JP2000055917A - Method for detection or measurement using antigen- antibody complex antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To permit the detection of a substance by immunological reaction superior in specificity and sensitivity by detecting the substance with antigenicity through the use of an antibody specific to an antigen-antibody complex. SOLUTION: An anti-antigen-antibody complex antibody specifically reacts with an antigen-antibody complex antibody and slightly reacts or does not react at all with a substance with antigenicity, an antibody to the substance, or the ligand of the substance if it exists. It is possible to use the anti-antigen- antibody complex antibody with such specificity in reaction for non-competitive methods such as immunoradiometric assay(IRMA), sandwich ELISA using a plurality of antibodies with different bonding sections. By this, it is possible to detect or measure the substance with antigenicity, specially an environmental pollutant. It is possible to create a reagent or a kit for detection or measurement through the use of the anti-antigen-antibody complex antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting or measuring a specific substance utilizing an immune reaction. With such a method, detection or measurement of environmental pollutants can be performed.

[Prior art]

[0002] Immunoassays (immunoassays) for many substances have been developed utilizing antigen-antibody reactions. The immunoassay uses one type of antibody, such as radioimmunoassay (RIA) or competitive enzyme immunoassay (EIA), and labels either the antigen or the antibody with a radioisotope or an enzyme. , A competitive assay method for measuring an antigen by using a competitive reaction with an unlabeled antigen or an unlabeled antibody and, for example, an immunoradiometric assay (IRMA) or a sandwich ELISA
zyme-Linked Immuno-Sorbent Assay), which can be roughly classified into two groups of non-competitive assay methods for detecting an antigen using a plurality of antibodies having different binding sites.

[0003] According to the radioimmunoassay, substances can be detected or measured in the following manner.

[0004] A certain amount of an antibody is reacted with a certain amount of a radioisotope-labeled antigen or hapten. When an antigen or hapten to be detected or measured is added, the amount of the labeled antigen or hapten bound to the antibody decreases.
From the degree of this decrease, the amount of the antigen or hapten, which is the substance to be measured, can be known. Separation of the labeled antigen or hapten bound to the antibody from the unbound one can be carried out by adsorption using dextran / charcoal or precipitation using polyethylene glycol, particularly when the hapten or antigen is a small molecule. . Generally, separation can be performed by precipitation using an anti-immunoglobulin antibody (secondary antibody), which may be insolubilized in a solid phase.

In the competitive enzyme immunoassay (EIA), unlike a radioimmunoassay, an enzyme is used as a label instead of a radioisotope.

An enzyme is bound as a label to either an antigenic substance (antigen substance) or an antibody, and the amount of the labeled antigen substance or the labeled antibody is determined by the amount of the product (coloring ). For example, when an antigen substance to be measured and an enzyme-labeled antigen substance are reacted with a certain limited concentration of antibody, the amount of the enzyme-labeled antigen substance bound to the antibody is almost inversely proportional to the concentration of the substance. .

[0007] Separation of the antigen-antibody complex from the free antigen substance or antigen can be easily carried out by using a solid-phased antibody. For example, when an unlabeled antigen substance and an enzyme-labeled antigen substance are reacted with an immobilized antibody, the free antigen substance can be removed by a washing operation. Antigen material that has reacted with the antibody is easily separated.

[0008] The homogeneous enzyme assay is based on the fact that the enzyme reaction of an enzyme label is generally inhibited by the binding of an antibody and an antigenic substance.
Although the color develops due to the enzymatic reaction with the substrate, the color that is bound to the antibody does not develop due to the weakening or disappearance of the enzymatic reaction. This method does not require a separation operation.

However, since the competitive assay uses only one type of antibody, it has limitations such as lower specificity and sensitivity than the non-competitive assay using a plurality of antibodies.

[0010] Non-competitive ELISAs have advantages over competitive ELISAs in specificity and sensitivity because they use two types of antigen-antibody reactions (Christopher, P. Pr.
ice, David, J. Newman (Eds.), 1991, Principles and
Practice of Immunoassay, Stockton Press, New York, U
SA, see). Although a variety of non-competitive ELISAs have been developed, the most prevalent is the sandwich EL
It is an ISA and uses two types of antibodies that bind to the analyte. The antibody used in the sandwich ELISA is most preferably two types of monoclonal antibodies (MAbs) recognizing different parts of the same antigenic substance. However, the binding of the first monoclonal antibody to the antigen allows the second monoclonal antibody to bind to the antigen. It is often difficult to produce a second monoclonal antibody suitable for constructing a sandwich ELISA, since it is necessary that the binding of the antibody be sterically hindered. In addition, in the case of low-molecular substances such as drugs and haptens, the size of the antigen site that can bind to the antibody is limited, so that it is in principle impossible to obtain a second monoclonal antibody suitable for constructing a sandwich ELISA. It is possible. Therefore, for low molecular substances such as drugs, toxins, and haptens, methods such as IRMA and sandwich ELISA, which are methods using multiple antibodies with different binding sites, cannot be developed. I have been.

