Development of a lateral flow immunoassay strip for rapid detection of rotavirus in fecal samples - Do Thi Thu Ha

TAP CHI SINH HOC 2015, 37(1se): 12-17 DOI: 10.15625/0866-7160/v37n1se. DEVELOPMENT OF A LATERAL FLOW IMMUNOASSAY STRIP FOR RAPID DETECTION OF ROTAVIRUS IN FECAL SAMPLES Do Thi Thu Ha1, Ngo Thu Huong2, Nguyen Dang Hien2, Le Thi Luan2, Luong Trinh Thuy Linh1, Truong Quoc Phong1* 1Hanoi University of Science and Technology, Ministry of Education and Training, *phong.truongquoc@hust.edu.vn 2Center for Research and Production of Vaccines and Biologicals, Ministry of Health ABSTRACT: Rotavirus is the most common cause of severe, dehydrating diarrhea in children worldwide. Without treatment rotavirus infection may result in severe illness with dehydration and disturbances of the body’s normal electrolyte balance, especially in babies and preschool children. Rotavirus is the cause of up to 50% of the hospitalized cases of diarrheal illness in infants and young children and a major cause of infant mortality in the developing countries. Rapid detection of rotavirus infection could help in effective treatment of diarrheal patients. In this paper, we reported the results of the development of a lateral flow immunoassay (LFA) strip for rapid detection of rotavirus. Rabbit and guinea pig anti-rotavirus IgGs were successfully purified from serum by protein A affinity chromatography. For making the conjugate pad, optimal amount of rabbit anti-rotavirus antibody was 0.25 µg/pad. The optimal amount of guinea pig anti-rotavirus antibody for immobilization on nitrocellulose membrane was 0.1 µg at the test line. The cellulose membrane could be processed at 4oC and 25oC overnight or 42oC for 30 mins after antibody immobilization. The detection limit of the generated LFA strip was 1.6 ´ 104 virus particles. The produced LFA strips were tested with 30 fecal specimens and compared with ELISA assay. Keywords: diarrhea, feces, lateral flow immunoassay, rapid detection, rotavirus. INTRODUCTION Rotaviruses are one of the major causes of pediatric gastroenteritis and diarrhea. Untreated, rotavirus infection may results in severe illness with dehydration and disturbance of the body’s normal electrolyte balance, and a major cause of infant mortality in the developing countries [2]. From 1999, surveillance system of rotavirus diarrhea has been established in Asian countries including Vietnam. The report of surveillance showed that rotavirus infection explains for up to 55-60% of the hospitalized cases of diarrheal illness in infants and young children [3-5]. Estimated by WHO, 20% of deaths in children under 5 years old was due to rotavirus infection. Rotavirus diarrhea has become a burden for all countries in the world [1, 6]. Together with epidemiological surveillance for producing an efficient vaccine, diagnosis of rotavirus infection is absolutely necessary for doctor to provide an appropriate and efficient method of treatment. Several methods for detection of rotavirus are available such as electron microscope, detection of rotavirus antigens (ELISA, latex agglutination, immunochromatography), detection of nucleic acid (polyacrylamide gel electrophoresis-PAGE, RT-PCR, nucleotide sequencing) [8-10]. However, each method is presenting advantages and disadvantages in such application. Almost these methods are time consuming and suitable for detection of rotavirus in well equipped laboratories. Detection of rotavirus infection in patients requires a quick, simple method and could be performed on site. Among them, immunochromatographic lateral flow assay (LFA) is considered as the best way for rotavirus detection from patients [7, 11]. Comercial kits were using monoclonal anibodies, whereas rotaviruses are antigenically complex with multiple serotypes. Therefore, diagnosis in some cases is incorrect. In this study, we present the development of a lateral flow immunoassay for detection of rotavirus using polyclonal antibodies. MATERIALS AND METHODS Standard rotavirus sample was from ProSpecTTM Rotavirus Microplate Assay (Oxoid Ltd, UK). Serum samples from rotavirus infected rabbits and guinea pigs were from POLYVAC-Ministry of Heath. Materials for making lateral flow strip were purchased from Shanghai JY-BiotechTM. Protein A-Sepharose 4B conjugate was purchased from Invitrogen (USA). Chemicals for IgGs purification, polyacrylamide gel electrophoresis and buffers for immunochromatographic lateral flow assay were purchased from Sigma, Merck, Fermentas. Procedure for IgG purification was performed according to the instruction of Protein A-Sepharose kit using AKTA FPLC system (GE Healthcare, USA). Conjugation of IgG with colloidal gold and conjugate