
Times immunization). After 30 days, the mouse was intraperitoneally administered with 200 μl of the same mixture as described above (second immunization).
21 days after the second immunization, digoxin-DIG-1
Diluted with an equal volume of physiological saline solution of the immunogen solution (1.0 mg / ml),
200 μl of the diluted solution was intravenously administered to the mouse (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse, and the anti-digoxin specific antibody DIG-
Cell fusion was performed in the same manner as in Method 1. On the 12th day after the cell fusion treatment, a hybridoma producing an antibody that specifically reacts with the digoxin-DIG-1 complex was screened by ELISA.

That is, the digoxin-was added to each well of a 96-well ELISA plate (ImmulonII, Nippon Dynatech Co., Ltd.).
Dispense 50 μg / ml of the BSA conjugate solution in 50 μl
It was left at 5 ° C. for 2 hours (plate C). Similarly, 50 μl of DIG-1 and digoxin-DIG-1 complex were respectively dispensed at a concentration of 50 μg / ml on separate plates. (Plate D, Plate E). Next, after washing three times with 0.05% Tween 20-physiological saline, 50 μl of the culture supernatant was added to each well and reacted at 25 ° C. for 1 hour.

Next, Tween 20-a peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold with saline
μl was added to each well. After completion of the reaction, 0.05% Tween20
Washing each well three times with saline, adding 250 μl of a solution containing 0.5 mM aminoantipyrine, 10 mM phenol and 0.005% hydrogen peroxide to each well, reacting for 30 minutes at 25 ° C., and reacting each well at 490 nm The absorbance was measured.

As a result, wells that reacted on plate E but did not react on plate C and plate D were observed in 2 of 288 wells. The hybridoma in the well was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 7 days. Then, again by the ELISA method,
After confirming the presence or absence of the production of an antibody that does not react with the digoxin-BSA conjugate and DIG-1, but reacts with the digoxin-DIG-1 complex, cloning was performed by the limiting dilution method. In the limiting dilution method, a cell suspension diluted to 5 hybridomas / ml in HT medium
100 μl of ALB / C mouse peritoneal cells were dispensed to each well of a 96-well plate in which 2 × 10 ⁴ per well cells were dispensed. 12 days later, anti-digoxin-DIG
Hybridoma clones producing the -1 complex-specific antibody were screened. As a result, for each hybridoma, out of 20 to 40 antibody-producing clones, a clone having good growth potential, high antibody secretion ability, and a stable clone was selected and recloned in the same manner as described above. A hybridoma DIG-2 producing an anti-digoxin-DIG-1 complex-specific antibody was established.

(8) Production of monoclonal antibody and identification of immunoglobulin class Production of monoclonal antibody DIG-2 and identification of the subclass were performed in the same manner as described above. As a result, the immunoglobulin subclass of monoclonal antibody DIG-2 was IgG1. .

The monoclonal antibody DIG- examined by the aforementioned ELISA method
The specificity of 2 is shown in FIG. In FIG. 2, (a) shows a plate of digoxin-BSA conjugate, (b) shows a plate of monoclonal antibody DIG-1, and [(a) + (b)] shows a plate of digoxin-DIG-1 complex. Show.

(9) Binding of antibody to insoluble carrier Latex solution (2%, Dow Chemical: particle size 0.482μ)
m) 2 ml and 2 ml of an aqueous solution containing 2.0 mg / ml of DIG-2 antibody
And stirred for about 1 hour. After centrifugation (20,000 g, 10
Min) and the precipitate was suspended in a 0.1% BSA solution and stirred for about 1 hour. After centrifugation again (20,000 g, 10 minutes), the precipitate was suspended in water and stirred for about 2 hours. Thus, a DIG-2 antibody-latex complex-containing liquid was obtained.

(10) Quantification by slide agglutination reaction 30 μl of the DIG-2 antibody-latex complex-containing solution prepared in (9) above, 30 μl of an aqueous solution containing various concentrations of digoxin, and 30 μl of an aqueous solution containing a certain amount of DIG-1 Was mixed and shaken in the form of a glass slide, and after 3 minutes, the aggregated image was visually judged. The results are shown in Table 1 below.

(11) Measurement by Spectroscopic Method Using the DIG-2 antibody-latex complex prepared in (9) above, the dependence of the agglutination reaction rate on the concentration of sigoxin was examined by an automatic analyzer (LPIA: Mitsubishi Kasei). A fixed amount of DIG-1 and digoxin solutions having various concentrations of 0.125 to 32 ng / ml were dispensed into the reactor, and a DIG-2 antibody-latex-containing solution was added thereto.

