Selected Article 2 「SmartAssay（スマートアッセイ）シリーズ」は，農産物中の残留農薬測定に適用可能との評価を得，農産物の出荷前検 製した。まずハプテン設計や抗体調製の基礎的検討を行い，モノクローナル抗体が対象農薬と高い反応性を示すほか， 査に利用されている。 農産物の収穫直前に散布される主な農薬（殺虫剤と殺菌剤）21種類について，直接競合ELISAを利用した測定キットを開 有機溶媒耐性など農薬分析に有効な反応特性を示すことを見出した。これらの知見に基づいて開発した農薬測定キット 発した。農薬などの低分子性物質は，それ自体に抗体産生能がないが，免疫原性を持つタンパク質表面に共有結合し接 種することで抗体を産生する場合がある。このような物質をハプテンといい，農薬に対する抗体もこの性質を利用して調 the pesticide residues exceed the standard values and the the and values standard the exceed residues pesticide the which in cases the dispel to difficult is it However, Act. Regulation Chemicals Agricultural by provided are users the for usages adequate and system registration the under used are pesticides All fungicides. or insecticides pests using control to indispensable is it Therefore, products. offarm value commercial the reduce they period, of a harvest in appear kinds they if Especially, various appear. pests humid, agricultural and hot is it where Japan In Introduction test pesticide for before shipment. used are kits The products. farm in analysis pesticide residual to applicable as researchers by evaluated been have knowledge a such on based developed series” “SmartAssay kits test analysis residue Pesticide solvent. organic to tolerance as such analysis pesticide to property reaction antibody efficient and and antibodies design hapten of polyclonal than pesticides the examinations to reactivity higher showed basic antibodies monoclonal the that found was it In preparation, behavior. this by anti-pesticide and prepared hapten also called were are antibodies compounds These innoculate. to to protein covalently immunogenics binding of by surface antibody the produce them of some However, themselves. producibility have by not do antibody an pesticides including compounds molecular Low harvests. before just applied are pesticides The fungicides. and insecticides Twenty ELISA werecompetitive for test developed checking ofkits direct kinds one 三宅 司郎 Shiro MIYAKE 「SmartAssayシリーズ」の開発 イムノアッセイによる農薬測定キット Series”“SmartAssay for Pesticide Analysis Development ofImmunoassay Test Kit Selected Article English Edition No.16 February 2012 English Edition No.16February For pesticide residue analysis, instrument analysis using using analysis instrument analysis, residue pesticide For for inspection by growers. residue pesticide acceptance for requirement high a leads That completed. was per products farm in residue ofpesticide product environment farm regulatory the Thus, each chemical. for set agricultural is residue pesticide of value standard a and 2006 May since operation into come List Positive has Foods in Act, Residues Chemical Agricultural for System Sanitation Food in hand, other On the detected. are pesticides unregistered which in cases Technical Reports

