Monoclonal antibody for immunoassay of detergents and their degradation products and its use

A hybridoma obtainable by fusing a spleen cell or lymphocyte of an animal immunized by a complex of a surfactant compound for synthetic detergent and protein with a myeloma cell, which produces a monoclonal antibody against the compound or its degradation product. A monoclonal antibody against a surfactant compound for synthetic detergent or its degradation product which is produced by the hybridoma, a kit for immunoassay of detergent, its degradation product or a mixture thereof containing as an essential constitutional component the monoclonal antibody, and an immunoassay method, in particular, ELISA of detergent, its degradation product or a mixture thereof in a specimen by reacting the specimen with the monoclonal antibody supported on a carrier and the detergent, its degradation product or the mixture thereof labeled with a labeling agent are also disclosed.

FIELD OF THE INVENTION
 The present invention relates to a monoclonal antibody for an immunoassay
 of detergents and their degradation products, and its use. More
 specifically, it relates to a hybridoma which produces the monoclonal
 antibody, the monoclonal antibody produced by the hybridoma, and a kit and
 a method for immunoassay of detergents, their degradation products or a
 mixture thereof.
 BACKGROUND OF THE INVENTION
 Among surfactants for synthetic detergents, a linear alkylbenzenesulfonate
 (hereinafter sometimes abbreviated to LAS), which is an anionic
 surfactant, has been sold and used since 1967 instead of a branched
 alkylbenzenesulfonate (ABS), which has poor biodegradability, according to
 the recommendation of the administrative. Since LAS is cheap and its
 toxicity is low, it is still using as a main component of household
 synthetic detergents. However, recently, water pollution by LAS and its
 degradation products have been a social problem because of the discharge
 of drainage from living into rivers, etc. Then, the tap water standard of
 anionic surfactants has been established (not more than 0.2 mg/liter). In
 addition, nonionic surfactants are used as about 40% of surfactant
 compounds for synthetic detergents and establishment of its tap water
 standard is being studied.
 Under these circumstances, synthetic detergents and their degradation
 products in an environment are determined and analyzed and the results are
 utilized for environmental preservation. As methods for such determination
 and analysis, for example, high-performance liquid chromatography (HPLC)
 and gas chromatography (GC) are employed for quantitative determination of
 detergents and their degradation products in tap water, rivers, swamps,
 lakes or sewege etc. However, the problems of these methods are such that
 they require expensive apparatus or devices, pretreatment and the
 operation with great skill. As official methods for measuring anionic
 surfactants and nonionic surfactants, there are methylene blue absorption
 method and potassium zinc (II) tetrathiocyanate method, respectively.
 However, these methods require, for example, extraction with organic
 solvents which are harmful to human beings and complicated concentration
 steps and therefore, improvement of these methods is expected.
 In view of these problems, regarding anionic and nonionic surfactants, it
 is desired to develop a method for determination thereof which can
 maintain present high sensitivity and can be readily and quickly operated
 with high specificity at a low cost.
 As one means for solving these problems, an enzyme-linked immunosorbent
 assay (hereinafter sometimes abbreviated to ELISA) can be considered.
 Assay systems and kits utilizing ELISA have been constructed for various
 other environmental pollutants. For example, WO 94/12536 discloses ELISA
 for detecting petroleum fuel in an environment and a kit for such ELISA.
 The kit comprises an enzyme and an antibody, and the measurement can be
 completed very quickly, usually, within several hours. And, the operation
 is very simple in comparison with conventional HPLC and GC, and, in most
 cases, no complicated pretreatment required in such conventional methods
 is needed. Further, a very specific determination can be carried out by
 using an antibody, in particular, a monoclonal antibody. HPLC and GC
 require very expensive apparatus and devices and their maintenance is also
 expensive, while ELISA has no such problem.
 However, any immunoassay method which can be used for detection and
 determination of synthetic detergents including LAS, in particular, ELISA
 has not yet been established.
 OBJECTS OF THE INVENTION
 In order to establish an immunoassay method for detection and determination
 of synthetic detergents, one object of the present invention is to provide
 a hybridoma which can produce a monoclonal antibody useful for an
 immunoassay of synthetic detergents, their degradation products and a
 mixture thereof.