As a control experiment, the same operation was performed except for DIG-1. The results obtained are shown in FIG. In FIG. 3,
Shows the results of the experiment by the method of the present invention in which DIG-1 is present,
△ indicates the result of the control experiment excluding DIG-1, and ● indicates the DIG-1 antibody-latex obtained by sensitizing the DIG-1 antibody to the latex in the same manner as in the above (9). Shows the results obtained by reacting DIG-2 with various concentrations of digoxin solutions in the absence of DIG-2 antibody-latex. As is clear from FIG. 3, the digoxin concentration shows a good correlation with the agglutination reaction rate (V value: change in transmittance per unit time).

Example 2 (1) Preparation of calcitonin-ovalbumin conjugate (complex) According to the glutaraldehyde method (Clin. Chem; 31: 430, 1985), human calcitonin (molecular weight: 3500) is covalently bound to carrier protein ovalbumin. I let it. That is,
Human calcitonin (hereinafter simply referred to as calcitonin)
3 mg and 10 mg of ovalbumin were dissolved in 2.0 ml of 0.9% sodium chloride solution, 0.2 ml of 1% (v / v) glutaraldehyde was added to the solution, and the mixture was reacted at room temperature for 2 hours. The solution was dialyzed (4 ° C.). The obtained dialysate was diluted with a 0.9% sodium chloride solution to a concentration of 1.0 mg / ml, and the diluted solution was used as an immunizing antigen for preparing a monoclonal antibody against calcitonin.
It was used as an antigen for ELISA for selecting hybridomas producing anti-calcitonin monoclonal antibody. Coupling efficiency was about 40%.

(2) Preparation of Immunized Splenocytes Calcitonin-ovalbumin conjugate immunogen solution (1.0 mg / ml) is mixed with an equal volume of Freund's complete adjuvant until emulsified, and 200 μl of the mixture is intraperitoneally administered to mice. Immunization (first immunization). After 30 days, add the same mixture of 20
0 μl was administered intraperitoneally to mice (second immunization). Second
Twenty-one days after the first immunization, a calcitonin-ovalbumin conjugate immunogen solution (1.0 mg / ml) was diluted with an equal volume of physiological saline, and 200 μl of the diluted solution was intravenously administered to the mouse (final immunization). ). Three days after the final immunization, the spleen was aseptically removed from the mouse and used for the next cell fusion step.

(3) Cell fusion step The above spleen aseptically removed was placed in a Petri dish containing 5 ml of DME medium containing 15% fetal bovine serum. Next, the spleen was refluxed with about 15 ml of DME medium containing 15% fetal calf serum to drain spleen cells, and the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50 ml centrifuge tube and centrifuged at 500 × g for 10 minutes. The pellet thus obtained was subjected to a hemolyzing solution (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 1 mM
4 ml of Na ₂ EDTA (pH 7.0) was added and suspended. It was left at 0 ° C. for 5 minutes to destroy red blood cells in the suspension. DME medium containing 10 ml of 15% fetal bovine serum was added, followed by centrifugation. The cell pellet thus obtained was washed with a DME medium by a centrifugation method, and the number of living spleen cells was measured.

On the other hand, 1 × 10 ⁸ spleen cells were added to about 2 × 10 ⁷ mouse emiroma cells (myeloma cells) SP 2 / O-Ag-14 (RIKEN Genebank Cell Bank) which had been cultured in advance. In addition, mix well with DME medium injection and centrifuge (500 ×
g, 10 minutes). The supernatant is aspirated, the pellet is thoroughly disentangled, 0.5 ml of a 40% polyethylene glycol 4000 solution (preserved at 38 ° C) is dropped, and the polyethylene glycol solution and the cell pellet are gently rotated by hand for 1 minute by hand. Mixed. Next, 1 ml of DME medium kept at 38 ° C. was added every 30 seconds, and the tube was gently rotated. After repeating this operation 10 times, DME medium containing 20 ml of 15% fetal bovine serum was added, and centrifugation (500 ×
g for 10 minutes). After removing the supernatant, the cell pellet was converted to a HAT medium containing 15% fetal bovine serum (4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine, and 1 × 10 ⁻⁴ M thymidine in DME medium). Was washed twice by centrifugation, and suspended in 40 ml of the HAT medium. 200 ml of this cell suspension was dispensed into each well of a 96-well cell culture plate, and culture was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. About 100 μl of the medium in each well was removed at intervals of 2 to 3 days after the culture injection, and 100 μl of the above-mentioned HAT medium was newly added to select hybridomas that grew in the HAT medium injection. From the 7th day, the medium was replaced with a HAT medium containing 15% fetal bovine serum (DME medium supplemented with thymidine at 1.6 × 10 ⁻⁵ M and hypoxanthine at 1 × 10 ⁻⁴ M), and hybridoma growth was observed. On the 11th day, digoxin antibody-producing hybridomas were screened by the ELISA method described later.

