Kết quả thử nghiệm bột kít thử nhanh aflatoxin B1 và M1

BỘ NÔNG NGHIỆP VÀ PHÁT TRIỂN NÔNG THÔN VIỆN THÚ Y & QUY TRÌNH ĐỀ TÀI Tên quy trình: Quy trình chế tạo KIT phát nhanh Aflatoxin B1 thức ăn chăn nuôi Aflatoxin M1 sữa tươi Thuộc đề tài: “Nghiên cứu chế tạo KIT phát nhanh Aflatoxin B1 thức ăn chăn nuôi Aflatoxin M1 sữa tƣơi.” Chủ trì đề tài: TS Phạm Thị Ngọc Đơn vị chủ trì: Viện Thú y HÀ NỘI - 2019 I QUY TRÌNH CHẾ TẠO KIT PHÁT HIỆN NHANH AFLATOXIN B1 TRONG THỨC ĂN CHĂN NI (AFB1) 1.1 Mục đích Aflatoxin chất trao đổi bậc có độc tính gây ung thư tạo hai loài nấm mốc chủ yếu Aspergillus flavus Aspergillus parasiticus có mặt loại nông sản thực phẩm lạc, hạt ngô, ngũ cốc, thức ăn chăn nuôi Trong số aflatoxin đề cập aflatoxin B1 loại phổ biến có mặt 75% mẫu thực phẩm thức ăn chăn ni kiểm tra có nhiễm Aflatoxin (Hussein et al., 2001; Cullen et al., 1993; Ayub et al., 1997) Đây nhóm độc tố nấm mốc nguy hiểm gây độc cho người động vật, gây bệnh cấp tính (tổn thương gan, viêm gan, phù nề, hoại tử xuất huyết) ung thư biểu mơ mãn tính (ung thư gan, phổi, thận ức chế miễn dịch), chí với liều lượng cao dẫn tới tử vong Cơ quan Nghiên cứu Ung thư Quốc tế (IARC) phân loại Aflatoxin B1 hợp chất gây ung thư quan trọng (nhóm 1), đặc biệt liên quan đến ung thư gan, khả gây quái thai, gây đột biến Tại Việt Nam, nhiều kiểm nghiệm cho thấy có mặt aflatoxin mẫu nơng sản Việt Nam cao từ 60% - 65%, đặc biệt ngơ Sự có mặt loại độc tố, Aflatoxin, nhiễm vào thức ăn chăn nuôi (TĂCN) ngũ cốc, gây ảnh hưởng lớn người tiêu dùng, người chăn nuôi người sản xuất TĂCN Hiện có nhiều phương pháp phát triển để phát Aflatoxin B1với độ nhạy cao TLC, HPTLC, HPLC, LC-MS/MS, FTIR, RIA, ELISA, SPR, điện hóa, que thử miễn dịch (Ordaz et al., 2013; Liu et al., 2013; Arduini et al., 2007; Sobolev et al., 2007) Đa số phương pháp đòi hỏi người phân tích phải có kỹ tốt, phải tiền xử lý mẫu, tốn thời gian sử dụng thiết bị đắt tiền, phù hợp để thực phân tích aflatoxin điều kiện phịng thí nghiệm Những năm gần phương pháp que thử miễn dịch với nhiều ưu điểm dễ dàng thao tác, tiết kiệm thời gian phân tích, phù hợp sử dụng ngồi trường trọng phát triển để tiếp cận nhiều đối tượng người tiêu dùng Do đó, việc tạo que thử nhanh với độ nhạy độ đặc hiệu cao, sử dụng trường cần thiết Que thử thiết kế dựa nguyên lý sắc ký miễn dịch cạnh tranh nhằm phát có mặt độc tố aflatoxin B1 thức ăn chăn ni 1.2 Phạm vi áp dụng Quy trình áp dụng cho sở chăn nuôi sở chế biến thức ăn cho gia súc, gia cầm 1.3 Định nghĩa chữ viết tắt Cụm từ đầy đủ Chữ viết tắt AFB1 Aflatoxin B1 AFM1 Aflatoxin M1 ELISA Enyme Link Immunoglobin Sandwich Assay FTIR Fourier transform infrared HPTLC High-performance thin layer chromatography LC-MS/MS Liquid Chromatography - Mass Spectrometry/ Mass Spectrometry RIA Radioimmunoassaay SPR Surface Plasmon Resonance TĂCN Thức ăn chăn nuôi TLC Thin layer chromatographic 1.4 Trang thiết bị nguyên vật liệu 1.4.1 Trang thiết bị CƠNG NGH ENZYME VÀ HỐ SINH the signal band intensity is still clearly visible The optimal amount of anti-AFB1 antibody was determined as ng/10 µl of AuNP Optimization of pH for conjugation reaction In order to investigate the effect of pH on the conjugation efficiency of gold nanoparticles and antibodies, the conjugation reactions were carried out at the different pH values of 7.0 – 10.0 The obtained results showed that the signal band intensities were decreased when the pH was increased up 10.0 (Fig 5) The same signal intensities were observed at pH 7.0 and 7.5 and higher than that of pH range of 8.0-10.0 Theoretically, the surface of gold nanoparticle is maximally absorbed by protein at isoelectric point (pI) of molecule or 0.5 pH unit higher (Zhang et al., 2009) Obtained result demonstrated that the suitable pH value for conjugation was 7.0-7.5 pH 7.0 % 8.63 pH 7.5 pH 8.0 pH 8.5 pH 9.0 8.33 8.23 8.24 8.48 pH 9.5 8.35 pH 10.0 8.30 Figure Conjugation of IgG and AuNP at different pH conditions.