 Another object of the present invention is to provide the monoclonal
 antibody produced by the hybridoma of the present invention.
 Another object of the present invention is to provide a kit for an
 immunoassay of synthetic detergents, their degradation products and a
 mixture thereof.
 Still another object of the present invention is to provide a method for
 immunoassay of synthetic detergents, their degradation products and a
 mixture thereof.
 These objects as well as other objects and advantages of the present
 invention will become apparent to a person skilled in the art from the
 following description with reference to the accompanying drawings.

SUMMARY OF THE INVENTION
 The present inventors have studied to establish a method for immunoassay of
 LAS, APE and their degradation products, in particular, a method for
 detection and determination thereof by ELISA, intensively. As a result, it
 has been found that anti-LAS or anti-APE monoclonal antibody-producing
 hybridoma can be obtained by using SPC, which is an analogue of LAS, or a
 half ester of nonylphenol ethoxylate and succinic anhydride (hereinafter,
 sometimes, abbreviated to NP7EC), which is an analogue of APE, combining
 it with a carrier protein to obtain a complex, immunizing a mouse with the
 complex and isolating the hybridoma from its spleen. In addition, it has
 also been found that LAS, APE and their degradation products can be
 determined with high sensitivity by using the above monoclonal antibody
 and labeled LAS, APE and their degradation products. Thus, the present
 invention has been completed.
 That is, according to the present invention, there is provided a hybridoma
 obtainable by fusing a spleen cell or lymphocyte of an animal immunized by
 a complex of a surfactant compound for synthetic detergent and a protein
 together with a myeloma cell, which produces a monoclonal antibody against
 the compound or its degradation product.
 The present invention also provides a monoclonal antibody against a
 surfactant compound for synthetic detergent or its degradation product
 which is produced by the hybridoma of the present invention.
 Further, the present invention provides a kit for immunoassay of detergent,
 its degradation product or a mixture thereof comprising as an essential
 constitutional component the monoclonal antibody of the present invention.
 Furthermore, the present invention provides a method for immunoassay, in
 particular, ELISA of detergent, its degradation product or a mixture
 thereof in a specimen which comprises reacting the specimen with the
 monoclonal antibody of the present invention held on a carrier and the
 detergent, its degradation product or the mixture thereof labeled with a
 labeling agent, and then measuring the activity of either the labeling
 agent held on the carrier or the free labeling agent.
 The present invention is applicable to detergents containing surfactants
 for synthetic detergents, for example, anionic surfactants and nonionic
 surfactants and, according to the present invention, such detergents,
 their degradation products or a mixture thereof can be analyzed
 immunologically with high sensitivity and specificity.
 DETAILED DESCRIPTION OF THE INVENTION
 The hybridoma of the present invention can be obtained according to a per
 se known method by using a complex of a surfactant compound for synthetic
 detergent and a protein (carrier protein).
 Examples of the surfactant compounds for synthetic detergents to be used
 include anionionic surfactant compounds such as alkylsulfuric acid,
 alkylbenzenesulfonic acid, alkylnaphthalenesulfonic acid,
 naphthalenesulfonate-formaldehyde condensate and their salts (e.g., alkali
 metal salts, etc.); and nonionic surfactant compounds such as
 polyoxyethylene alkylphenyl ether, polyoxyethylene alkyl ether,
 polyoxyether polystyrylphenyl ether, polyoxyethylene styrenated phenyl
 ether and their salts (e.g., phosphate, sulfate, etc.). Examples of the
 "alkyl" used herein includes that having 2 to 18 carbon atoms. The
 surfactant compound can be appropriately selected according to the
 detergent to be analyzed and its degradation products. However, from the
 viewpoint of environmental preservation, to select LAS, APE or its salt is
 particularly desired.
 As the carrier protein, for example, there are bovine serum albumin
 (hereinafter abbreviated to BSA), ovalbumin (hereinafter abbreviated to
 OVA) bovine thyroglobulin (hereinafter abbreviated to BTG) and the like.
