Hybridoma, an antibody resistant to an interfering substance, and a kit for immunological analysis

The present invention provides for a method of selecting an antibody against a target substance to be measured, which comprises selecting the antibody against the target substance by antigen-antibody reaction in the presence of a substance interfering with the antigen-antibody reaction. That is, an antigen and a labeled antigen are reacted with the antibody in the presence of an interfering substance, such as an environment pollutant, and on the basis of the degree of reaction thereof, the antibody against the target substance, which is highly resistant to the interfering substance, is selected. Thereby, even if a test sample is contaminated with a substance interfering with antigen-antibody reaction, the antibody, which is highly resistant to the substance interfering with antigen-antibody reaction, is not influenced by the interfering substance and gives a correct value in the quantification. The present invention further provides for antibodies, which are highly resistant to the substance interfering with the antigen-antibody reaction, and hybridomas producing the antibodies.

FIELD Of THE INVENTION

This invention relates to an antibody having a property of reacting with an antigen while being hardly influenced even in the presence of an substance interfering with antigen-antibody reaction (hereinafter also called an interfering substance), that is, an antibody resistant to or tolerant of an interfering substance, a method of selecting the antibody, a hybridoma producing the antibody, and use of the antibody.

BACKGROUND OF THE INVENTION

In recent years, in the fields of environmental quantification etc., immunoassays (hereinafter abbreviated to IA) are frequently used because expensive devices and proficiency in the operation are not necessary.

However, the antibody used in IA, which acts specifically on a target substance to be measured, is a high-molecular protein, and thus the activity of the antibody is influenced by various substances exerting an influence on the protein, and as a result, quantifications are inaccurate in some cases.

On one hand, prior to measurement of a target substance existing in a very small amount in the order of ppb (part per billion) and ppt (part per trillion), concentrating the sample at high degrees may be necessary, and in such cases, the sample concentrated by a solid-phase extraction method (hereinafter also called solid-phase concentration) or by extraction with a solvent (hereinafter also called solvent concentration) is used to measure the target substance.

However, the test sample contains a wide variety of compounds in addition to the target substance, and thus when the test sample is concentrated by solid-phase concentration or solvent concentration, a compound exerting an influence on the antibody, that is, a substance interfering with the antigen-antibody reaction is also concentrated, and as a result, the activity of the antibody is influenced, resulting in abnormal values of quantifications in some cases.

One object of this invention is to provide a method of selecting an antibody which in measurement of the antigen by IA, is hardly influenced by an interfering substance with which a sample may possibly be contaminated, whereby occurrence of incorrect measurement in IA is prevented.

Another problem of this invention is to provide an IA kit with little occurrence of incorrect values by utilizing an antibody tolerant of or resistant to a substance interfering with the antigen-antibody reaction.

SUMMARY OF THE INVENTION

The present inventors made extensive study for establishing a method of detecting and measuring various substances by IA with minimum influence of contaminated interfering substances. As a result, the present inventors found that, for selection of an antibody used in IA, an antigen and a labeled antigen are reacted with the antibody in the presence of an interfering substance such as an environment pollutant, a decomposed product thereof, a germicidal disinfectant or a solvent, and on the basis of the degree of reaction thereof, the antibody against the target substance highly resistant to the interfering substance is selected, thereby making it possible to detect and measure a sample even concentrated at a high degree because of a small amount of a target substance to be measured. As a result of further extensive study on the basis of such finding, the present inventors have completed the invention.

That is, this invention provides:

(1) A method of selecting an antibody against a target substance to be measured which comprises selecting the antibody against the target substance by antigen-antibody reaction in the presence of a substance interfering with the antigen-antibody reaction.

(2) The method of selecting an antibody according to (1), wherein the target substance to be measured is an environmental pollutant.

(3) The method of selecting an antibody according to (1), wherein the target substance to be measured is a hormone.

(4) The method of selecting an antibody according to (1), wherein the antibody against the target substance is a monoclonal antibody.

(5) The method of selecting an antibody according to (1), wherein the substance interfering with the antigen-antibody reaction is an environmental pollutant, a decomposed product thereof, a germicidal disinfectant or a solvent.