% = Iband/It*100, in which Iband is the intensity of target signal band, It is total intensity of inspection window The temperature for conjugation 37oC The adsorption efficiency of antibody on the surface of gold nanoparticles is temperature dependent The conjugation reaction o o o was carried out at three different temperatures (4 C, 25 C and 37 C) for 120 minutes and the result indicated that the slight difference of o o signal band intensities were observed at C and 25 C but significant o difference at 37 C The highest signal intensity was achived at 25oC o Therefore, the room temperature (25 C) was chosen for conjugation of antibody and gold nanoparticles 25 oC 4oC Figure Conjugation of IgG and AuNP at the different temperatures (4, 25 and 37oC) Time for conjugation Antibody absorption on the surface of gold nanoparticles is also time dependent The coupling reaction was carried out at four different times of 30, 60, 90 and 120 minutes The result showed that no significant difference in signal intensity between 30 and 120 minutes reaction (Fig 7) It means that 30 is enough for coupling reaction between anti-AFB1 antibody and gold nanoparticles 30 60 90 120 Figure Coupling reaction of anti-AFB1 antibody and AuNP for 30, 60, 90 and 120 minutes The capture reagent buffer The capture reagent buffer has the function for solubilizing the anti-AFB1 antibody/AuNP conjugate before loading onto glass fibre pad Therefore, the chemical composition of buffer can affect the stability of anti-AFB1 antibody/AuNP conjugate during storage and solubility of the conjugate from the conjugate pad when loading sample solution Buffer components also affect the migrating flow of conjugate throught the membrane In the present study, seven different formulations of the capture reagent buffers (CB1: 20mM Sodium borate; CB2: 20mM Sodium borate, 3% sucrose; CB3: 20mM Sodium borate, 3% sucrose, 1% BSA; CB4: 20mM Sodium borate, 3% sucrose, 1% Glycerol; CB5: 20mM Sodium borate, 3% sucrose, 1% BSA, 1% Glycerol; CB6: 20mM Sodium borate, 3% sucrose, 1% BSA, 1% Glycerol, 2mM CaCl 2; CB7: 20mM Sodium borate, 3% sucrose, 1% 304 H I NGH KHOA H C CÔNG NGH SINH H C TOÀN QU C 2018 BSA, 0.6 M NaCl, 0.2% Tween-20) were investigated and showed that the highest signal was observed with the formulation of CB6 (Fig 7) CB1 CB2 CB3 CB4 CB5 CB6 CB7 Figure Seven different capture reagent formulations for preparation of conjugate pad Optimization of conditions for immobilizing AFB1-BSA on the membrane Membrane types In the test strip, the membrane is one of the most important component as it relates to the sensitivity, specificity, and stability of the test strips The capillary flow rate of the membrane flow in the test strips affects the interaction between the antibody and the antigen The different types of nitrocellulose membranes are in terms of thickness, capillary speed, membrane size, etc In this study, the AFB1-BSA conjugate was immobilized on fives different types of the membrane The result indicated that the membrane of Vivid170 from Pall showed the strongest signal band than others (Fig 8) AE99 Vivid 170 NC-B101 NC-B102 NC-B110 Figure Different types of the membrane material for lateral flow strip Determination of AFB1-BSA amount for immobilizing on the