 The formation of the complex of the surfactant compound for synthetic
 detergent or its analogous compound and the protein can be carried out by
 combining a compound of the formula (I):
EQU A--R (I)
 [wherein R is COOH, NH.sub.2 or SH; and A is a group which forms the
 surfactant compound for synthetic detergent or its analogous compound by
 removal of R] to the protein according to a per se known method.
 The compound of the formula (I) can also be synthesized chemically by
 introducing or forming a carboxyl group, amino group or sulfhydryl group
 in a suitable starting material according to a per se known method.
 For example, the compound of the formula (I) wherein R is COOH and A is a
 group which forms LAS can be produced by sulfonating a
 phenylalkylcarboxylic: acid, for example, phenyl valeric acid [J.
 Chromato., 178, 259 (1979)].
 The compound of the formula (I) wherein R is COOH and A is a group which
 forms a polyoxyethylene alkylphenyl ether can be produced by subjecting a
 polyoxyethylene alkylphenyl ether and succinic anhydride to
 dehydration-condensation (formation of half ester) [Cancer Biochem.
 Biophys., 7, 175 (1984)].
 The compound of the formula (I) wherein R is NH.sub.2 and A is a group
 which forms LAS can be produced by sulfonating a chloroalkylbenzene, for
 example, 2-chloroethylbenzene [J. Chromato., 178, 259 (1979)], followed by
 treatment with ammonia (Organic Functional Group Preparations, Vol. 1, p.
 382).
 The compound of the formula (I) wherein R is NH2 and A is a group which
 forms a polyoxyethylene alkylphenyl ether can be produced by chlorinating
 the hydroxyl group of the polyoxyethylene alkylphenyl ether with thionyl
 chloride [J. Am. Chem. Soc., 60, 2540 (1938)], followed by treatment with
 ammonia (Organic Functional Group Preparations, Vol. 1, p. 382].
 The compound of the formula (I) wherein R is SH and A is a group which
 forms LAS can be produced by sulfonating a chloroalkylbenzene, for
 example, 2-chloroethylbenzene [J. Chromato., 178, 259 (1979)], followed by
 reaction with sodium hydrosulfide [J. Am. Che. Soc., 72, 1843 (1950)].
 The compound of the formula (I) wherein R is SH and A is a group which
 forms a polyoxyethylene alkylphenyl ether can be produced by chlorinating
 hydroxyl group of the polyoxyethylene alkylphenyl ether with thionyl
 chloride [J. Am. Che. Soc., 60, 2540 (1938)], followed by reaction with
 sodium hydrosulfide [J. Am. Chem. Soc., 72, 1843 (1950)).
 For immunization of an animal, the animal is inoculated with the complex
 thus obtained. Examples of animals to be inoculated include goat, sheep,
 rabbit, rat, mouse, guinea pig, chicken and the like. In case that a
 monoclonal antibody (hereinafter, sometimes, abbreviated to MoAb) against
 LAS or APE is desired, a mouse is particularly preferred.
 The inoculation can be carried out according to a conventional method. For
 example, a mouse is inoculated with 1 to 100 .mu.g, preferably 50 to 100
 .mu.g of the immunogen which is emulsified with equal amounts (0.1 ml) of
 physiological saline and Freund's complete adjuvant or RIBI adjuvant
 system per once at the back or abdomen subcutaneously or
 intraperitoneally. The mouse receives the inoculation 3 to 6 times at 2 to
 3 week intervals.
 Among the immunized animals, for example, mice, the individual having a
 high antibody titer is selected and, 3 to 5 days after the final
 immunization, the spleen or lymph node is collected. Then, the antibody
 producing cells contained therein are fused together with myeloma cells.
 The fusion can be carried out by a known method and as a fusion promoting
 agent, for example, polyethylene glycol (hereinafter abbreviated to PEG)
 or Sendai virus (HVJ) can be used. PEG is preferred.