pad preparation were performed according to Zhang et al. (2009) [11]. Briefly, the colloidal gold suspension is adjusted the pH to 9.0 with 0.2 M K2CO3. Appropriate amount of antibody is added to the colloidal solution, mixed rapidly and then incubated at room temperature for 30 min. One ten volume of 10% BSA is added and mixed rapidly, then incubated for further 15 min at room temperature. Unbound IgG is removed by centrifugation. The pellet is washed twice with 20 mM sodium borate containing 1% BSA. Finally, the pellet is suspended in 20 mM sodium borate containing 2% BSA, 3% sucrose, 0.6 M NaCl, 0.2% Tween 20 and loaded on the conjugate pad. Then the conjugate pad is dried at 42oC for 30 min in air drying oven. Preparation of blotting membrane: the nitrocellulose membrane was cut into pieces with appropriate sizes and assembled on the CAMAG Linomat 5 automatic sampler platform. The antibody solution was dispensed onto the membrane at 1.5 µl/cm. The blotted membrane then is dried at 42oC in an air drying oven. RESULTS AND DISCUSSION Purification of anti-rotavirus IgG Protein A can bind specifically to Fc regions of many mammalian immunoglobulins and commonly used as affinity absorbents to purify antibodies from serum. In present study, protein A - sepharose 4B conjugate was used for affinity purification of anti-rotavirus IgGs from sera of rabbit and guinea pig. The result of rabbit IgG purification shows that there were only two separate peaks on the chromatographic chart with retention times of 3.93 and 86.41 min, respectively (fig. 1A). The first peak was present just after loading sample (3.93 min) indicating that proteins in this fraction were unbound proteins. The second peak was present just after adding the low pH glycine solution (pH 2.7) indicating that proteins in this fraction were protein A specific binding IgGs. Under low pH condition the weak linkages between protein A and IgGs will be broken to release IgGs in eluent fraction. The protein pattern of eluent fraction was analyzed by SDS-PAGE and showed the presence of two protein bands with size of approximately 50 and 25 kDa (fig. 1, lane 3). These bands were the IgG heavy chain and light chain, respectively. Observed results demonstrated that IgG was purified from serum using protein A-sepharose gel. After PBS pH 7.4 buffer exchange using Amicon Ultra-4 30K, the amount of purified IgG was determined as 1.4 mg/ml serum by bradford assay. For guinea pig IgG purification, the similar results were observed and showed in fig. 1C, D. The amount of purified IgG was determined as 1.86 mg/ml serum. Obtained IgGs were stored at -20oC for further experiments. Optimization of LFA strip generating conditions Conjugation of antibody with colloidal gold In general, a protein maximally absorbs on the gold nanoparticle (GNP) surface at the isoelectric point (pI) of the molecule or 0.5 pH units higher [11]. For optimal binding of the antibody while retaining a high degree of specific activity, the pH value of the gold solution must be adjusted to slightly higher than the pI of the coating antibody prior to conjugation. In this study, polyclonal antibody was used for conjugation therefore the pH value of colloidal solution was adjusted higher than 9.0. The appearance of a clear band on the strip after loading rotavirus (fig. 2) indicated that antibodies were well bound to gold nanoparticles to generate an antibody-GNP conjugate. A C B D Figure 1. Anti-rotavirus IgGs affinity purification using Protein A - epharose beads Chromatographic chart (A, C) and protein pattern (B, C) of purified IgGs from rabbit and guinea pig sera, respectively. M, protein marker; 1. whole serum; 2. Flow through (peak 1); 3. Eluted IgG fraction (peak 2). Figure 2. Conjugation of rabbit anti-rotavirus antibody with gold nanoparticles Optimization of IgG amount for conjugating with GNPs The amount of antibody will influence in the number of antibody molecules absorping on the surface of GNP. The optimal amount of antibody may be application-dependent. Therefore, it is necessary to optimize the amount of antibody for conjugation. The optimal ratio of antibody concentration to colloidal gold solution is determined prior to conjugation by adding several dilutions of antibody into a given amount of colloidal gold concentration. The color of samples gradually changes from brilliant red to blue as the concentration of protein decreases. The highest dilution of the solution with no change of color contains the optimum antibody for colloidal gold labeling. In this study, serially twofold dilution of rabbit anti-rotavirus antibody was added to 10 µl of GNP solution and resulted in no change of color at