nitrocellulose membrane The loading capacity of a protein on a given surface area depends on the protein compactness of structure and its effective diameter The amount of antigen immobilized on the membrane should be higher than the amount of antibody in the conjugate pad In this study, five different amounts of antigens: 10, 50, 100, 200 and 300 ng were loaded on the nitrocellulose membrane The result showed that a significant signal was observed at 100 ng AFB1BSA immobilized (Fig 9), implicating that the appropriate amount of AFB1-BSA for immobilization on the nitrocellulose membrane was approximate 100 ng Suitable temperature and time for immobilizing AFB1-BSA o conjugate on the nitrocellulose membrane were 37 C and 30 (data not shown) 10 ng 50 ng 100 ng 200 ng 300 ng Figure 10 Optimization of AFB1-BSA conjugate amount on Nitrocellulose Determination of detection ability of the generated test strip The generated test strip in this work contained an AFB1-BSA conjugate acted as the coating antigen, anti-AFB1 antibody/AuNP conjugate located in conjugate pad served as the probe, and commercial AFB1 acted as the competitive antigen The result showed there was a significant signal in negative sample (control) while no signal was observed in positive sample containing ppb aflatoxin B1 (Fig 10) The limit of detection was equal to that of commercial kits (Afla-Check, VICAM) Obtained result from positive sample indicated that aflatoxin B1 bound to anti-AFB1 antibody and compete binding space for AFB1-BSA antigen leading to no anti-AFB1 antibody/AuNP conjugates was captured at the target line on the nitrocellulose membrane 305 CƠNG NGH ENZYME VÀ HỐ SINH Figure 11 Immunochromatographic detection of AFB1 CONCLUSION Critical component of LFA-based test strip including polyclonal antibodies and was generated The LFA-based strip was successfully constructed in this study with optimal conditions The test strip was checked to detect aflatoxin B1 with LOD of ppb The generated test strip can be used to detect aflatoxin B1 in feed samples, plant products and contaminated others Acknowledgement: The research funding from Ministry of Agriculture and Rural Development (Researching on the development of KIT for rapid detection of Aflatoxin B1 in feed and Aflatoxin M1 in milk) was acknowledged REFERRENTS Abdel-Hadi AM, Caley DP, Carter DR, Magan N (2011) Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene Toxins (Basel) 3:647-659 Fakruddin M, Chowdhury A, Hossain MN, Ahmed MM (2015) Characterization of aflatoxin producing Aspergillus flavus from food and feed samples Springerplus 4:159 Kim SH, Cha SH, Karyn B, Park SW, Son SW, Kang HG (2011) Production of group specific monoclonal antibody to aflatoxins and its application to enzyme-linked immunosorbent assay Toxicol Res 27:125-131 Krska R, Molinelli A (2009) Rapid test strips for analysis of mycotoxins in food and feed Anal Bioanal Chem 393:67-71 Rajasekaran K, Sayler RJ, Sickler CM, Majumdar R, Jaynes JM, Cary JW (2018) Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182 Plant Sci 270:150-156 Wacoo AP, Wendiro D, Vuzi PC, Hawumba JF (2014) Methods for detection of aflatoxins in agricultural food crops Journal of Applied Chemistry 2014:15 Zhang G, Guo J, Wang X (2009) Immunochromatographic lateral flow strip tests Methods Mol Biol 504:169-183 NGHIÊN CỨU TẠO VÀ ỨNG DỤNG KHÁNG THỂ KHÁNG AFLATOXIN B1 ĐỂ PHÁT TRIỂN QUE THỬ NHANH 1* Nguyễn Thị Ngọc Ánh , Phạm Thị Ngọc , Ngô hu Hƣờng , rƣơng Quốc Phong Viện Công nghệ Sinh học Công nghệ Thực phẩm, Trường Đại học Bách khoa Hà Nội Viện Thú y Trung tâm Nghiên