(4) Establishment of hybridoma ELISA for the presence or absence of produced antibody in the culture supernatant of hybridoma
It was measured by the method. 96-well ELISA plate (Immulon
II, Nippon Dynatech Co., Ltd.), 50 μl of the calcitonin-ovalbumin conjugate immunogen solution (50 μg / ml, diluted with physiological saline) was dispensed into each well and allowed to stand at 25 ° C. for 2 hours ( Plate F).

Similarly, add ovalbumin (50μg / ml) to another plate
(Plate G). Next, 0.05% Tw
After washing three times with een20-physiological saline, 50 μl of the culture supernatant was added to each well and reacted at 25 ° C. for 1 hour.

Next, Tween 20-a peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold with saline
μl was added to each well. After completion of the reaction, 0.05% Tween20
Washing each well three times with saline, adding 250 μl of a solution containing 0.5 mM aminoantipyrine, 10 mM phenol and 0.005% hydrogen peroxide to each well, reacting for 30 minutes at 25 ° C., and reacting each well at 490 nm The absorbance was measured (search for wells that reacted on plate F and did not react on plate G). As a result, anti-calcitonin antibody production was observed in 3 of the 192 wells. The hybridoma in the well was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 4 to 5 days. Then EL again
The presence or absence of the production of an anti-calcitonin antibody that does not react with the carrier protein (ovalbumin) was confirmed by the ISA method, and then cloned by the limiting dilution method. In the limiting dilution method, a 96-well cell suspension in which 2 × 10 ⁴ peritoneal cells of a normal BALB / C mouse are pre-dispensed per well of a cell suspension diluted to 5 hybridomas / ml in HT medium 100 μl was dispensed into each well of the plate. 8 days later, E
Hybridoma clones producing anti-digoxin-specific antibodies were screened by the LISA method. As a result, for each hybridoma, out of 30 to 50 antibody-producing clones, a clone having good growth ability, high antibody secretion ability, and a stable clone was selected and recloned in the same manner as described above. , An anti-digoxin-specific antibody-producing hybridoma CAL-1 was established.

(5) Production of monoclonal antibody (a) In vitro method Mouse hybridoma CAL-1 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. Centrifuge the culture (10000x
g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. Mix the mixture under ice cooling for 30 minutes.
After stirring for 60 minutes, leave for 60 minutes and centrifuge (10000 ×
g, 10 minutes), the resulting precipitate was dissolved in a small amount of 10 mM phosphate buffer (pH 8.0), and dialyzed against a 1000-fold amount of 10 mM phosphate buffer. This was loaded onto a DEAE-cellulose column already equilibrated with 10 mM phosphate buffer. Elution of monoclonal antibody is performed with 10 mM phosphate buffer (pH 8.0) and 0.2 MN
The measurement was performed by a concentration gradient method between 10 mM phosphate buffer (pH 8.0) containing aCl. The eluted monoclonal antibody was concentrated by an ultrafiltration method, and dialyzed against 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG-Sepharose 4B to remove 1 gG of bovine serum. Next, the permeate was packed in a protein A-Sepharose 4B column equilibrated with 0.1 M phosphate buffer (pH 8.0). The column was eluted with a buffer solution of pH 3.5 to obtain a solution of purified calcitonin-specific antibody CAL-1.

(B) In vivo method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to BALB / c mice of 10 to 12 weeks of age, and after 14 to 20 days, Hybridoma CAL-1 grown in vitro in the abdominal cavity of a mouse was inoculated to give 2 × 10 ⁶ cells per mouse.