 As myeloma cells, for example, NS-1, P3U1 or Sp2/O can be used and, in
 particular, P3U1 is preferred. For example, the preferred proportion of
 the spleen cells : myeloma cells is 1:1 to 10:1 and PEG having a molecular
 weight of 1,000 to 6,000 is added thereto in concentration of 10 to 80%.
 Desirably, the mixture is incubated at 20 to 37.degree. C., preferably, at
 30 to 37.degree. C. for 3 to 10 minutes.
 For screening of the desired hybridoma, various methods can be employed.
 For example, there is ELISA, wherein a supernatant of a hybridoma culture
 is added to a microplate on which OVA combined with SPC or NP7EC has been
 adsorbed and then an anti-mouse immunoglobulin antibody labeled with
 horseradish peroxidase (hereinafter abbreviated to HRP) is added thereto
 to detect the antibody combined to the solid phase of the plate. A
 hybridoma having positive antibody activity is subjected to cloning
 immediately. Normally, this can be readily carried out by limiting
 dilution analysis or the like.
 The antibody titer of cloned hybridomas is determined by the above method
 and a hybridoma which constantly produces an antibody having high titer is
 selected to obtain the desired monoclonal hybridoma.
 Examples of the antibody producing hybridoma obtained by the above
 described method include mouse hybridomas GOT930-9, GOT935-54 and MOF3-139
 as described in Examples hereinafter.
 The monoclonal antibodies against surfactant compounds for synthetic
 detergents or degradation products thereof which are produced by the
 hybridomas of the present invention are also included in the scope of the
 present invention.
 The production of the monoclonal antibody by the hybridoma and the
 purification thereof can also be carried out by a per se known method. The
 monoclonal antibody thus obtained can serve as not only an antibody
 against the surfactant compound for synthetic detergents or their
 degradation products but also the compound of the formula (I).
 The monoclonal antibody thus obtained can be prepared in the form of a
 solid phase antibody by a known method such as bromocyanate method
 described in "Taisha", Vol. 8, 696 (1971), glutaraldehyde (hereinafter
 abbreviated to GLA) and the like. Further, as preferred simpler and easier
 methods, there are a method wherein the antibody is physically adsorbed
 on, for example, a microplate, tube or polystyrene particles, a method
 utilizing immunochromatography, and the like.
 They can be combined with enzymes for ELISA, buffers and the like to obtain
 a kit for immunoassay of detergents, their degradation products or a
 mixture thereof and such a kit is also included in the scope of the
 present invention.
 In the immunoassay of the present invention, a so-called competitive assay
 is employed. That is, the quantitative determination can be carried out by
 quantitatively measuring competition between a specimen (e.g., water and
 soil samples containing detergents, their degradation products or a
 mixture thereof) and a known amount of labeled detergent, its degradation
 product or a mixture thereof for linking to the monoclonal antibody of the
 present invention. In such competitive assay, a given amount of an
 antibody supported on a carrier is added to a specimen solution containing
 an unknown antigen and further a given amount of an the antigen labeled
 with a labeling agent. The amount of the labeling agent supported by the
 carrier or the amount of the labeling agent which does not supported by
 the carrier is measured. Desirably, the specimen and the labeled antigen
 are added almost at once.
 In general, in comparison with sandwich method or the like, the competitive
 assay can be carried out by a simpler and easier operation within a
 shorter period of time.
 Examples of the agent for labeling the antigen include radioisotopes
 (hereinafter abbreviated to RI), enzymes, enzyme substrates, fluorescent
 materials, biotin and the like. For linking the labeling agent to the
 antigen, maleimide method [J. Biochem., 79, 233 (1976)], active biotin
 method [J. Am. Chem. Soc., 100, 3585 (1978)] and the like can be used.
 For example, in the case of LAS, a specific immunochemical determination by
 the competitive assay can be carried out by adding a solid phase, to which
 the antibody is linked physically or chemically by a know conventional
 means, to a sample to be tested, which contains an unknown amount of LAS,
 to allow to react. At the same time, a given amount of an antigen labeled
 with a labeling agent is added thereto to allow to react. Then, normally,
 the solid phase is washed thoroughly and the activity of the labeling
 agent linked to the solid phase is measured. When the labeling agent is
 RI, the measurement is carried out with a well counter or a liquid
 scintillation counter. When the labeling agent is an enzyme, its substrate
 is added, the mixture is allowed to stand and then the enzymatic activity
 is measured by colorimetry or fluorometry. When the labeling agent is a
 fluorescent material or a luminous material, it is measured according to a
 known method.