cứu sản xuất vắcxin sinh phẩm y tế, Bộ Y tế TÓM TẮT Aflatoxin sản phẩm phụ sinh tr nh sinh trưởng nấm Aspergillus flavus Aspergillus parasiticus tìm thấy loạt sản phẩm thực vật hạt ngô, đậu phộng, ngũ cốc Aflatoxin tìm thấy sản phẩm sữa, trứng thịt ăn phải aflatoxin từ thức ăn nhiễm nấm flatoxin phân loại thành số nhóm nhóm quan trọng aflatoxin B1, B2, G1 G2 Trong số aflatoxin, aflatoxin B1 (AFB1) hợp chất độc người động vật Phát aflatoxin thực số phương pháp T C, H T C, HPLC, LC-MS / MS, FTIR, RI , E IS , S R, điện hóa, que thử miễn dịch Mỗi phương pháp có ưu điểm nhược điểm riêng Tuy nhiên, có phương pháp que thử nhanh phù hợp sử dụng ngồi trường Do đó, mục 306 H I NGH KHOA H C CƠNG NGH SINH H C TỒN QU C 2018 tiêu nghiên cứu tạo kháng thể đặc hiệu AFB1 xác định điều kiện tối ưu để phát triển que thử phát nhanh aflatoxin B1 Cộng hợp AFB1-K H tiêm vào thỏ để tạo kháng thể đặc hiệu Kháng thể đa dòng đặc hiệu F tinh xác định đặc tính SDS-PAGE, Dot-blot E IS Các điều kiện tối ưu để cộng hợp kháng thể kháng aflatoxin B1 với hạt nano vàng ( u ) xác định: lượng kháng thể ng, kích thước hạt nano vàng 40 nm, pH 7,5, nhiệt độ 25oC, thời gian 30 phút Cộng hợp kháng thể anti-AFB1/ u chuẩn bị đệm tối ưu (20 mM sodium borate, 3% sucrose, 1% BSA, 1% glycerol mM CaCl2) hấp phụ lên miếng thấm 37oC 30 phút để tạo miếng cộng hợp Cộng hợp AFB1-BSA kháng thể dê kháng IgG thỏ cố định màng nitrocellulose để tạo que thử Que thử kiểm tra để phát aflatoxin B1 với LOD ppb Que thử nghiệm tạo sử dụng để phát aflatoxin B1 mẫu thức ăn, sản phẩm thực vật bị nhiễm bẩn Từ khóa: Aflatoxin B1, cộng hợp aflatoxin B1- BSA, cộng hợp aflatoxin B1- KLH, kháng thể kháng aflatoxin B1, ngưng kết miễn dịch hạt nhựa, que thử nhanh 307 Vietnam Journal of Science and Technology 56 (4A) (2018) 190-198 EFFICIENT CONJUGATION OF AFLATOXIN B1 WITH BOVINE SERUM ALBUMIN FOR THE DEVELOPMENT OF AFLATOXIN B1 QUICK TEST Truong Quoc Phong1, *, Pham Thi Ngoc2, Nguyen Dieu Huong1, Nguyen Thi Ngoc Anh1 School of Biotechnology and Food Technology, Hanoi University of Science and Technology, No.1 Dai Co Viet, Hai Ba Trung, Ha Noi National Institute of Veterinary Research, No.86 Truong Chinh, Ha Noi * Email: phong.truongquoc@hust.edu.vn Recieved: 23 July 2018; Accepted for publication: October 2018 ABSTRACT Among several methods to detect the toxic and cancerous aflatoxins in agricultural products used in foods and animal feeds, only the lateral flow immunodipstick method is suitable for field usage The aim of this study is to determine optimal conditions for the conjugation of aflatoxin B1 (AFB1) with bovine serum albumin (BSA) to develop a lateral flow immunoassay test strip for detection of AFB1 Optimal conditions for the generation of the intermediate compound AFB1-CMO were: AFB1/CMO ratio of 1:2, AFB1 concentration of mM, reflux temperature of 80 oC, reaction time of hour The optimal conditions for further conjugation of AFB1-CMO with bovine serum albumin (BSA) were: AFB1-CMO/BSA ratio of 40:1 in bicarbonate buffer pH 9.5, reaction temperature of 25 oC, reaction time of hours The generated conjugate AFB1-BSA was used to construct a lateral flow immunoassay test strip for the detection of aflatoxin B1 Keywords: aflatoxin B1, aflatoxin B1- BSA conjugate, quick test INTRODUCTION Aflatoxins are carcinogen secondary metabolites produced mainly by the fungi Aspergillus flavus and Aspergillus parasiticus [1] These fungi contaminate a wide range of agricultural products, such as peanuts, corn, rice, and animal feed [2] Due to appropriate climate for fungi growth, the rate of aflatoxin contaminated