Table 1 SmartAssay Serices Line up Measuring Range (ppb) Measuring Range (ppb) Measurement Item (Insecticides) Measurement Item (Fungicides) Selected Article Lower Limit Limit Lower Limit Limit Acetamiprid 0.30 4.0 Iprodione 1.5 30 imidacloprid 2.0 100 Isoprothiolane 6.0 100 Fenitrothion 0.15 2.0 Myclobutanil 0.2 2.0 Isoxathion 1.0 20 Enilconazole 5.0 50 Chlorfenapyr 2.0 10 Flutolanil 1.0 8.0 Malathion 15 250 Bitertanol 9.0 50 Carbaryl 1.5 30 Triflumizole 2.0 20 Development of Immunoassay Test Kit Clothianidin 1.5 15 Chlorothalonil 0.15 1.5 Dinotefuran 1.5 30 Azoxystrobin 10 200 Emamectin 0.30 3.0 “SmartAssay Series” for Pesticide Analysis Thiamethoxam 0.30 3.0 イムノアッセイによる農薬測定キット Nitenpyram 5.0 100 「SmartAssayシリーズ」の開発 gas chromatography (GC) or high-performance liquid Preparation of Antibody chromatography (HPLC) is generally used. Instrument Shiro MIYAKE analysis is superior in sensitivity and accuracy, and has Low-molecular substances such as pesticides are only the advantage of analyzing simultaneously many metabolized and excreted, and never generate any 三宅 司郎 compounds in combination with a mass spectrometer. antibody even if they are inoculated into animals. However, as it requires expensive instruments, dedicated However, if they are covalently-bound to the surface of installation sites and high analytical technique including immunogenic protein before inoculation (immunity), not the pretreatment of farm products, it was not considered only the protein but the antibody for the pesticide part Twenty one kinds of direct competitive ELISA test kits were developed for checking insecticides and fungicides. to be suitable for an acceptance inspection in a production may be generated. Such a substance is called hapten and The pesticides are applied just before harvests. Low molecular compounds including pesticides do not have site. the antibodies against pesticides are also prepared using an antibody by producibility themselves. However, some of them produce antibody by binding covalently to this behavior. Some pesticides have the functional group the surface of immunogenics protein to innoculate. These compounds are called hapten and anti-pesticide Compared with instrument analysis, immunoassay that enables a direct binding with protein, however, in antibodies were also prepared by this behavior. In the basic examinations of hapten design and antibody (collective term of analysis method using antibody) using many cases, the design of hapten such as newly preparation, it was found that monoclonal antibodies showed higher reactivity to the pesticides than polyclonal antibody is known as a rapid, easy and economical introducing a functional group (example: carboxyl group) antibodies and efficient reaction property to pesticide analysis such as tolerance to organic solvent. Pesticide method. It is used for a routine inspection work in the is required. The successful example of preparing the residue analysis test kits “SmartAssay series” developed based on such a knowledge have been evaluated by healthcare field and contributes in diagnosing the clinical antibody that has high reactivity for subject pesticides by researchers as applicable to residual pesticide analysis in farm products. The kits are used for pesticide test condition of patients. Although it was developed also for devising the design of hapten is shown in Figure 1. before shipment. pesticides, it was used for environmental analysis such as Although the design of hapten differs depending on groundwater contamination and was rarely used for the pesticides, there are several effective designs: (1) like the 農産物の収穫直前に散布される主な農薬（殺虫剤と殺菌剤）21種類について，直接競合ELISAを利用した測定キットを開 acceptance inspection. This arises from inaccurate results case of oxamyl, the linker chain in calboxyl group 発した。農薬などの低分子性物質は，それ自体に抗体産生能がないが，免疫原性を持つタンパク質表面に共有結合し接 of existing immunoassay caused by matrix influence. introduction part is stretched to inhibit the interaction 種することで抗体を産生する場合がある。このような物質をハプテンといい，農薬に対する抗体もこの性質を利用して調 with the surface of protein, (2) like the case of malathion, 製した。まずハプテン設計や抗体調製の基礎的検討を行い，モノクローナル抗体が対象農薬と高い反応性を示すほか， In such a condition, HORIBA, Ltd. found that a direct in the living body, an unstable chemical structure is competitive ELISA (abbreviation of Enzyme-Linked modified and stabilized and (3) like pyrethroid 有機溶媒耐性など農薬分析に有効な反応特性を示すことを見出した。これらの知見に基づいて開発した農薬測定キット ImmunoSorbent Assay: an immunoassay technique or an insecticides that have chrysanthemic acid in common, in 「SmartAssay（スマートアッセイ）シリーズ」は，農産物中の残留農薬測定に適用可能との評価を得，農産物の出荷前検 analysis method, in which one side of antigen or antibody preparation of group specific antibody, common structure 査に利用されている。 is bound to solid phase and the liquid phase antigen or of structural related substance group is bound to antibody is enzyme-labeled for antigen antibody reaction. protein.[2-4] By washing unreacted substances for removal after the reaction, the enzyme activity bound to solid phase is Antibody is divided into polyclonal antibody (PoAb) and detected for highly sensitive analysis. ), using monoclonal monoclonal antibody (MoAb). PoAb is the purified antibody was insusceptible to matrix and was applicable antibody component of prepared serum from an to the inspection of farm products. The 21 kinds of immunized animal blood and consists of multiple pesticide residue test kits developed on the basis of the antibody molecules that have diverse reactions for subject knowledge (Refer to Table 1) have been spreading into the pesticides. On the other hand, MoAb is the prepared acceptance inspection of farm products by growers. In antibody derived from an antibody producing-cell in a this article, I state the preparation method of antibody for living body and consists of antibody molecules of which pesticides, development of direct competition ELISA, the reactivity to subject pesticides is uniform. As a result commercialization of kits and applicability to analysis of comparing the reactivity of PoAb and MoAb in several samples. pesticides, MoAb always showed higher reactivity than