(6) The method of selecting an antibody according to (5), wherein the environmental pollutant as a substance interfering with the antigen-antibody reaction is a surfactant, environment water, a concentrate thereof or a humic substance.

(7) The method of selecting an antibody according to (6), wherein the surfactant is an anionic surfactant, a cationic surfactant or a nonionic surfactant.

(8) The method of selecting an antibody according to (6), wherein the environment water or a concentrate thereof is river water, lake water, seawater, water in a tap water-treatment process, water in a waste water treatment process or a concentrate thereof.

(9) The method of selecting an antibody according to (5), wherein the germicidal disinfectant is a chlorine agent.

(10) The method of selecting an antibody according to (5), wherein the solvent is an alcohol, nitrile, ketone or ester.

(11) The method of selecting an antibody according to (1), wherein a polyclonal antibody [obtained from an animal immunized with a target substance to be measured or a conjugate between a hapten of the target substance to be measured and a carrier protein as the antigen, or a monoclonal antibody obtained by culturing a hybridoma producing a monoclonal antibody against a target substance to be measured or a conjugate between a hapten of the target substance to be measured and a protein, and being obtained by fusing myeloma cells and spleen cells or lymph node cells from an animal immunized with the target substance to be measured or a conjugate between a hapten of the target substance to be measured and a carrier protein as the antigen, is reacted with an antigen-enzyme conjugate (labeled antigen), the target substance to be measured or a conjugate between a hapten of the target substance to be measured and a carrier protein (antigen) in the presence of an substance interfering with the antigen-antibody reaction, and on the basis of the degree of reaction thereof, an antibody resistant to the substance interfering with the antigen-antibody reaction is selected.

(12) A hybridoma producing the monoclonal antibody resistant to an substance interfering with the antigen-antibody reaction, which was selected according to the method of (11).

(14) An antibody resistant to a substance interfering with the antigen-antibody reaction, which was selected by the method of (11).

(15) A monoclonal antibody resistant to an interfering substance, which was produced by the hybridoma of (12) or (13).

(16) A kit for immunological analysis of a target substance to be measured, which comprises the antibody resistant to a substance interfering with the antigen-antibody reaction of (14) or (15).

(17) A kit for immunological concentration of a target substance to be measured, which comprises the antibody resistant to a substance interfering with the antigen-antibody reaction of (14) or (15).

EXAMPLES

The present invention is described in more detail by reference to the following Examples.

1. Preparation of a Monoclonal Antibody

1-1 Preparation of a Hapten

(1) Preparation of a Hapten for Ethynylestradiol Antibody

1.0 g of ethynylestradiol (EE2) and 0.76 g of sodium methylate were dissolved in 35 ml of ethanol, and then 0.41 g of monochloroacetic acid was further added to the mixture. The mixture was heated under reflux for 22 hours, concentrated under reduced pressure and partitioned by adding about 100 ml each of water and ethyl acetate. The aqueous layer was washed with 50 ml of ethyl acetate, and was acidified with conc. hydrochloric acid (pH 1–2). The solution was extracted with 100 ml and 50 ml of ethyl acetate, and the organic layer was washed with 30 ml of saturated sodium chloride solution and dehydrated over sodium sulfate anhydride. The solution was concentrated under reduced pressure, and the concentrate was left at −20° C. for 2 days, whereby it was partially crystallized. A part of the crystals was recovered and used as seed crystals. The concentrate was dissolved in a small amount of acetone, and crystals were precipitated by adding hexane and the seed crystals. The recovered crystals were dried under reduced pressure, whereby the desired product EE2-3-carboxymethyl ether (EE2-3CME) was obtained.

(2) Preparation of a Hapten for 17β-Estradiol Antibody

1.0 g of 17β-estradiol (E2) and 0.82 g of sodium methylate were dissolved in 35 ml of ethanol, and 0.45 g of monochloroacetic acid was further added to the mixture. The mixture was heated under reflux for 2 days, concentrated under reduced pressure and partitioned by adding about 300 ml of water and about 200 ml of ethyl acetate. The aqueous layer was washed with 50 ml of ethyl acetate, and the aqueous layer was acidified with conc. hydrochloric acid (pH 1–2). The solution was extracted twice with 100 ml of ethyl acetate, and the organic layer was washed with 30 ml of saturated sodium chloride solution and dehydrated over sodium sulfate anhydride. The solution was concentrated under reduced pressure, and the concentrate was dissolved in a small amount of acetone, and crystals were precipitated by adding isopropyl ether (IPE). The recovered crystals were washed with IPE and dried under reduced pressure, whereby the desired product E2-3-carboxymethyl ether (E2-3CME) was obtained.