agricultural products and food in Vietnam is very high (95.4 %) Twenty three to sixty seven percent of them exceeded the maximum levels set by the Ministry of Health [3-5] Aflatoxins are classified as group I carcinogens by the International Agency for Research on Cancer (IARC) [6] Aflatoxins are difuranocoumarines synthesized through the polyketide pathway [7] There are types of aflatoxins commonly found in food, including B1, B2, G1, and G2 Aflatoxin B1 (AFB1) is the most common compound and shows the highest toxicity [8] Efficient conjugation of aflatoxin B1 with bovine serum albumin applying for ……… Several methods have been proposed for the detection and determination of AFB1 in agricultural food crops and feeds, including thin layer chromatography (TLC) [9, 10], high performance liquid chromatography (HPLC) [11, 12], and immunoassays [13-16] TLC method is simple, easy and economic to perform, but the sensitivity and accuracy of TLC is low HPLC is widely accepted as an official method for aflatoxin detection However, HPLC method is expensive and time-consuming Both methods require skilled technician, pretreatment of sample and well-equiped laboratory Furthermore, they are unsuitable for the routine screening of large sample numbers and on-site analysis Immunoassay, especially the rapid test strip is becoming more widespread because of its sensitivity, specificity, rapidity, simplicity and costeffectiveness The rapid test strip is suitable for the on-site application in detection of aflatoxin The test strip is based on immunochromatographic principle and often called lateral flow assay This technique ultilizes antigen and antibody properties to rapidly detect an analyte For the development of a test strip to detect aflatoxin B1, two crucial components are requested, an AFB1-carrier conjugate and an anti-AFB1 antibody – gold nanoparticle conjugate This paper reports the generation of the AFB1-BSA carrier conjugate and application of this conjugate for the development of a test strip MATERIALS AND METHODS 2.1 Materials Aflatoxin B1 (AFB1) was purchased from Cayman Chemical, USA O-(Carboxymethyl) hydroxylamine hemihydrochloride (CMO), Pyridine anhydrous, Bovine serum albumin (BSA), goat anti-mouse IgG antibody alkaline phosphatase conjugate, 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), sulfo-N-hydroxysuccinimide (sulfo-NHS), 5bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were purchased from Sigma-Aldrich, USA Monoclonal anti-Aflatoxin B1 antibody [AFA-1] was purchased from Abcam, USA Other chemicals and solvents were purchased from Merck and SigmaAldrich 2.2 Synthesis of the aflatoxin B1-carboxymethyl hydroxylamine hemihydrochloride hapten (AFB1-CMO) The AFB1-CMO hapten was generated according to the method of Wicaksono with some modifications [17] Briefly, 60 µl of a mixture of methanol/pyridine/water (4:1:1) containing 16 µg of AFB1 and 16.8 µg of CMO was refluxed at 90 oC in the dark for hours, followed by evaporation at 65 oC, 150 mbar until dry to remove pyridine The obtained brown yellow pellet was dissolved in 200 µl of 0.1 M NaOH and neutralized by 20 µl of M HCl The AFB1-CMO was extracted by adding 220 µl of ethyl acetate The extraction was repeated for three times and the ethylacetate phase was collected The extract was evaporated until dry to remove ethylacetate The pellet was dissolved in DMF/H2O (6:9) and characterized using TLC and FTIR method 2.3 Synthesis of AFB1-BSA conjugate The conjugate AFB1-BSA was prepared by mixing 54 µg of AFB1-CMO with 80 µl of DMF in phosphate buffer pH 5.7 (6:9) Then µl of 0.1 