 Since the monoclonal antibody having a high specificity is used, the
 immunoassay of the present invention has such a very advantageous
 characteristic that the specific determination of LAS, APE and their
 degradation products can be carried out without an error due to cross
 reaction with compounds other than LAS, APE and their degradation
 products, for example, compounds having a benzene ring such as toluene,
 phenol, benzene and aniline, or other surfactants.
 Thus, in the present invention, by using the antibody having very high
 specificity for detergents and their degradation products, influence of
 other chemical substances in a specimen can be eliminated and therefore
 the determination can be simply and readily carried out with high
 sensitivity and specificity. Therefore, the present invention is useful
 for not only academic analyses but also investigation of influence of
 detergents and their degradation products on environmental preservation
 and the like.
 The following Examples further illustrate the present invention in detail
 but are not to be construed to limit the scope thereof.
 Although the following Examples are directed to LAS, SPC, SDS and APE, they
 are applicable to other surfactants for synthetic detergents in the same
 way. In addition, the mouse--mouse hybridomas GOT930-9 and GOT935-54 have
 been deposited at National Institute of Bioscience and Human-Technology
 (NIBH), Agency of Industrial Science & Technology, Ministry of
 International Trade & Industry since Sep.25, 1996 (date of the original
 deposit) under Budapest Treaty under the accession numbers of FERM BP-6065
 and FERM BP-6066, respectively. The hybridoma MOF3-139 has been deposited
 since Aug.13, 1997 under Budapest Treaty under the accession umber of FERM
 BP-6059.
 EXAMPLE 1
 Production of anti-hapten monoclonal antibody
 (1) Preparation of immunogen
 The haptens, SPC and NP7EC, were synthesized from phenyl valerate and
 nonylphenolethoxylate by a known method, respectively. An aqueous solution
 of the hapten (50 mg/ml) was added to an aqueous solution of BSA (50 mg/4
 ml). To the mixture was slowly added dropwise 1 ral of an aqueous solution
 of water-soluble carbodiimide (50 rag/ml) with ice-cooling. The resultant
 mixture was reacted for 5 hours. After dialyzing with 3 liter of distilled
 water 5 times, the reaction mixture was lyophilized and stored so as to
 use as an immunogen.
 (2) Immunization
 To the complex of the hapten and BSA (500 .mu.g/ml physiological saline)
 was added the equal amount of Freund's complete adjuvant and the mixture
 was thoroughly emulsified. Then, it was inoculated into BALB/c mice
 (female, 100 .mu.g/0.4 ml/mouse) at their abdomens and backs
 subcutaneously and booster immunization was conducted 2 week intervals.
 After immunizing booster 6 times, the same complex solution (30 .mu.g/0.1
 ml physiological saline/mouse) was administered to individuals which
 showed maximum serum antibody titers in 2 weeks intravenously.
 (3) Cell fusion
 Three days after the last immunization, the spleen was excised from the
 abdomen of each mouse and a suspension of spleen cells (about 10.sup.8
 cells) was prepared according to a conventional manner. Then, mouse
 myeloma cells (P3U1) (2.times.10.sup.7 cells) which had been washed with a
 serum-free culture medium three times were added thereto and subjected to
 cell fusion with PEG 6000 according to the method disclosed by Koehler and
 Milstein [Nature, 256, 495 (1975)].
 After completion of the fusion, the cell mixture was suspended in so-called
 HAT medium containing hypoxanthine, aminopterin and thymidine and
 cultivated for 10 days. Cells originating from the spleen died out during
 the 10 days cultivation period spontaneously and P3U1 strain which did not
 fused together with the spleen cells also died out due to HAT medium. The
 cells which survived in the medium were regarded as hybridomas and then
 cultivation was continued by replacing the medium with HT medium
 corresponding to aminopterin-free HAT medium.