English Edition No.16 February 2012 3 Selected Article 4 inhibition concentration to show the proportional proportional the 50% show its to around concentration mainly inhibition distributed, are molecules antibody PoAb, in that considered is reason The PoAb. Selected Article Solid-Phased Well ①An Antibody Figure 2 Measuring flow of Direct Competitive ELISA Competitive Direct of flow 2Measuring Figure Haptens and Pesticides of Structure 1The Figure (3) (2) (1) Pesticides (+) Pesticides (−) English Edition No.16 February 2012 English Edition No.16February H H H H H H H H H H H H 3 3 3 3 CO CO CO CO 3 3 3 3 3 3 3 3 C C C C C C C C N C C N Malathionhapten Oxamylhapten S P S P C O C H C O C H Antibody Malathion Oxamyl H N S SCH C C SCH CHCOOC CHCOOC CH CH CH CH NOCNH (CH 3 NOCNHCH 3 2 2 3 COOC COO (CH 3 CH CH COOH C O O O

3 3 O 2 2 Development ofImmunoassay Test Kit Series”“SmartAssay for Pesticide Analysis H 2 H (Room Temp.,after1hour) ②Competitive Reaction Allethrinhapten Allethrin H 5 5 5 2 3 ) 2 ) nCOOH 5 CH COOH The EnzymeLabeledHapten 3 O CH 2 CH CH 2 under the existence of complex and large quantity of of quantity large products. farm and from derived matrix complex of existence the under antibody, monoclonal of characteristic the way, by stable was It relatively a in analyzed be ELISA. could pesticides that expected competitive direct develop to solvents for organic tolerance and reactivity high has that antibody monoclonal the use to decided was it results, these From index, it was possible to select the antibody molecule. antibody it possible select the was to index, as solvents organic for tolerance the uses that screening the by MoAb, in hand, other the on among molecules, antibody varies tolerance the because antibodies tolerant highly prepare to to difficult PoAb,is it In solvents. tolerance organic a show that antibodies the analysis for many use as to effective is it compounds, Additionally, hydrophobic are pesticides selected. constantly be could range concentration low the at to reactivity high a show that MoAb, antibodies in the hand, other the on reactivity, Direct Competitive ELISA any pesticide is contained in the sample and the lower lower contained the is pesticide excess and that case the is flow analysis sample the in contained is pesticide any also were in shown analysis As ELISA. competitive direct the pesticide applying by developed for kits Immunoassay substances. molecule low diverse analyzing for used is it accurate, and sensitive highly is it and small is analysis at steps reaction of number the As them. for antibodies the with react competitively (enzyme) peroxidase horseradish and hapten its to hapten) (enzyme-labeled conjugate the and substance subject the after antibody to labeled bound hapten enzyme of activity enzyme using pesticides, of concentration the determines ELISA competitive Direct Figure 2 Figure ③Washed out , the upper analysis flow is the case that that flowthecase analysis is upper , the Pesticides No ColorDevelopment Color Development ④Coloring Reaction [1]

Technical Reports

Selected Article Development of Immunoassay Test Kit “SmartAssay Series” for Pesticide Analysis in the sample. One of the characteristics of direct competitive ELISA is that the coloring level is low depending on the concentration of pesticide if it is within the determination range. The steps of the analysis flow are explained as follows.