M EDC and µl of 0.1 M sulfo-NHS was added, followed by incubation for 15 Then the solution was added dropwise into the BSA solution (1 mg/ml in carbonate buffer pH 9.5) and incubated at 25 oC for hours The 191 Truong Quoc Phong, Pham Thi Ngoc, Nguyen Dieu Huong, Nguyen Thi Ngoc Anh conjugate AFB1-BSA was washed three times with PBS pH 7.4 and stored at -20 oC The result was checked by dot-blot method 2.4 Dot-blot method One µg of AFB1-BSA conjugate was dotted on the nitrocellulose membrane The membrane then was bloked with % non-fat dry milk for hour and washed three times with PBS pH 7.4 After washing, the membrane was incubated in solution of monoclonal anti-AFB1 antibody for hours After three washes, the membrane was incubated in solution of second antibody of anti-mouse IgG antibody alkaline phosphatase conjugate, followed by three washes The signal was developed by adding the solution of NBT and BCIP and quenched by adding H2SO4 2.5 Thin layer chromatography Thin layer chromatography - TLC was used to analyze aflatoxin B1 and AFB1-CMO TLC was performed by spotting the product and standard aflatoxin at position of cm from the base of a silica gel plate followed by running the TLC plate in a mixture of chloroform and acetone (9:1) The result was visualized by UV exposure RESULTS AND DISCUSSION 3.1 Synthesis of the AFB1-CMO hapten 3.1.1 Optimizing the AFB1 to CMO ratios and concentration of AFB1 AFB1 is a low molecular weight compound (312.27 g/mol) and considered as a hapten Therefore, in order to recognize this toxin by specific antibody, aflatoxin B1 needs to be conjugated with a protein carrier Since AFB1 lacks a reactive group for the coupling of the toxin to a protein carrier, AFB1 needs first to react with O-(Carboxymethyl) hydroxylamine hemihydrochloride (CMO) to generate the AFB1-CMO hapten (Fig 1) Aflatoxin B1 - CMO Aflatoxin B1 N CMO Figure The formation of the AFB1-CMO hapten Theoretically, aflatoxin molecule will be coupled to CMO with a molar ratio of 1:1 In this study, four different molar ratios (AFB1/CMO 1:1, 1:2, 1:3, 1:4) were investigated The results obtained from TLC showed that the product (AFB1-CMO) was more polar than aflatoxin B1 TLC results showed that there were two spots on the TLC pattern of reaction with molar ratio of 1:1, whereas there was only one spot on the others (Fig 2) The spot in the upper part of the lane (ratio 1:1) had the same Rf value as the standard aflatoxin B1 (Fig 2A, lane 1), and the spots with lower Rf value were the product AFB1-CMO (Fig 2A, lane 3-5) Obtained result indicated that AFB1 was completely converted to AFB1-CMO with a minimum AFB1/CMO ratio of 1:2 192 Efficient conjugation of aflatoxin B1 with bovine serum albumin applying for ……… Furthermore, four different concentrations of AFB1 were investigated, namely 1, 2, and mM The results showed that complete coupling reaction was observed at concentration of mM (Fig 2B) A B 5 Figure (A) TLC analysis of the products from reaction between AFB1 and CMO Lane 1: Standard aflatoxin B1; Lanes 2-5: AFB1/CMO ratios were 1:1, 1:2, 1:3 and 1:4, respectively (B) Effect of AFB1 concentration on the reaction between AFB1 and CMO Lanes 1-4, reaction products at AFB1 concentrations of 1, 2, 4, mM Lane 5, standard aflatoxin B1 3.1.2 Optimizing the reaction temperatures and times In the present study, the reaction of AFB1 with CMO was performed at five different temperatures of 70, 80, 90, 100 and 110 oC The reaction was incomplete at lower temperature (70 oC) (Fig 3, lane 2) and complete at temperature higher than 80 oC (Fig 3A, lane 3-6) This