 (4) Primary selection and cloning of hybridomas
 An antibody titer of the supernatant of the hybridoma culture was measured
 by ELISA using a microplate in which a complex of the hapten and OVA
 (hereinafter abbreviated to hapten-OVA) was adsorbed on the solid phase.
 From 10 days to 20 days after fusion, the appearance of hybridomas were
 observed and the appearance of an antibody which linked to the hapten-OVA
 was recognized. Hybridomas which produced antibodies showing especially
 high binding activity were subjected to cloning by limiting dilution
 analysis.
 (5) Secondary selection of hybridomas
 (5)-1 Selection of anti-LAS antibody, anti-SPC antibody and anti-SDS
 antibody-producing hybridomas
 A compound such as LAS, SPC, SDS, benzene sulfonate, toluene, benzene or
 phenol was added to the supernatant of the hybridoma culture obtained in
 the above (4) and the hybridoma was subjected to binding inhibitory
 examination wherein inhibition of linking to a SPC-OVA-adsorbing
 microplate was examined and a hybridoma whose linkage was inhibited by
 only LAS or LAS, SPC and SDS and was not influenced by other compounds was
 selected.
 As a result, hybridoma GOT935-54 which produced a monoclonal antibody
 specifically linking to LAS and hybridoma GOT930-9 which produced a
 monoclonal antibody specifically linking to LAS, SPC and SDS were
 obtained. According to the determination of ELISA, the immunoglobulin
 class and subclass of these monoclonal antibodies were IgG1.
 (5)-2 Selection of anti-APE antibody-producing hybridoma
 A compound such as APE, APE degradation products (hereinafter, sometimes,
 abbreviated to APEC) or PEG was added to the supernatant of the culture of
 hybridoma obtained in the above (4) and the hybridoma was subjected to
 linking inhibitory examination wherein inhibition of linking to a
 microplate on which OVA combined with NP7EC had been adsorbed was examined
 and a hybridoma whose linkage was inhibited by only APE and APEC and was
 not influenced by other compounds was selected.
 As a result, hybridoma MOF3-139 which produced a monoclonal antibody which
 specifically link to APE and APEC was obtained. According to the
 determination of ELISA, the immunoglobulin class and subclass of this
 monoclonal antibody was IgG1.
 The materials determined, haptens used, hybridomas obtained and their
 accession Nos. are summarized in Table 1.
 TABLE 1
 Material Hapten Hybridoma Accession No.
 LAS SPC GOT935-54 FERM BP-6066
 SPC SPC GOT930-9 FERM BP-6065
 SDS SPC GOT930-9 FERM BP-6065
 APE NP7EC MOF3-139 FERM BP-6059
 (6) Production of monoclonal antibody
 The cloned hybridoma was cultivated by incubating it in Daigo T medium
 (Nippon Seiyaku, Tokyo, Japan) containing 10% bovine fetal serum at
 37.degree. C. under 5% CO.sub.2 and the antibody was obtained from the
 supernatant of the culture.
 On the other hand, for obtaining a large amount of antibody, 0.5 ml of
 mineral oil was administered to a BALB/c mouse intraperitoneally and then
 5.times.10.sup.6 cells of hybridoma were inoculated in the mouse
 intraperitoneally. After about 10 days, the collection of ascites fluid
 was observed in the mouse that received inoculation of the cells and the
 ascites fluid was harvested from the peritoneal cavity by abdominal
 section of the mouse.
 The purification of the antibody was carried out by fractionating a
 fraction from the supernatant of cell culture or ascites fluid with 45 to
 50% saturated ammonium sulfate according to a conventional method and
 subjecting to column chromatography on a protein A column. 2 to 20 mg of
 the monoclonal antibody was obtained from 2 ml of the supernatant of
 ascites fluid depending upon the particular cell strain. When the purified
 monoclonal antibodies were subjected to electrophoresis on SDS
 polyacrylam:ide gel, they showed more than 96% purity.