About 10-15 ml from one mouse for each hybridoma
Of ascites was obtained. The antibody concentration was 2-10 mg / ml. Purification of the monoclonal antibody in the ascites fluid is carried out in the same manner as the above-mentioned in vitro purification method (except for goat anti-bovine serum).
Except for the operation of passing through a column of IgG-Sepharose 4B).

(6) Identification of Immunoglobulin Class and Specificity of Monoclonal Antibody Identification of the immunoglobulin class and specificity of the anti-digoxin-specific monoclonal antibody CAL-1 was performed by the octeroney immunodiffusion method and the enzyme immunoassay method. One immunoglobulin class was found to be IgG2a.

(7) Preparation of calcitonin-CAL-1 (anti-calcitonin monoclonal antibody) complex, preparation of immunized spleen cells, cell fusion and establishment of hybridoma 1 mg (1 mg / ml, 0.9% NaCl) of purified antibody CAL-1 Mix with 1 mg of calcitonin (1 mg / ml, 0.9% NaCl) and add
Incubated for hours. This mixture was applied to a Sephadex G-50 gel column to remove free calcitonin, thereby obtaining a calcitonin-CAL-1 complex. This calcitonin-C
Immunization was carried out by mixing the AL-1 complex (1.0 mg / ml, 0.9% NaCl) with an equal volume of Freund's complete adjuvant until emulsified, and administering 200 μl of the mixture intraperitoneally to the mouse (No. 1). Times immunization). After 30 days, the mouse was intraperitoneally administered with 200 μl of the same mixture as described above (second immunization). Twenty-one days after the second immunization, the calcitonin-CAL-1 complex immunogen solution (1.0 mg / ml) was diluted with an equal volume of physiological saline, and 200 μl of the diluted solution was intravenously administered to the mouse. (Final immunity). Three days after the final immunization, the spleen was aseptically removed from the mouse, and cell fusion was performed in the same manner as the method of the anti-calcitonin-specific antibody CAL-1. On the 14th day from the cell fusion treatment, a hybridoma producing an antibody that specifically reacts with the calcitonin-CAL-1 complex was screened by ELISA.

That is, 50 μg / ml of the above calcitonin-ovalbumin conjugate solution was added to each well of a 96-well ELISA plate (Immulon II, Nippon Dynatech).
The mixture was left at 25 ° C. for 2 hours (plate H). Similarly, 50 μl of CAL-1 and calcitonin-CAL-1 complex were respectively dispensed at a concentration of 50 μg / ml on separate plates. (Plate I, Plate J). Next, 0.05
After washing three times with% Tween 20-physiological saline, 50 μl of the culture supernatant was added to each well and reacted at 25 ° C. for 1 hour.

Next, Tween 20-a peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold with saline
μl was added to each well. After completion of the reaction, 0.05% Tween20
Washing each well three times with saline, adding 250 μl of a solution containing 0.5 mM aminoantipyrine, 10 mM phenol and 0.005% hydrogen peroxide to each well, reacting for 30 minutes at 25 ° C., and absorbance at 490 nm of each well Was measured.

As a result, the number of wells that reacted on plate J and did not react on plate H and plate I was determined in 2 of 384 wells. The hybridoma in the well was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 11 days. Then, again by the ELISA method,
Calcitonin-ovalbumin conjugate and CA
After confirming the production of an antibody that does not react with L-1 but reacts with the calcitonin-CAL-1 complex, cloning was performed by the limiting dilution method. In the limiting dilution method, a 96-well cell suspension in which 2 × 10 ⁴ peritoneal cells of a normal BALB / C mouse are pre-dispensed per well of a cell suspension diluted to 5 hybridomas / ml in HT medium 100 μl was dispensed into each well of the plate. Twelve days later, a hybridoma clone producing an anti-calcitonin-CAL-1 complex-specific antibody was screened by ELISA. As a result, for each hybridoma, out of 10 to 20 antibody-producing clones, a clone having good growth potential, high antibody secretion ability, and stable was selected and recloned in the same manner as described above. A hybridoma CAL-2 producing an anti-calcitonin-CAL-1 complex-specific antibody was established.

(8) Production of Monoclonal Antibody and Identification of Immunoglobulin Class The production of monoclonal antibody CAL-2 and identification of the subclass were performed in the same manner as described above. As a result, the immunoglobulin subclass of monoclonal antibody CAL-2 was IgG1. .