reaction was carried out at 86 oC by Wicaksono et al [17] and at 110 oC by Kim et al [18] We observed that the spot intensity of AFB1-CMO was decreased at 110 oC Therefore, suitable temperature for coupling reaction of AFB1 and CMO should be in a range of 80 - 100 oC B A Figure (A) Effect of temperatures on the reaction between AFB1 and CMO Lane 1, standard aflatoxin B1; lanes 2-6, coupling products at different temperatures of 70, 80, 90, 100 and 110 oC (B) Reaction time of AFB1 and CMO Lane 1, standard aflatoxin B1; lanes 2-5, coupling products at different times of 1, 2, 3, hours Four different reaction times of 1, 2, and hours were investigated to identify the suitable reaction time for coupling AFB1 and CMO The results showed that AFB1 has completely reacted with CMO after one hour at 80 oC (Fig 3B) This reaction was carried out for hours by Khademi et al [19] and even for hours by Wicaksono et al [17] 3.1.3 Characterization of the hapten AFB1-CMO In order to confirm the coupling of AFB1 to CMO, the reaction product was analyzed by TLC, HPLC and FT-IR The TLC results showed spots of the hapten seem to be not moving in chloroform – acetone (9:1) solvent system, while the standard aflatoxin B1 was reached near the 193 Truong Quoc Phong, Pham Thi Ngoc, Nguyen Dieu Huong, Nguyen Thi Ngoc Anh solvent front (Fig 3A) In addition, the product of the coupling reaction was analyzed by HPLC (Fig 4) The results showed that the free AFB1 peak appeared at 13.15 min, AFB1-CMO peak appeared at 29.33 B A Figure HPLC chromatogram of standard AFB1 (A) and AFB1-CMO (B) Characterization using FT-IR spectroscopy showed a peak at 1634 cm-1, indicating the absorption of the C=N groups (Fig 5) The peak was also observed in spectra of AFB1 due to C=C bonds The intensity of this peak increase as the C=N group was created after coupling CMO to AFB1 Furthermore, the presence of a broad peak at 3433 cm-1 indicated the absorption of the O-H groups The increase of peak intensity is due to the presence of carboxylic group after coupling CMO to AFB1 Figure Fourier-Transform Infrared Sprectra of the aflatoxin B1 (solid line) and the hapten of AFB1-CMO (dotted line) 3.2 Conjugation of AFB1-CMO to Bovine Serum Albumin (BSA) 3.2.1 Optimizing the AFB1-CMO to BSA ratios Aflatoxin B1 - BSA Aflatoxin B1-CMO N N Carrier protein - BSA EDC/Sulfo-NHS Figure Reaction scheme of AFB1-BSA synthesis 194 N BSA H Efficient conjugation of aflatoxin B1 with bovine serum albumin applying for ……… The AFB1-CMO hapten is conjugated to the protein carrier BSA as shown in Fig Theoretically, one BSA molecule can couple with a maximum of 35 molecules of AFB1-CMO based on the number of lysine and Ɛ-amine residues of BSA In order to determine the optimal ratio of AFB1-CMO to BSA, five molar ratios of 10:1, 20:1, 30:1, 40:1, 50:1 were investigated The conjugation efficiency was checked by dot blot method The result showed that the increase of molar ratio of AFB1-CMO/BSA resulted in the increase of conjugation efficiency (Fig 7) The significant efficiency was obtained at the molar ratio of 40:1 The results obtained from spectrometric method by Khademi et al showed that 12 moles of AFM1-CMO were successfully conjugated to mole of BSA [19] 250000 BSA 10:1 30:1 B1-BSA 20:1 40:1 Spot Intensity 200000 50:1 150000 100000 50000 10:1 20:1 30:1 40:1 50:1 Ratios of AFB1-CMO and BSA Figure Dot blot analysis of products from conjugation reactions of AFB1-CMO and BSA with different ratios (10:1, 20:1, 30:1, 40:1, 50:1) BSA, bovine serum albumin; B1-BSA, standard conjugate 3.2.2 Effect of pH, temperature and