The present inventors have previously produced an anti-microcystin monoclonal antibody (MC MAb) (Nagata
et al., Natural Toxins 3, 78 (1995) and
-89998), microcystin in environmental water (M
C) Developing a competitive ELISA for direct measurement of
In addition, a competitive ELISA utilizing an anti-idiotypic monoclonal antibody to the MC MAb was developed. (Tsutsumi et al., Toxin, in press (1997)). (Nagata et al., J. Assoc. Off. Anal. Che
m. Int. 80, 408 (1997); Ueno et al., Carcinogenesi
s 17, 1317 (1996), Natural Toxins 4, 271 (1996),
Tsutsumi et al., Toxicon 36, 235 (1998)

[0012] Microcystin (MC) is a group of hepatotoxins having a cyclic heptapeptide structure, and is a toxin produced by cyanobacteria, which often occurs frequently due to eutrophication of lakes and marshes (Carmichael, WW and Falconer, IR, in Falcon
er, IR (Ed.), Algal Toxins in Seafood and Drinki
ng Water. Academic Press, London, p. 187 (1993)), which has recently been found to have tumor promoter activity. (Nishiwaki-Matsushima, R. et a
l., Liver tumor promotion by the cyanobacterial cy
clic peptide toxin microcystin LR, J. Cancer Res.
Clin. Oncol. 118,420-424 (1992)) The potential health hazard of ingesting microcystin through drinking water is a major public health problem (Carmichael et al.). ., (1993) supra, Y
u, SZ, Hepatol. 10, 674 (1995) and Ueno et a
l., (1996) supra).

For example, in such a measurement of microcystin, the MC concentration in tap water, which may cause a health problem, is very low.
There is a need for a highly sensitive method of accurately detecting the level of the trace amount. The competitive ELISA method developed by the inventor previously was an insufficient measurement method for trace amounts of MC measurement in terms of accuracy and sensitivity.

It is known that some antibodies exhibit anti-antigen-antibody complex activity (Vosset et al., Mol. Immun
ol. 25, 751 (1988); Ullman et al., Proc. Natl. Aca.
d.Sci. USA 90, 1184 (1993); Weidner et al., Mol. Im.
munol. 30, 1003 (1993)), such an antibody has hardly been noticed as an antibody in an immunoassay-based assay such as a non-competitive assay such as a sandwich ELISA. Recently, such anti-antigen-antibody complex antibodies have been
Only three studies have been reported for application to SA. (Self et al., Clin. Chem. 40, 203
5 (1994); Towbin et al., J. Immunol. Methods 181,
167 (1995), J. Immunoassay 17, 353 (1996))

[0015]

An object of the present invention is to provide a method for detecting or measuring a substance having excellent specificity and sensitivity by an immune reaction.

[0016]

SUMMARY OF THE INVENTION The present invention provides a method for detecting or measuring an environmental pollutant using an antibody against a complex of an environmental pollutant and an antibody against the environmental pollutant, that is, a method for detecting a complex of an antigen and an antibody. Is a method for detecting or measuring a substance having antigenicity using an antibody specific to According to this method, it is also possible to construct a non-competitive measurement method for low-molecular substances, which was impossible until now, and has the high reliability, sensitivity and high specificity in the low concentration range, which are the advantages of the non-competitive method A measuring method is provided.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION An anti-antigen-antibody complex antibody specifically reacts with an antigen-antibody complex and has a substance having antigenicity, an antibody against this substance, or, if present, this substance. Reacts slightly or not at all. An anti-antigen-antibody complex antibody specifically distinguishes between an antigen-antibody complex (immune complex) formed by these substances and an antibody against the substance, and a single antibody or this substance alone.