A subject pesticide and 10% methanol solution of enzyme-labeled hapten are added in each well of an Absorbance (450 nm ) antibody solid-phased 96 well micro-titer plate for competitive reaction at room temperature for one hour. The concentration of antibody is preliminarily adjusted so Dinotefuran Conc. (ppb) Standards Curve Calibration Curve of Kit that required determination range and absorbance are (2 Points calibration) obtained. The determination range usually depends on the reactivity of antibody and the absorbance depends on the Figure 3 Calibration Curve (Dinotefuran Test Kit) concentration of enzyme-labeled hapten. Commercialization of Kits After competitive reaction, the wells are washed with normal saline 3 times to remove the enzyme-labeled If the direct competitive ELISA is made in a laboratory hapten and the pesticide that did not bind to the solid- and used for analysis, many preparations are required phase antibody. Via the antibody, the solution including before analysis. First of all, an antibody diluted to the tetramethylbenzidine is added as the chromogenic specified concentration is dispensed to the wells of a 96 substrate of the enzyme bound to the solid phase of the well-micro titer plate and the plate is left at 4˚C for a night wells. After the chromogenic reaction at room so that the antibody is solid phased and an antibody plate temperature for 10 minutes, 0.5M sulfuric acid is added is formed via blocking. That requires, in general, a day. to stop the reaction. At this time, enzyme is inactivated Concerning enzyme-labeled hapten, concentrated one by by diluted sulfuric acid, the degradation of pH changes 100 times is dispensed into vials and frozen. For analysis, the color of chromogenic agent from blue to yellow and it is fused and diluted. The pesticide standard solution and then the chromogenic reaction stops. chromogenic substrate used for drawing calibration curve are easily degraded and should be prepared for each The absorbance after the stop of chromogenic reaction is analysis. Moreover, the difference between preparations determined with the dedicated spectrophotometer for is often large, which causes inaccurate results. micro plate. If the concentration of the pesticide contained in the sample is high, the quantity of the enzyme-labeled The kits (See Figure 4) are commercialized for the hapten bound to the antibody is small and the level of purpose of omitting these preparations and also chromogenic reaction becomes low. If the concentration stabilizing the reagent. Using the kits, pesticide residue is of the pesticide is low, the chromogenic reaction is strong. inspected without a cumbersome preparation for analysis. A calibration curve is drawn with standard pesticide As the product performance is guaranteed in the specified concentration on the x-axis and the corresponding range, the difference between preparations is considered absorbance on the y-axis, and the absorbance of the sample is applied to calculate the pesticide concentration. In Figure 3, the calibration curve (2 point calibration) of dinotefuran test kit and the standard curve by 5 point calibration are illustrated by examples. For a standard curve that is, in general, a sigmoid curve, multiple point calibration is used in most of the cases. However, in a pesticide analysis test kit “SmartAssay Series”, the determination range is the linearity range by 2 point calibration limited to the higher linearity. This is for the purpose of minimizing the number of wells used for calibration curve and increasing the number of samples for analysis. As clarified in the Figures, 2 point calibration ensures the practical accuracy.

Figure 4 SmartAssay Series Nitenpyram Test Kit

English Edition No.16 February 2012 5 Selected Article 6 to maximize the potential. Smart Assay Series have been been have Series Assay Smart potential. the maximize to kits the enables features the the understanding after that use better summarizes Amano column. activated mini a or with carbons sample the up clean (3) and ultrafiltration by membrane sample the filter (2) with again methanol, 10% sample the The dilute (1) are description. methods technical effective the in detail in written are avoidthem to how and effects The meetings. scientific in reported have been reaction anormal interrupts pesticides with extracted matrix derived products farm the which in cases the Series, Assay Smart in even hand, other the On a is immunoassay test. screening rapid and easy as means that promising consider others and Watanabe products developed, the degradation developed in the the in developed ofprocesses. many consists ofthat GC pretreatment degradation farm the in developed, pesticide products a unstable an is d e c a chlorothalonil l as p that considered is r emphasis e t e m disproportionate a o placed r slope t c e p s it s why the reason (r=0.986). The side, s correlativity showedhigh a a immunoassay m the on e emphasis h t d disproportionate n a C G f o combination the of result GC/MS the and line regression slope of the although kit, test chlorothalonil in hand, other than r=0.979 was shown for cucumbers, eggplants and and in lettuces. eggplants shown cucumbers, for shown was was r=0.979 higher than of peppers correlativity the However, its kit. k test green for Imidacloprid several 0.913 = ng r i n HPLC, concer with difference slight a result, the As Series. analysis SmartAssay with ment u r correlativity nst the i examined others and Watanabe products. farm an in residue for sample pesticide analysis as used normally is solution diluted This 10% equivalent. to diluted is sample the in methanol the operation, this By water. purified with 7.5 folds to diluted is filtrate the and filtered is extract The extraction. for minutes 30 tube, for shaken is mixture the and added centrifuge is a methanol of 25mL to taken is sample the of gram Five homogenization. for minute one approximately for a by mixer ground and chopped are samples all, of first fruits, not and farm for vegetables condition of pretreatment the In does products. pretreatment the in cleanup a immunoassay require basically analysis, instrument Unlike Sample Analysis is condition. analysis the and analyzer the analysis, the accuracy the of charge in ofperson technique Therefore, the verifying by ensured range. guaranteed the in Selected Article than r=0.989 was shown for apples and for for shown pears. applesand was r=0.989 than English Edition No.16 February 2012 English Edition No.16February [5] In chlorfenapyr test kit, the reactivitiy higher higher reactivitiy the kit, test chlorfenapyr In