time on the conjugation efficiency The conjugation reaction of AFB-CMO and BSA is catalyzed by 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (Sulfo-NHS) EDC reacts with carboxylic acid group of CMO moiety of AFB1-CMO to form reactive intermediate follows by reacting with Sulfo-NHS to form a stable active ester In the presence of amine nucleophiles from protein (BSA), the sulfo-NHS ester is rapidly hydrolyzed, allowing the formation of an amide bond between BSA and AFB1-CMO The activation reaction with EDC and Sulfo-NHS is pH dependent In this study, the reaction was carried out at three different pH values of 4.5, 7.4 and 9.5 The result showed that the highest conjugation efficiency was observed in carbonate buffer pH 9.5 (Fig 8) 350000 300000 Spot Intensity 250000 BSA Ace Car 200000 150000 100000 50000 B1-BSA PBS Acetate Buffer, pH 4.5 PBS, pH 7.4 Carbonate Buffer, pH 9.5 Figure Dot blot analysis of products from conjugation reactions of AFB1-CMO and BSA in different buffers: acetate buffer pH 4.5 (Ace), phosphate bufferred saline (PBS) pH 7.4, carbonate buffer pH 9.5 (Car) BSA, bovine serum albumi; B1-BSA, standard conjugate 195 Truong Quoc Phong, Pham Thi Ngoc, Nguyen Dieu Huong, Nguyen Thi Ngoc Anh 400000 Spot Intensity 350000 BSA 300000 250000 o 25 C 200000 o 150000 37 C 100000 50000 B1-BSA 25 37 Temperature (oC) Figure Dot blot analysis of products from conjugation reactions of AFB1-CMO and BSA at 25oC and 37oC BSA, bovine serum albumin; B1-BSA, standard conjugate The conjugation reaction was carried at 25 oC and 37 oC and showed that the conjugation reaction at 25 oC was better than 37 oC (Fig 9) The conjugation reaction was carried out for 0.5, 1.0, 2.0, 3.0 and 4.0 hours The results indicated that the highest conjugation efficiency was obtained for hours incubation (Fig 10) 400000 Spot Intensity 350000 300000 BSA 250000 0.5 2.0 B1-BSA 1.0 3.0 4.0 200000 150000 100000 50000 0.5 1.0 2.0 Time (hours) 3.0 4.0 Figure 10 Dot blot analysis of products from conjugation reactions of AFB1-CMO and BSA for 0.5, 1.0, 2.0, 3.0 and 4.0 hours BSA, bovine serum albumin; B1-BSA, standard conjugate 3.3 Preliminary application of AFB1-BSA for generating a lateral flow immunoassay test strip Figure 11 A format of the lateral flow test strip generated by the combinantion of AFB1-BSA immobilized membrane and anti-AFB1 antibody – gold nanoparticles conjugate Signal band is indicated by arrow The generated AFB1-BSA conjugate was immobilized on a nitrocellulose membrane and assembled with anti-AFB1 antibody – gold nanoparticles conjugate to prepare a lateral flow 196 Efficient conjugation of aflatoxin B1 with bovine serum albumin applying for ……… immunoassay test strip The result showed there was a clear band on the observation window of the test strip (Fig 11) The presence of a signal band indicated that AFB1-specific antibody in conjugate with gold nanoparticles recognized the hapten of AFB1 on the surface of protein carrier BSA which was immobilized on the nitrocellulose membrane CONCLUSIONS Aflatoxin B1 (AFB1) was shown to form the hapten AFB1-CMO by condensation with O(carboxymethyl) hydroxylamine hemihydrochloride (CMO) The optimal reaction conditions were found as follows: AFB1/CMO ratio of 1:2, AFB1 concentration of mM, reflux temperature of 100 oC, reaction time of hours Successful condensation of AFB1 with CMO was confirmed by TLC, HPLC and FT-IR methods The optimal conditions for further conjugation of AFB1-CMO with bovine serum albumin (BSA) were found as follows: AFB1CMO/BSA ratio 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