(3) Preparation of a Hapten for Estrone Antibody 20 g of estradiol-3,17-diacetate (E2-3,17-diAce) was dissolved in 25 ml of acetic acid, and ⅘ part of a mixture of 9 g of chromic acid anhydride, 27 ml of acetic acid and 4 ml of water was gradually added to the above solution under cooling. The mixture was stirred overnight, then water was added, and the reaction solution was extracted with chloroform, washed with a solution of potassium carbonate, dehydrated over sodium sulfate and concentrated under reduced pressure. The concentrate was dissolved in benzene, subjected to column chromatography (Wako gel 2w) and eluted with hot benzene to separate crude 6-oxo-E2-3,17-diAce (the starting materials were removed under monitoring with TLC). This substance was dissolved in 2-methoxy-ethanol, and an equal volume of 20 % NaOH was added, and the mixture was reacted at 100° C. for 1 hour. The reaction solution was acidified with dilute hydrochloric acid, eluted with ethyl acetate, dehydrated over sodium sulfate, concentrated under reduced pressure, and 6-oxo-E2 was recrystallized from methanol. 300 mg of 6-oxo-E2 was dissolved in 2 ml of ethanol, 50 mg of fine powder of potassium carbonate was added and 50 μl of benzyl bromide was added. The mixture was stirred at 70° C. for 2 hours and the concentrate was recrystallized from methanol to obtain 6-oxo-E2-3-benzyl ether. The 6-oxo-E2-3-benzyl ether was dissolved in methanol, and carboxymethoxyl amine-½ HCl and sodium methoxide (1.2 moles) were dissolved in a small amount of water, and then diluted with methanol and added to the above solution. The mixture was reacted at room temperature for 1 hour, and after ⅔ part of the reaction solution was evaporated, the solution was acidified with water and HCl, extracted with ethyl acetate and dehydrated over sodium sulfate. The product was dissolved in hexane, then purified by column chromatography on silica gel followed by elution with methanol, and recrystallized from methanol. 0.6 g of the recrystallized substance was dissolved in 2 ml of acetone, and half of a mixed solution of 1 g of chromic acid anhydride, 5 ml of acetic acid and 1 ml of water was added, and after the exothermic reaction was finished, the reaction solution was extracted with ethyl acetate and evaporated to dryness to obtain 6-oxo-E1-3-benzyl ether. 400 mg of 6-oxo-E1-3-benzyl ether was dissolved in 5 ml of methanol, and 100 mg of 5% Pd-C was added, and the mixture was stirred while introducing hydrogen. After the reaction for 1 hour, the reaction solution was evaporated to dryness and recrystallized from methanol, whereby the desired product 6-oxo-E1-6 carboxymethyl oxime (E1-6CMO) was obtained.
1-2 Preparation of an Immunogen

0.1 mol of each hapten out of the 3 haptens obtained in the above 1-1, 0.14 mol of water-soluble carbodiimide, and 0.14 mol of N-hydroxysuccinimide were reacted overnight in 2 ml of dimethyl sulfoxide to form an activated ester. Then, 10 mg of keyhole limpet hemocyanin (KLH) was dissolved in 0.13 mol of sodium bicarbonate (NaHCO3) solution, and 200 μl of the activated ester was added and reacted overnight at 4° C. The reaction solution was dialyzed against Dulbecco's phosphate buffer (PBS) to remove unreacted reagents, and frozen and stored as an immunogen.

Each immunogen obtained in the above 1-2 was dissolved at 500 μg/ml in PBS, and added to an equal volume of Freund adjuvant or RIBI adjuvant system. The mixture was sufficiently emulsified and then administered subcutaneously to BALB/C mice (female) in a dose of 100 μg/mouse, and immunization with a booster was conducted at 2-weeks (Freund) or 3-weeks (RIBI) intervals. After the immunization with a booster was conducted 5 to 6 times, a mouse showing the maximum serum antibody titer was intravenously given the same immunogen (50 μg/0. 1 ml PBS/mouse) to complete the final immunization.