The anti-antigen-antibody complex antibody having such reaction specificity can be used in a non-competitive method such as IRMA or sandwich ELISA using a plurality of antibodies having different binding sites. It is possible to detect or measure a substance having antigenicity, particularly an environmental pollutant. An anti-antigen-antibody complex antibody can be used to create a reagent or kit for detection or measurement.

Immunoradiometric assay (IRM)
In the case of A), a substance can be detected or measured as follows.

The antibody is insolubilized in the solid phase, and
The substance having antigenicity is trapped on the solid phase, and further reacted with an anti-antigen-antibody complex antibody labeled with a radioisotope. The amount of the labeled antibody bound to the substance having antigenicity in the sample is measured. The immobilized antibody, the substance having antigenicity, and the labeled antibody may be reacted simultaneously, or the labeled antibody and the substance having antigenicity may be reacted first, and then reacted with the immobilized antibody. The detection limit of a substance differs depending on the affinity of the antibody used. When the affinity is high, the detection limit is 1 ×
10 ^- a ^{1 5} M about. This method is more specific and sensitive than the competition method.

[0021] The labeling of these methods, ^{1 2 5} I,
^{1 3 1} I, ^{1 4} C, ³ H radioactive isotopes such substances and properties object, can be used depending on the detection or measurement purposes.

A substance having antigenicity (antigen substance) can be detected or measured by a sandwich ELISA method using an anti-antigen-antibody complex antibody.

Usually, a 96-well microtiter plate is used. First, the primary antibody is adsorbed on the bottom of the well, and then the antigen substance is reacted with the immobilized antibody thus obtained. Then, an enzyme-labeled secondary antibody in which an enzyme is bound to the antibody is added. Thereafter, a substrate is added, and an enzyme reaction is performed with the complex enzyme-labeled antibody. The color of the product obtained by the reaction is qualitatively or quantitatively determined. For quantification, make serial dilutions of a known antigenic substance,
Using it, a calibration curve is made to quantify the target substance. Anti-antigen-antibody complex antibodies can be used as both primary and secondary antibodies.

As the labeling enzyme, horseradish peroxidase, alkaline phosphatase and β-galactosidase can be used. Other enzymes include lysozyme, maleate dehydrogenase, glucose-6-
Phosphate dehydrogenase and glucose oxidase can be mentioned. Enzyme substrates include horseradish peroxidase substrates such as 3,3 ', 5,5'-tetramethylbenzidine (TMBA), o-phenylenediamine, 2,2-azino-bis (3-ethylbenzthiazoline-6). -As sulfonic acid and alkaline phosphatase substrates, mention may be made of p-nitrophenyl phosphate, and as β-galactosidase substrates, mention may be made of nitrophenyl-β-D-galactoside and 4-methylumbelliferyl-β-D-galactoside. Can be.

The immobilization can be carried out by adsorbing various materials or shapes such as plastic beads and test tube walls.

The secondary antibody is biotinylated and, for example, sandwich E using an avidin-enzyme complex is performed as follows.
LISA can be performed. A primary antibody solution diluted with phosphate buffered saline (PBS) or the like is added to a microtiter plate, and the primary antibody is immobilized by allowing it to stand at about 4 ° C. overnight. A blocking solution (eg, a PBS solution containing gelatin) is added to the antibody-immobilized plate thus prepared to block the wells. After pre-incubating the sample and the biotinylated secondary antibody, add the primary antibody to the solid-phased well,
For example, incubate at about 4 ° C. overnight. Next, after the enzyme is added and incubated, the enzyme solution is started by adding the substrate solution. After reacting for an appropriate time, the reaction is stopped by adding a reaction stopping solution (for example, sulfuric acid), and the absorbance of the reaction solution is measured. Sandwich ELI
The SA method can measure a trace amount with high sensitivity, the operation is simple and fast, and the sample preparation is simple. Moreover, the quantification and specificity in the low concentration range are extremely high.

When an antibody is used as a solid phase, it is characterized by having versatility that all substances exhibiting antigenicity can be measured as long as the antibody is obtained. The sample volume is usually 50μ
L. About 0.1 to 10 ^{- 1 5} M antigen substance can be quantified. Such non-competitive methods are excellent in sensitivity and accuracy, and also allow detection or measurement of trace levels of substances.

An anti-antigen-antibody complex antibody can also be used for the fluorescent antibody method using a fluorescent dye as a label.
Fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate can be used as fluorescent dyes. To observe immunofluorescence, a fluorescence microscope can be used. A flow cytometer can also be used for quantitative analysis of immunofluorescence.