Development ofImmunoassay Test Kit Series”“SmartAssay for Pesticide Analysis [6] On the the On [7]

users have to verify the effects and how to avoid them them avoid to how beforehand. and effects the verify to have users The matrix. the by affected are products farm and kits of However, derived matrix. some that combinations it isbeen true has products products- farm of farm the effects the byin inhibiting realized analysis residue pesticide result, a As tolerance. solvent organic and reactivity high the shows that antibody monoclonal the using developed the readers that pesticides are rapidly and easily easily and rapidly analysis. for pesticide kits the use to users the are for informative is and sensitivity high at pesticides determined that readers the understand makes article direct this that author of the pleasure a the by kits of is It outlined. are applicability its and ELISA preparation competitive development the antibody with design, together hapten the above, As or test efficacy in residual as such monitoring. environment pesticides, of clarified, test are efficacy pesticides In target the markets. where wholesale or fields the in used be to started have products the addition, cooperatives mainly places, the agricultural in diffused been have Series Assay Smart using shipment before tests fact, In products. farm of shipment before tested are samples many where areas shipping and consolidating the in test residue pesticide the for effective is it Therefore, analyzed. simultaneously are samples Many (4) required. not is knowledge advanced An (3) rapid., and easy is pretreatment Its (2) required., not are instruments (1) as has Expensive such advantages the analysis, instrument with compared Immunoassay, Conclusion [6] [5] [4] [3] [2] [1] Japanese JPesticide Sci. ,35(2),S. Miyake 176-180 (2010) in Ohkawa: Pestic. Sci. 54, 189-194Ohkawa: (1998) H. & Kaneko H. Y. Yamaguchi, Beppu, R. Miyake, S. 10-17 (2000) 25, Sci. Y. & J.Pesticide Yuasa: Takewaki S. Kawata, M. Y. Ohde, K. Yamaguchi, Morimune, K. Miyake, S. Sci. 28, 301-309 (2003) Pesticide J. Ohkawa: H. & Kawata M. Morimune, K. Miyake, S. Ohde, K. Nakata, M. Y. Nishi, K. Imajuku, Ueji &S. Endo: J. A1074, Chlomatogr. M. 145-153Y. Ishii, (2005) Arao, T. Eun, H. Baba, K. Watanabe, E. E. Watanabe, H. Eun, K. Baba, T. Arao, Y. Ishii, S. S. 521, Acta &M. Ueji:Endo Chim. Anal. 45-51Y.Ishii, (2004) Arao, T. Baba, K. Eun, H. Watanabe, E. References Technical Reports

Selected Article Development of Immunoassay Test Kit “SmartAssay Series” for Pesticide Analysis [7] E. Watanabe, S. Miyake, S. Ito, K. Baba, H. Eun, M. Ishizaka & S. Endo: J. Chlomatogr. A 1129, 273-282 (2006) [8] A. Amano J Pesticide Sci., 35(3), 396-400 (2010) in Japanese

Shiro MIYAKE 三宅 司郎 Chemical & Bio Sensor Dept. Advanced R&D Center HORIBA Ltd.

English Edition No.16 February 2012 7