Monoclonal antibody CAL- examined by the aforementioned ELISA method
The specificity of 2 is shown in FIG. In FIG. 4, (a) shows a plate of calcitonin-ovalbumin conjugate, (b) a plate of monoclonal antibody CAL-1,
[(A) + (b)] shows a plate of calcitonin-CAL-1 complex.

(9) Binding of antibody to insoluble carrier Latex solution (2%, Dow Chemical: particle size 0.482μ)
m) 2 ml and 2 ml of an aqueous solution containing 2.0 mg / ml of CAL-2 antibody
And stirred for about 1 hour. After centrifugation (20,000 g, 10
Min) and the precipitate was suspended in a 0.1% BSA solution and stirred for about 1 hour. After centrifugation again (2000 g, 10 minutes), the precipitate was suspended in water and stirred for about 2 hours. Thus, a liquid containing the CAL-2 antibody-latex complex was obtained.

In addition, a latex solution (2%, Dow Chemical: particle size
0.091 μm) and 2.0 ml of an aqueous solution containing 4.0 mg / ml of CAL-1 antibody were mixed and stirred for about 1 hour. After centrifugation (200
00g, 10 minutes), and the precipitate was suspended in a 0.1% BSA solution and stirred for about 1 hour. After centrifugation again (20,000 g, 10 minutes), the precipitate was suspended in water and stirred for about 2 hours. Thus, a CAL-1 antibody-latex complex-containing liquid was obtained.

(10) Quantification by slide agglutination reaction 30 μl of the CAL-2 antibody-latex complex-containing solution prepared in (9) above, 30 μl of an aqueous solution containing various concentrations of calcitonin, and an aqueous solution containing a certain amount of CAL-1 antibody latex 30 μl of the mixture was mixed in a glass slide and rocked, and after 3 minutes, the aggregated image was visually determined. The result is the second
It is shown in the table.

(11) Measurement by Spectroscopic Method Using the CAL-2 antibody-latex complex prepared in the above section (9), the dependence of the agglutination reaction rate on calcitonin concentration was examined by an automatic analyzer (LPIA: Mitsubishi Kasei). In a reactor, a fixed amount of CAL-1 antibody latex and 0.125 to 32 ng / ml
Aliquots of calcitonin solutions of various concentrations
-2 antibody-latex containing solution was added.

As a control experiment, the same operation was performed except for the CAL-1 antibody latex. The results obtained are shown in FIG. Fifth
In the figure, ○ indicates the result of the experiment according to the method of the present invention in which CAL-1 is present, Δ indicates the result of a control experiment excluding the CAL-1 antibody latex, and ● indicates the non-existence of CAL-2 antibody-latex. The results obtained by reacting the CAL-1 antibody latex with various concentrations of calcitonin solutions in the presence are shown.
As is clear from FIG. 5, the calcitonin concentration shows a good correlation with the agglutination reaction rate (V value: change in transmittance per unit time).

[Effects of the Invention] According to the present invention, an agglutination reaction can be performed even on a test control substance, particularly a low molecular weight compound, in which it was conventionally impossible or difficult to utilize agglutination by an antigen-antibody reaction. Therefore, a clinical test technique utilizing an agglutination reaction which has been conventionally developed for a high molecular compound can be used as it is for a low molecular compound.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing the principle of the present invention, FIG. 2 is a graph showing the specificity of the secondary antibody DIG-2 of the present invention, and FIG. Fig. 4 is a graph showing that the digoxin concentration and the agglutination reaction rate have good correlation in the method of the present invention. Fig. 4 is a graph showing the specificity of CAL-2 of the secondary antibody of the present invention. And FIG. 5 is a graph showing that the calcitonin concentration and the agglutination reaction rate have a good correlation in the method of the present invention.

Claims (2)

(57) [Claims] (1) an insoluble carrier carrying a secondary antibody to an antigen-antibody reaction complex of a compound to be tested and a primary antibody to the compound to be tested; (ii) the primary antibody; Or (iii) bringing a test sample into contact with an insoluble or soluble carrier carrying the primary antibody, and then contacting the test compound in the test sample with:
Measuring the agglutination reaction between the secondary antibody-carrying carrier and the primary antibody or the primary antibody-carrying carrier,
Immunological measurement method. 2. An insoluble carrier carrying a secondary antibody to an antigen-antibody reaction complex of the compound to be tested and a primary antibody to the compound to be tested; and (ii) the primary antibody or a primary antibody thereof. A reagent for immunological measurement, comprising an insoluble or soluble carrier carrying a primary antibody.