A chemiluminescent method using a chemiluminescent agent as a label can also be performed. As such a chemiluminescent agent, 2,6-dimethyl-4- (N-succinimidyloxycarbonyl) phenyl 10-methylacridinium-9-carboxylate meso sulfate can be used. Chemiluminescence can be measured on a microtiter plate luminometer. The chemiluminescence reaction is started by injecting the reaction reagents, the plate is washed after incubation, and the chemiluminescence is measured.

The detection or measurement reagent used in these methods can be composed of a first antibody immobilized on a solid phase and a labeled second antibody. Further, such a reagent can be used as a kit including various auxiliary agents other than the antibody. The anti-antigen-antibody complex antibody can be used as any antibody. As such an auxiliary, a standard product for preparing a calibration curve, a substrate for measuring an enzyme activity, and the like can be combined.

In a detection or measurement method using an anti-antigen-antibody complex antibody, a substance having reactivity with an antibody, that is, a substance having antigenicity can be used as a target for the detection or measurement. Such a substance is an antigen or a hapten, and can also be an antibody. Environmental pollutants such as water pollutants such as microcystine and toxic substances shown in Examples can be detected or measured. Examples of the environmental pollutants include pesticides, environmental hormones, specific chemical substances, viruses, antigens or antibodies in infectious diseases, infectious pathogens, and the like.
For each substance, an anti-antigen-antibody complex antibody is prepared, and the antibody can be used in a method for detecting or measuring these substances depending on the specific reactivity of the antibody to the antigen-antibody complex. When constructing a non-competitive method, based on this method, high sensitivity, reliability at a low concentration range, and excellent specificity by utilizing two types of immune reactions, not only substances that are normally present, It is possible to detect or measure a substance that is present only in a trace amount in a specimen.

For example, with respect to the detection or measurement of microcystin (MC) shown in the Examples,
A sandwich ELISA using an antibody conjugate antibody is:
Its minimum detection limit is 2 pg / ml (FIG. 3), which is 10 times more sensitive than the previously reported competitive ELISA using the anti-microcystin antibody MC MAb. In addition, 2
Satisfactory with accuracy and precision at -100 pg / ml. According to the cross-reactivity data (Table 2)
In this sandwich ELISA method, the tertiary structure of Adda, which is a common structure of toxic microcystin and is an important structure (Harada et al., Chem. Res. Toxicol., 3, 473-4)
81 (1990); Nishiwaki-Matsushima et al., Jpn. J. Ca
ncer Res. 82, 993 (1991); Stotts et al., Toxicon 3
1, 783 (1993)), and at the same time, most of the toxic MCs were detected. Such extensive cross-reactivity is due to the competitive ELISA reported previously.
The same as in the case of A. Such a measurement method can be applied to direct measurement of MC in tap water samples where trace level monitoring is required.

The detection limit in the assay is governed by the background level. The lower the background level, the lower the detection limit (higher sensitivity). Secondary antibody 3F7, shown in the examples
Shows that dissociation (Kdiss) from the anti-microcystin antibody MC MAb alone (background) is about 300 times greater than that from the antigen-antibody complex with this antibody and antigen.
Twice faster. Thus, this reduces the background level, resulting in high sensitivity.

Further, from the viewpoint of the assay format, the anti-antigen-antibody complex antibody is used for sandwich ELISA.
Is advantageous over conventional sandwich ELISAs. ICM is an anti-antigen-antibody complex antibody
Ab is the antigen microcystin-LR (MCL
R) and does not react (FIG. 1C). This prevents the hook effect observed in co-incubation with antigen excess.

For use in the detection or measurement method of the present invention,
The anti-antigen-antibody complex antibody can be produced using a conventional antibody production method. A specific substance is selected as a substance having antigenicity, and an anti-antigen-antibody complex is prepared. It can be prepared by mixing and incubating a substance having antigenicity with an antibody. Incubation conditions are, for example, room temperature for 2 hours. A polyclonal antibody can be obtained by administering and immunizing the obtained anti-antigen-antibody complex, that is, the antigen-antibody complex, to an animal.

In the case of a monoclonal antibody, a hybridoma is formed by cell fusion of spleen cells from an animal thus immunized. By screening using RIA, ELISA, or the like, hybridomas producing antibodies specific to the antigen-antibody complex are selected based on their ability to bind to the antigen-antibody complex.