 EXAMPLE 2
 Determination of LAS and LAS degradation products
 (1) Preparation of HRP labeled SPC
 HRP was dissolved in distilled water (4 mg/0.2 ml) and mixed with SPC (10
 mg/0.2 ml) and to the mixture was added dropwise water-soluble
 carbodiimide solution (15 mg/0.1 ml) with stirring on ice. The mixture was
 allowed to react on ice for about 8 hours and then dialyzed sufficiently
 against PBS (-) to remove unreacted SPC and WSC.
 (2) Preparation of solid phase monoclonal antibody
 The antibody obtained in Example 1 (5)-1 was dissolved in 25 mM bis-Tris
 buffer at concentration of 5 .mu.g/ml and distributed in each well of a
 96-well immunoplate (manufactured by Nunc, Denmark). The plate was allowed
 to stand at 4.degree. C. overnight to prepare the solid phase monoclonal
 antibody. For blocking the binding group, 150 .mu.l aliquots of a Block
 Ace solution (manufactured by Snow Brand Milk Products, Tokyo) containing
 0.01% sodium merthiolate (Sigma, U.S.A.) were distributed to the solid
 phase antibody. The antibody was stored at 4.degree. C. until it was used.
 (3) Determination of LAS and LAS degradation products
 LAS, SPC and SDS diluted with distilled water were mixed with the equal
 amount of HRP labelled SPC prepared in the above (1) (2 .mu.g/ml) (0.02%
 Tween 20--PBS) and the mixture was added to each well of the
 antibody-binding microplate prepared in the above (2). After reaction at
 room temperature for 90 minutes, the plate was washed with PBS and 100
 .mu.l of the enzyme substrate (TMBZ 0.1 mg/ml, 40 mM citrate buffer
 containing urea hydrogen peroxide 0.35 mg/ml, pH 5.0) was added. The color
 developing reaction was conducted for 15 minutes and the reaction was
 stopped with iN phosphoric acid. Then, absorbance at 450 nm was measured.
 The calibration curves of LAS, SPC and SDS are shown in FIGS. 1, 2 and 3,
 respectively. In this system, about 20 to 500 ppb of LAS and about 50 to
 500 ppb of SPC and SDS were able to be determined.
 EXAMPLE 3
 Determination of APE
 (1) Preparation of HRP labelled NP7EC
 HRP was dissolved in 0.13 M aqueous NaHCO.sub.3 solution (3.3 mg/2 ml) and
 mixed with NP7EC (300 .mu.g/5 .mu.l DMSO) which formed its activated ester
 by a known method. The mixture was allowed to react overnight at 4.degree.
 C. and then dialyzed sufficiently against PBS (-) to remove unreacted
 NP7EC.
 (2) Preparation of solid phase monoclonal antibody
 The antibody obtained in Example 1 (5)-2 was dissolved in PBS (-) at
 concentration of 5 .mu.g/ml and, according to the same manner as described
 in Example 2 (2), the solid phase thereof was formed on the 96-well
 immunoplate.
 (3) Determination of APE and APE degradation products
 APE (Tokyo Kasei) diluted with 60% aqueous methanol was mixed with the
 equal amount of HRP labeled NP7EC prepared in the above (1) (0.3 .mu.g/ml)
 (0.1% Tween 20--double concentrated PBS) and the mixture was added to each
 well of the antibody-binding microplate prepared in the above (2). After
 reaction at room temperature for 90 minutes, the plate was washed with PBS
 containing 0.05% Tween 20 and 100 .mu.l of the enzyme substrate (TMBZ 0.1
 mg/ml, 40 mM citrate buffer containing urea hydrogen peroxide 0.35 mg/ml,
 pH 5.0) was added. The color developing reaction was conducted for 15
 minutes and the reaction was stopped with 1N phosphoric acid. Then,
 absorbance at 450 nm was measured.
 The calibration curve of APE is shown in FIG. 4. In this system, about 50
 to 500 ppb of APE was able to be determined.
 As described hereinabove, according to the present invention, detergents
 and their degradation products can be simply and readily determined with
 high sensitivity and specificity, and such determination can be utilized
 not only in academic area but also in environmental preservation.