1-4 Cell fusion

From the mouse subjected to the final immunization in the above 1-3, the spleen was extracted 3 days after the final immunization to prepare spleen cells. Mouse myeloma cells separately cultured and the spleen cells were contacted in the ratio of 1:5 in the presence of polyethyleneglycol with average molecular weight 4000, whereby cell fusion was effected to give fused cells (hybridoma). This hybridoma was suspended in HAT medium, put to each well of a 96-wells microplate and cultured in a CO2gas incubator (37° C., 5% CO2).

1-5 Screening of the Hybridoma

(1) Preparation of an Assay Plate

A goat anti-mouse IgAGM antibody (#55461 produced by ICN/Cappel) was dissolved at 50 μg/ml in PBS, added at 100 μl/well to a microplate and reacted at 4° C. overnight. The microplate was washed twice with 300 μl/well of T-PBS (PBS containing 0.05% Tween 20), and Block Ace (Snow Brand Milk Products Co., Ltd., Tokyo) diluted 4-fold with PBS was added at 200 μl/well to the plate. After the sample was reacted at 4° C. overnight or longer and stored in a refrigerator until use.

0.1 mol of each hapten out of the 3 haptens obtained in the above 1-1, 0.14 mol of water-soluble carbodiimide, and 0.14 mol of N-hydroxysuccinimide were reacted overnight in 2 ml of dimethyl sulfoxide to form an activated ester. Then, 10 mg of horseradish peroxidase (HRP) was dissolved in 10 ml of 0.13 mol sodium bicarbonate (NaHCO3) solution, and 15 μl of the activated ester was added and reacted overnight at 4° C. Unreacted reagents were removed by ultrafiltration, and the solution was stored at a concentration of 3 mg/ml in a refrigerator.

100 μl of the culture on a well wherein cell growth had been confirmed in the microplate to which the cells had been put in the above 1-4 was added to the assay plate (after washing twice with 300 μl/well of PBS (or T-PBS) before use) prepared in (1). After the reaction for 1 hour at room temperature, the plate was washed 3 times with 300 μl/well of T-PBS, and the labeled antigen prepared in 1-5 (2) to the antibody against the target substance was diluted at 5000-fold with T-PBS and added to the microplate. After the reaction for 1 hour at room temperature, the plate was washed 3 times with 300 μl/well of T-PBS, and a TMB peroxidase substrate kit (Nippon Bio-Rad Laboratories, Tokyo, #172-1066: hereinafter referred to as “the coloration substrate”) was added in a volume of 100 μl/well. After the reaction for 30 minutes at room temperature, the coloration reaction was stopped by adding 100 μl/well of 1 N phosphoric acid. The absorbance at a wavelength of 450 nm was read, and cells showing an absorbance of higher than 1 were then put on a 24-wells microplate for scaled up cultivation.

Out of the cells put to a 24-wells microplate in the above 1-5 (3), a culture of cells on a well wherein sufficient cell growth had been confirmed was added to wells in a volume of 100 μl/well in the assay plate (after washing twice with 300 μl/well of PBS (or T-PBS) before use) prepared in (1). After the reaction at room temperature for 1 hour, the plate was washed 3 times with 300 μl/well of T-PBS, and the female hormone as the subject of quantification (1 ng/ml in 10% methanol (MeOH)) or 10% MeOH only (control) and the labeled antigen diluted at 5000-fold with T-PBS to the antibody against the target substance were mixed in equal volumes and added to the microplate. After the reaction at room temperature for 1 hour, the plate was washed 3 times with 300 μl/well of T-PBS, and the coloration substrate was added in a volume of 100 μl/well. After the reaction for 30 minutes at room temperature, the coloration reaction was stopped by adding 100 μl/well of 1 N phosphoric acid. The absorbance was read at a wavelength of 450 nm, and those cells confirmed to reduce the absorbance by 20% or more relative to that of the control in the presence of the female hormone were subjected to cloning in a usual manner, and candidates for hybridomas producing the desired antibody were obtained as shown below.