The animal to be immunized is determined by the tumor cell line used for cell fusion. Generally, rats and mice are frequently used. Among the mouse types, the BALB / c line, in which a tumor cell line that does not produce immunoglobulin is established, is often used.

The antigen-antibody complex was used in one mouse.
It is preferable to administer 10 μg to 300 μg at a time. Immunization is divided into several times, but the first immunization is often administered together with an adjuvant. As the adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, and the like can be used. Subsequent immunity is 2
Perform several times at 4-week intervals. In order to sufficiently stimulate the antigen, an antigen-antibody complex dissolved in physiological saline or the like is administered without using an adjuvant, particularly for the final immunization. Administration can be subcutaneous, intraperitoneal or intravenous. Spleen cells are subjected to cell fusion 2 to 4 days after the last administration.

The tumor cell line used for cell fusion includes P3-X63-Ag8-U, which does not produce immunoglobulin itself.
1, P3-NS-1, Sp2 / O-Ag14 (Sp2 / O), P3-X63-Ag8 ・ 653 (653)
Can be used.

The prepared spleen cells were mixed with tumor cells at a mixing ratio of 1
Mix at 0: 1 to 2: 1 and centrifuge to pellet. After loosening the pellet, cell fusion is performed with Sendai virus or polyethylene glycol (PEG). It is common to use 0.5-2 ml of a 40-60% solution of 1000-8000 PEG molecular weight.

After performing the cell fusion reaction for about 1 to 10 minutes, the DMEM medium or the RPMI1640 medium is added for 10 to 10 minutes.
The reaction is stopped by gradually adding 50 ml. Thereafter, the supernatant is removed by centrifugation. D containing 5-20% fetal bovine serum
MEM medium, IMDM medium and RPMI1640 medium are added and dispensed to the culture plate. As feeder cells,
It is preferable to add rat or mouse thymocytes, spleen cells and the like. Next, the medium is replaced with a HAT medium.

The cell culture supernatant in the well in which the growth of the hybridoma was confirmed was analyzed by various analytical methods (for example, ELISA
A, RIA, agglutination reaction, etc.) to select hybridomas producing the desired antibody. After obtaining the hybridoma, cloning is performed using a limiting dilution method or the like using mouse thymocytes as feeder cells. Once the clone is established, it can be cultured in vitro, or the antibody can be produced by in vivo culture such as injecting a hybridoma into a mouse or the like and obtaining a monoclonal antibody from ascites.

[0043]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Method 1- (1) Generation of anti-antigen-antibody complex antibody Microcystin (M) which is a toxin produced by cyanobacteria
Microcystin-LR (MCLR, having a molecular weight of about 100)
0) as antigen, anti-MC monoclonal antibody, M8H
5 and an antigen-antibody complex, 100 μg of which was mixed with Freund's complete adjuvant to form BALB / c
Mice (female, 10 weeks old) were administered subcutaneously, after which the antigen-antibody complex was mixed with Freund's incomplete adjuvant and administered subcutaneously. Further, a total of 50 μl of the antigen-antibody complex was intraperitoneally administered every 14 to 60 days for a total of 6 times. Three days after the final administration, the spleen of the mouse was subjected to cell fusion with Sp2 / 0 mouse myeloma, and the culture supernatant of the hybridoma was measured by screening ELISA.

1- (2) Screening of anti-antigen-antibody complex antibody ELISA Fc-specific anti-mouse Ig diluted 1: 120 with PBS
The G antibody was allowed to stand at 4 ° C. overnight and immobilized on each well of a 96-well microtiter plate. 0.5% in this well
A PBS solution containing gelatin (hereinafter referred to as a blocking buffer) was added to block the wells so that non-specific adsorption of proteins did not occur. The hybridoma culture supernatant obtained as described above was added to each well and incubated at room temperature for 2 hours.

Excess anti-mouse sites were blocked with normal mouse serum, and the biotinylated anti-MC monoclonal antibody Fab was added to each well in the presence or absence of excess MCLR.
(Prepared to 100 ng / ml with blocking buffer) for 2 hours at room temperature. 10,000 in PBS containing 0.5% gelatin, 0.005% Tween 20
After adding 0-fold diluted avidinated peroxidase and incubating at room temperature for 2 hours, 0.005% H ₂
A substrate solution of 0.1 M sodium acetate buffer (pH 5.0) containing O ₂ and 100 μg / ml TMBZ was added to start the enzyme reaction, and the reaction was stopped with 2N H ₂ SO ₄ . 450n
The absorbance at m was measured with a microtiter plate reader.

1- (3) Indirect ELISA The cross-reactivity of the anti-antigen-antibody complex antibody (IC MAb) to the anti-microcystin antibody (MC MAb) was tested by indirect ELISA. Antigen-antibody complex and MC
MAbs (1.7-53.3 nM) were immobilized on microtiter plates at various concentrations. Blocked with blocking buffer, 10 ng / ml biotinylated IC MAb was added to each well and incubated overnight at 4 ° C.
The bound biotinylated IC MAb was measured using avidinated peroxidase in the same manner as in the screening ELISA below.

1- (4) Competitive ELISA The cross-reactivity of IC MAbs to MCLR was determined by competitive EL.
Tested by ISA. P on microtiter plate
The antigen-antibody complex at 300 ng / ml in BS was immobilized and blocked with a blocking buffer. Serially diluted antigen-antibody complex or MCLR is biotinylated I
It was pre-incubated with CMAb (4 ng / ml) and reacted with the immobilized antigen-antibody complex. This biotinylated I
CMAbs were measured using avidinated peroxidase as in the screening ELISA.

1- (5) BIAcore Measurement The affinity constant (Kaff) of the IC MAb for the antigen-antibody complex and MCMA was determined using a biosensor assay (BIAcor
e, Pharmacia, Uppsala). The sensor chip (CM5) is an IC MAb (about 2,000R)
U). Antigen-antibody complex (150 mM NaC
1, containing 3.4 mM EDTA, 0.05% Tween-20
0 mM pH 7.4 50-500 nM in HEPES), MC MA
b (25-1,000 nM in PBS), control Ig
G1 (500 nM in HBS) showed an interaction on the fixed chip. The ratio of this association (Kass) and dissociation (Kdiss) was evaluated using BIAcore software.

1- (6) Construction of IC MAb Solid Phase Sandwich ELISA Using IC MAb as a solid phase, biotinylated M
The conditions of the sandwich ELISA using the C MAb are effective. Different concentrations of biotinylated MC MAb (1-100 ng / ml) in the presence and absence of excess MCLR and different concentrations of immobilized IC MAb (0.5, 1.0, 2....).
0, 4.0 μl) were tested. Under each solid phase concentration condition, the biotinylated MC was determined as a background value that was 5% or less of the absorbance when MCLR was present.
The highest concentration of MAb was chosen. The optimal combination of the solid phase and the concentration of the biotinylated MAb under the selected conditions is described below.

1- (7) Measurement procedure The microtiter plate was placed on an IC MAb (4.0 μg / m
Immobilized in l) and blocked with a blocking buffer. Various concentrations of MCLR were added to the same amount of biotinylated MC
Pre-incubation for 2 hours at room temperature with MAb (25 μg / ml) was added to the wells on which the IC MAb was immobilized. Incubate overnight at 4 ° C. and bind biotinylated MC as in screening ELISA
MAbs were measured.

1- (8) Measurement In measurement, a measured value from which non-specific binding was subtracted was calculated. Non-specific binding was determined using an incubated mixture containing the biotinylated MC MAb diluted in dilution buffer. The obtained standard curve was calculated from a graph with the logarithm of the amount of MCLR on the horizontal axis and the absorbance percentage on the vertical axis. Absorbance percent was expressed as the formula B / B100 × 100. B and B100 represent the average absorbance in the presence of each sample and the average absorbance of the highest standard amount. The fit between the standard curve and the calculated value of MC in the water sample was performed by using a second-order polynomial regression curve after log-logit conversion.

1- (9) Construction of Biotinylated Sandwich ELISA for IC MAb Using MC MAb as a solid phase, biotinylated I
Using the C MAb, all other conditions are 1-
The sandwich ELISAs used in (6) to (8) were also tested and measured.

1- (10) Recovery test This ELISA was used to evaluate that the four sources of tap water used in the recovery experiment were MC negative (<2 pg / ml). 2-100pg / m for this sample selected in Japan and China
l was added. This mixture is passed through a glass filter,
C content was measured directly by ELISA.

1- (11) Sandwich ELISA for MC in tap water IC MAb solid phase sandwich ELISA from Japan and China
A was used as the test material for A and was previously confirmed by competitive ELISA.
Seven tap water samples were selected. This sample was passed through a glass fiber filter, and the content of MC was adjusted to two ELs.
Measured directly by ISA. All data are MC as standard
It was expressed as the average of two measurements of LR.