1-6 Preparation of a Purified Antibody

The cell culture supernatant was fractionated with 45 to 50% saturated ammonium sulfate and then subjected in a usual manner to protein G affinity chromatography, while the mouse ascites fluid was directly subjected to protein G affinity chromatography, to give each purified antibody.

2. Selection of an Antibody Resistant to an Interfering Substance

The culture supernatant obtained in the above 1-5 (4) was used to conduct a test on resistance of each antibody to an interfering substance. As the interfering substance, a surfactant and a humic substance which occur at a high concentration in the environment and can be concentrated together with a substance to be measured in the step of pre-treating the substance to be measured were used.

Experimental Method 1

According to 1-5 (4), the degree of dilution of the culture supernatant, at which the antibody attained the highest sensitivity in quantification of the female hormone, was determined. Then, to an anti-mouse IgG immobilized plate to which the antibody in the supernatant had been bound at that concentration was added 100 μl of a mixture consisting of equal amounts of the following sample and the labeled antigen (diluted 5000-fold with T-PBS) to the antibody against a target substance to be measured. The sample used was a solution containing or not containing the female hormone as the subject of quantification at a concentration of 0.5 μg/L in (0, 10, 100, 1000 mg/L in 10% MeOH) sodium linear-alkylbenzene sulfonate (LAS), alkylphenol ethoxylate (APE) or alkyl ethoxylate (AE) or in (0, 1, 10, 100 mg/L in 10% MeOH) sodium humate. According to the above 1-5 (4), ELISA quantification was conducted, and the concentration of the female hormone in each sample was calculated by comparison with a standard curve prepared by standard solutions of known concentrations. The calculated value was divided by the concentration of the female hormone added (0.5 μ/L) and multiplied by 100 to calculate the recovery.

Results

Out of the anti-EE2 antibodies, the anti-EE2-227 antibody selected in this method showed about 50 to 200% recovery even if the concentration of the interfering substance was about 10 to 100 times as high as that for the anti-EE2-8 antibody (control) obtained without using this selection method, and thus a mouse hybridoma EE2-227 strain (FERM BP-7567) producing the antibody highly resistant to the interfering substance was selected.

Out of the anti-E2 antibodies, the anti-E2-73 antibody selected in this selection method showed 50 to 200% recovery even if the concentration of the interfering substance, the surfactant was about 10 to 100 times as high as that for the 3 antibodies (E2-CC, E2-NG and E2-RB) used in the commercial ELISA kit and obtained without using this selection method, so as a mouse hybridoma producing the antibody highly resistant to the interfering substance, the E2-73 strain (FERM BP-7569) was selected.

Further, the anti-E1-420 antibody selected in this selection method had high resistance to the surfactant, which was comparable with that of the anti-E2-73 antibody, so as a mouse hybridoma producing the anti-E1-420 antibody, the E1-420 strain (FERM-BP-7568) was selected.

Preparation of an EE2-ELISA Kit

(1) Preparation of an “Immobilized Plate”

A goat anti-mouse IgG antibody (Code No. 55479, ICN/Cappel Ltd.) dissolved in Dulbecco's PBS(−) (Code No. 041-20211, Wako Pure Chemical Industries, Ltd.) was pipetted into an immobilizing plate (Costar, EIA/RIA plate strip 8, #2592) in an amount of 0.5 μg/well, and the plate was left at 4° C. overnight, and then washed 3 times with 300 μl of washing solution (T-PBS). A blocking solution (1% BlocAce by Snow Brand Milk Products Co., Ltd. +0.05% Slaoff 72N (Slamonia 28 N) by Takada Chemical Industries, Ltd. in PBS) was added in a volume of 200 μL/well, and the plate was left at 4° C. overnight, then washed 3 times with 300 μL of washing solution (T-PBS). Then, the ethynylestradiol antibody (EE2-227) dissolved in PBS (containing 0.05% Slaoff 72N +0.1% BSA) was added in an amount of 0.002 μg/well, and the plate was left at 4° C. overnight, and then washed 3 times with 300 μl of washing solution (T-PBS). A blocking solution (1% BlocAce by Snow Brand Milk Products Co., Ltd. +0.05% Slaoff 72N by Takada Chemical Industries, Ltd. in PBS) was added in a volume of 200 μL/well, and the plate was left at 4° C. overnight, and the whole solution was sucked with an aspirator, and the residual solution was removed by tapping. The immobilizing plate was dried, placed in an aluminum bag, degassed by a vacuum dryer, sealed and stored in a refrigerator at 2 to 8° C. (2) Preparation of “EE2 standard stock solution”

Preparation of Stock Solution 1 (1000 mg EE2/L):

100 mg of an ethynylestradiol standard (content: 100%) (Code No. 055-05011, Wako Pure Chemical Industries, Ltd.) was accurately weighed, placed in a 100 ml measuring flask and adjusted to 100 ml with methanol.