2. Results 2- (1) Production of anti-antigen-antibody complex antibody By screening ELISA, not MC MAb,
A hybridoma producing a monoclonal antibody called IC MAb 3F7 was selected for its ability to bind to the antigen-antibody complex. A volume dependent binding curve for 3F7 was seen for the antigen-antibody complex but not for the MC MAb (FIG. 1A). Furthermore, the specific binding of 3F7 to the antigen-antibody complex that characterizes the MAb was determined by this ELISA and BI
Confirmed by Acore assay. First, the binding ability of the immobilized complex and biotinylated 3F7 to the MC MAb for the purpose of testing the cross-reactivity of this 3F7 to the MC MAb was measured by indirect ELISA. As a result, 3F7
Shows volume-dependent binding to the antigen-antibody complex, MC
Weak binding to MAb (FIG. 1B). Second, MC
Cross-reactivity of 3F7 to LR was measured in a competition ELISA. Free MCLR is the antigen-immobilized
Even at high concentration (100 nM) testing against antibody conjugates
There was no inhibition of F7 binding (FIG. 1C). On the other hand, the antigen-antibody complex was found to be less than 3/100 at this high concentration.
F7 binding was inhibited (FIG. 1C). Third, the dynamic constant of 3F7 for the antigen-antibody complex and MC MAb was
Determined by core assay. Average Kass of 3 concentration tests
Is 1.56 x 10 for the antigen-antibody complex
⁵ M ^{- 1.} S ^{- 1,} for the MC MAb, 1.49 × 10 ⁵
M ^{- 5} . S ^{- 1} was. On the other hand, the average Kdiss the antigen - for the antibody complex, 1.09 × 10 ^- For ^{1, MC MAb, 329 × 10} - - 4 S 4 S - was ¹ (Table 1). As a result, the Kaff of 3F7 was MC MAb (4.59 × 1 on average).
0 ⁶ M ^{- 1} antigen than to) - antibody complex (mean 14.
5 × 10 ⁸ M ^{- 1)} it is found that approximately 100 times higher. This difference is due to the low dissociation constant (Table 1).

[0056]

[Table 1]

2- (2) IC MAb (3F7) immobilized sandwich ELISA 2- (2) -a) Standard curve and detection limit From the six measurements performed on different days, the standard curve of MCLR is shown in FIG. Type2. This curve is 1-500 pg / m
1 showed a typical sigmoidal response from the MCLR concentration. This curve shows high reproducibility between wells and plates as indicated by low SD. The detection limit of this ELISA was 2 pg / ml indicating a concentration showing an absorbance twice that of the background value. These measurements are two EL
This corresponds to a measured value of the absorbance of the background value in ISA of 8 SD or more. In addition, MC is immobilized MC M
A different system of assays (2) that reacts with Ab and forms an antigen-antibody complex that is then detected by biotinylated 3F7
The sensitivity of the step sandwich ELISA was measured. This type is slightly less sensitive than the conventional form (5pg / m
l).

2- (2) -b) Specificity To determine the specificity of this ELISA, various MC
Was measured for cross-reactivity. Table 2 shows the results. The concentration of each compound required for 50% of the most efficient binding was determined by MC
It was compared with the concentration of LR. M as important as MCLR
CRR MCRR and MCYR were cross-reacted in this ELISA. MCLR, 3-desmethyl MCLR, MCLR GSH with a structure related to MC containing Adda protein
The ELISA showed a similar cross-reactivity to the conjugate, Me-MCLR. In contrast, the geometrical isomers of the Adda moiety, 6 (Z) -MCLR and 6 (Z) -MCRR, which are unique and minor constituents found in the environment, are represented by M
Cross-reactivity was less than CLR (<8% MCLR).
Thus, most of the MCs found in the environment can be measured equally by the developed ELISA.

[0059]

[Table 2]

2- (2) -c) Accuracy In order to measure the accuracy of ELISA quantification, a standard MCLR was used.
8 serial dilutions of the same plate in different wells or
Measurements were taken on triplet plates on different days.
Type 1 is a case where the IC MAb is biotinylated, and Type 2 is a case where the IC MAb is immobilized. As shown in Table 3 below, the measurement error was small for both types, but 2
In the low concentration range of ５5 pg / ml, Type 2 showed slightly better results than Type 1. For Type 1 and Type 2, the measurement error was small at 5 to 100 pg / ml and 2 to 100 pg / ml, respectively. In addition, CVs (coefficients of variation) in the same measurement range are 0.3 to 12.3% and 4.9, respectively.
It was a small value of about 10.8%. From these results, E
The quantification range of LISA is 5 to 100 pg / m for Type 1
For l, type 2, 2 to 100 pg / ml was selected.

[0061]

[Table 3]

2- (3) Recovery of MC addition from tap water sample A recovery test of MCLR in tap water was performed. Table 4 shows the results. A known concentration of MCLR (2-100 pg / ml) was added to 4 samples obtained from various places, and ELISA was performed.
It measured using. The recovery of the four tests was CV2.1-8.
At 8%, the recovery was 90-106%. Therefore,
This addition recovery can be fully reproduced. The respective results indicate that the presence of normal substances in tap water does not interfere with this assay.

[0063]

[Table 4]

2- (4) ELI of MC in tap water sample
The SA17 sample was the test material for the sandwich ELISA, which was previously confirmed in a competitive ELISA. In the sandwich ELISA, 2 out of 17 samples showed MC positivity in the range of 4.9-13 pg / ml, and competitive E
According to LISA, one sample out of 17 samples is 21 pg / ml
At a level of MC positive. In addition, sandwich E
Two positive samples by LISA were evaluated by a spike recovery test. A known concentration of MCLR (100 pg / ml) was added to 9 times the amount of the positive sample, and measured by this ELISA. The average recovery of this sample was 97%. From these results, the possibility of false positives is small.

[0065]

The detection or measurement method of the present invention can be applied to various detection or measurement methods. It is also possible to establish a non-competitive immunization method for small molecules such as environmental pollutants, which has been impossible so far, and it is possible to significantly expand the application range of a non-competitive method such as sandwich ELISA which is excellent in principle. it can.

[Brief description of the drawings]

FIG. 1 shows the ability of ELISA to identify the antibody MAb produced between the antigen-antibody complex and the anti-microcystin antibody (MC MAb) or microcystin-LR (MCLR). (A) Titering of IC MAbs against antigen-antibody complexes and MC MAbs in screening ELISA Various concentrations of IC MAbs were added to microtiter wells pre-coated with sheep anti-mouse antibody. After blocking with normal mouse serum, a biotinylated Fab fragment of MC MAb (100 ng / ml) was added in the presence (solid circle) or absence (open circle) of excess MCLR. Biotinylated MAbs are used to convert HRP-SA to a second
It was detected as a reagent. (B) Direct binding of IC MAbs to coated antigen-antibody complexes and coated MC MAbs in indirect ELISA Microtiter plates were coated with various concentrations of antigen-antibody complexes or MC MAbs (0.208-5).
(3.3 nM) and then reacted with biotinylated IC MAb (10 ng / ml). Bound biotinylated IC MAbs
Was detected using HRP-SA as the second reagent. (C) Effect of MCLR on binding of IC MAb to coated antigen-antibody complex in competitive ELISA Microtiter plates were coated with antigen-antibody complex (300 ng / ml) and then biotinylated IC MAb
(4 ng / ml) was added to each well along with equal volumes of various concentrations of MC (open circles) or antigen-antibody complex (solid circles). The bound biotinylated MAb is converted to H2 as a second reagent.
Detection was performed using RP-SA. Absorbance (450nm)
Expressed as a percentage of absorbance in the absence of competitor.

FIG. 2 is a standard curve of a sandwich ELISA based on IC MAb for MCLR. Circles represent the mean ± SD absorbance values (450 nm) of the results of measurements by another assay performed on another day. S. D. Vertical lines may not be visible due to the markings. Each point was performed three times in each assay. The numbers next to the circles represent the average of the SD values between the wells of six separate assays. Type1 is I
When C MAb is biotinylated, Type 2 is ICMA
This is the case where b was immobilized.

Claims (4)

[Claims] 1. A method for detecting or measuring an environmental pollutant using an antibody against a complex of the environmental pollutant and an antibody against the environmental pollutant. 2. The method according to claim 1, wherein the environmental pollutant is microcystin. 3. A reagent for detecting or measuring environmental pollutants, comprising an antibody against a complex of an environmental pollutant and an antibody against environmental pollutants. 4. A kit for detecting or measuring environmental pollutants, comprising the reagent for detecting or measuring environmental pollutants according to claim 3.