Preparation of Stock Solution 2 (10 mg EE2/L):

Accurately 1 ml of the stock solution 1 was introduced via a constant delivery pipette into a 100 ml measuring flask and adjusted to 100 ml with methanol.

Preparation of a Standard Stock Solution (0.1 mg EE2/L):

Accurately 1 ml of the stock solution 2 was introduced via a constant delivery pipette into a 100 ml measuring flask and adjusted to 100 ml with methanol and distilled water to prepare 10% methanol solution. 4 ml of the standard stock solution was placed in a suitable vessel and stored in a refrigerator at 2 to 8° C.

26 mg of water-soluble carbodiimide (WSC for peptide synthesis, Code No. 348-03631, by Wako Pure Chemical Industries, Ltd.) and 16 mg N-hydroxysuccinimide (NHSI for peptide synthesis, Code No. 089-04032, by Wako Pure Chemical Industries, Ltd.) were dissolved in 2 ml of dimethyl sulfoxide (DMSO, special grade, by Wako Pure Chemical Industries, Ltd.). An aliquot thereof, 0.5 ml, and 10 mg of the hapten (EE2-3CME) prepared in Example 1 were dissolved in 1 ml of DMSO and reacted at room temperature overnight. The resultant reaction solution, 13 AL, and 10 mg of peroxidase (POD for EIA, Code No. 814393, Boehringer) previously dissolved in 10 ml of 1.1% NaHCO3were reacted at 4° C. overnight under stirring, and the resultant reaction solution was filtered through an ultrafiltration membrane having a fractionation molecular weight of 30,000, and then adjusted finally to 30 ml with PBS containing 0.05% Slaoff 72N to give an antigen-enzyme conjugate solution.

200 μl of the solution was pipetted into a suitable vessel, then capped and stored in a refrigerator at 2 to 8° C.

13.4 mg of 5,5′-tetramethylbenzidine (TMBZ, for test and research, Code No. 346-04030 1, by Wako Pure Chemical Industries, Ltd.) was dissolved in 1 ml of dimethyl formamide (DMF, special grade, Code No. 045-02916, by Wako Pure Chemical Industries, Ltd.), and then mixed with 100 ml of 0.1 M sodium acetate buffer (pH 5.5) containing 0.1 g/L TWEEN-20 ((Atras Chemical Industries, Inc.): Poly(oxyethylene) sorbitan monolaurate), pipetted into suitable brown vessels in a volume of 10 ml/vessel, capped and stored in a refrigerator at 2 to 8° C.

30% aqueous hydrogen peroxide (Code No. 081-04215, special grade, Wako Pure Chemical Industries, Ltd.) was diluted to 0.1 g/L with distilled water, pipetted into suitable vessels in a volume of 5 ml/vessel, capped and stored in a refrigerator at 2 to 8° C.

1 N phosphoric acid solution was prepared, pipetted into suitable vessels in a volume of 15 ml/vessel, capped and stored at room temperature.

The kit constituents in (1) to (8) prepared above are packed in a box to complete an EE2-ELISA kit.

Quantification by the EE2-ELISA Kit

Quantification Using the EE2-ELISA Kit Prepared in Example 2 was as Follows:

The EE2 standard stock solution (0.1 mg/L solution in 10% methanol) was diluted with methanol and distilled water to prepare EE2 standard solutions at necessary concentrations (for example, 0, 0.05, 0.1, 0.3, 1.0, 3.0 μg/L in 10% methanol).

7 mL of the “antigen-enzyme conjugate solution” was added to 14 μL of the “antigen-enzyme conjugate solution” to prepare an “antigen-enzyme conjugate solution”.

100 μL/well of a quantification sample or the “EE2 standard solution” (both are 10% methanol solutions) and 100 μL/well of the “antigen-enzyme conjugate solution” were mixed on a “mixing microplate”.

The “mixed solution” prepared in (3) was pipetted into the “anti-EE2 monoclonal antibody-immobilized plate” in a volume of 100 μl/well and reacted at room temperature for 60 minutes.

During the antigen-antibody reaction, the “6-fold conc. washing solution” and distilled water were mixed in the ratio of 1:5, to prepare a “washing solution”.

(6) Removal of Unreacted Materials

The antigen-antibody reaction solution was discarded, and each well was washed 3 times with 300 μL/well of the “washing solution”.

The “coloration substrate solution-A” and “coloration substrate solution-B” were mixed in the ratio of 3:1, to prepare a “coloration reagent”.

(8) Coloration Reaction/Stop of the Reaction

The “coloration reagent” prepared in (7) was added in the volume of 100 μL/well, the mixture was reacted at room temperature for 30 minutes, and the “coloration-stop solution” was added in a volume of 100 μL/well to stop the reaction.

(9) Colorimetry and Calculation of the Concentration

The absorbance was measured at a wavelength of 450 nm with a plate reader, and the concentration of EE2 in the sample was determined from a standard curve.

Preparation of a Standard Curve by the EE2-ELISA Kit

A standard curve prepared according to Example 3 is shown inFIG. 1. The EE2 concentration was plotted on the ordinate [sic], and the ratio of the absorbance at each EE2 concentration to the absorbance at the concentration of 0 μg/L EE2 (degree of inhibition, B/BO %) was plotted on the abscissa [sic]. From this result, it was estimated that the quantification range of EE2 lay in about 0.05 to 3 μg/L.

For the estrogens shown in [Table 2], the respective standard curves were prepared in the same manner as in Example 4, and the EE2 concentration (IC50) at which the degree of inhibition was 50% was determined and the cross-reactivity was determined from the following equation, and the results are shown in [Table 2].
Cross-reactivity (%)=IC50of estrogen determined/IC50of EE2×100

The anti-EE2-227 antibody had high specificity for EE2, and hardly reacted with other estrogens.

Comparison Between LC-MS/MS and ELISA in Quantifications of Waste Water Treatment Process (WWTP) Water

10 L of WWTP water was filtered through a glass fiber filter and adjusted to pH 5 with 1 M acetate buffer (pH 5.0). Then, the water was passed through a C-18 solid phase cartridge previously conditioned with 5 ml methanol and 10 ml distilled water, and then the cartridge was washed with 5 ml each of distilled water and hexane. EE2 was eluted with 5 ml dichloromethane from the C-18 solid phase cartridge, and the eluent was evaporated into dryness in a nitrogen stream (40° C.), then dissolved in 10 % MeOH, and the concentration of EE2 in the sample was quantified according to Example 3.

For ELISA quantification, a commercial ELISA kit for EE2 (#04330, by R-Biopharm GmbH, German) was also used as the control, and quantification was carried out according to the accompanying instructions.

Further, a sample treated in the same manner was also analyzed by LC-MS/MS according to the method of Tsujimura et al. (Memorial Lecture in the 50th anniversary of the founding of Chemicals Evaluation and Research Institute, Japan, and Abstracts of Lectures in the 4th Research Presentation, p.17–26, 1999), and the result was compared with that in ELISA. The results are shown in [Table 3].

The quantification by the ELISA kit using the EE2-227 antibody obtained and selected as the interfering substance-resistant antibody in this selection method was similar to that by LC-MS/MS, but the quantification by the commercial ELISA kit using an antibody obtained without using this selection method was about 8 times as high as that by LC-MS/MS, and this quantification was considered to be influenced by an interfering substance present in the concentrate.

INDUSTRIAL APPLICABILITY

Even if a test sample containing a target substance to be measured is contaminated with a substance interfering with antigen-antibody reaction, the antibody resistant to a substance interfering with antigen-antibody reaction selected by the method of this invention, is not influenced by the interfering substance and can analyze and measure the target substance, and further it is a useful antibody which can also be used for concentrating the target substance to be measured.