antibody amount of 0.25 µg (fig. 3a). The optimal amount of antibody was determined as 0.25 µg/10 µl of AuNP with OD530 ~ 10.0 (fig. 3b). Optimization of IgG amount for immobilization 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. For IgG, the approximate loading capacity is 1 µg/cm2. Multiplying the loading capacity of IgG by the surface area ratio of the membrane (50-200) produces an approximate IgG binding capacity of 50-200 µg/cm2. In a typical test strip, the test line is 0.03 cm2, the amount of IgG that can be bound is 1.5-6 µg. This is normally greater than required for most assays. In this study, the different amounts of guinea pig anti-rotavirus antibody (0.1, 0.33, 0.66, 1.0 µg) were loaded on the nitrocellulose membrane to determine the appropriate IgG amount for detection of rotavirus. The result showed that a significant signal was observed at 0.1 µg IgG immobilized (fig. 4a) indicated that the appropriate amount of IgG for immobilization on the nitrocellulose membrane was approximate 0.1 µg. Red Red Red Dark red Dark red 1.0 0.5 0.25 0.125 0.0625 µg a b 0.1 µg 0.33 µg 0.66 µg 1.0 µg 1.6 x 106 1.6 x 106 a 4oC 25oC 42oC 1.6 x 106 1.6 x 106 b c Figure 4. Immobilization of different IgG amounts (a) on the nitrocellulose membrane at the different temperature conditions (b) and after 10 days at room temperature (c) Figure 3. Optimization of antibody amount for conjugation with gold nanoparticles a. colour checking of mixtures between serial dilutions of antibody and gold nanoparticle solution; b. The testing strips for rotavirus detection using conjugate pad with different amounts of antibody. It is critical to dry the membrane completely to fix IgGs to the nitrocellulose. Failure to dry the membrane completely may lead to the loss of antibodies from the capture lines. Drying efficacy is a function of temperature, humidity, airflow and time; therefore in this study we performed drying the dispensed membrane at 4oC and 25oC overning and 42oC for 30 mins. The results indicated that drying efficacy were the same in the different temperature conditions (fig 4b & c). The membrane drying should be performed at 42oC for 30 min for fixingantibody to nitrocellulose membrane. The limit of rotavirus detection To test the ability of the generated strip to detect low concentrations of rotaviruses, we performed a 10-fold serial dilution of a standard rotavirus sample and then subjected each dilution to strip. The results indicated that the limit of detection of the generated strip was 1.6×104 virus particles/ml (fig. 5). This virus titer was far below those found during the active phase of the disease (107-1011 virus particles/ml). NC 1.6 × 103 1.6 × 104 1.6 × 105 1.6 × 106 1.6 x 106 1.6 x 106 Figure 5. The limit of detection of test strip for detection of rotavirus. Figure 6. Detection of facel samples by generated strips. Samples labeled with number 1, 2, 3, 7 were negative and with other numbers were positive Detection of rotavirus in fecal samples Unlike the standard sample the fecal specimens may contain several inhibitor components that can affect on the working of strip. The strips were tested with thirty fecal samples (4 negative and 26 positive samples tested by ELISA) of patients collected from National Children Hospital (NCH) (fg. 6). Those samples were tested with our strips and showed the similar result in comparison with commercial ELISA kit. Obtained results indicate that LFA strips produced in this study are compatible for detection of rotavirus in fecal samples from diarrheal children patients. However for further confirmation of strip in stability, sensitivity, specificity, it is necessary to be tested with a large number of samples. CONCLUSION Lateral flow immunoassay-based test is considered as powerful tool for detection of rotavirus in fecal samples at the hospitals and medical centers as well. Critical components of LFA-based strip including polyclonal antibodies and gold nanoparticles were generated. The LFA-based strip was successfully constructed in this study with optimal conditions. The strip developed in this study is useful in rapid detection of low rotavirus concentration and compatible to detect rotavirus in fecal samples of diarrheal children patients. Acknowledgement: The research was supported by project “Research on the generation of LFA strip for rotavirus detection in the diarrheal children patients” with No.B2014-01-66. REFERENCES Bresee J. S., Glass R. I., Ivanoff B., Gentsch J. R., 1999. Current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries. Vaccine, 17: 2207-2222. CDC, 2009. 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Immunochromatographic Lateral Flow Strip Tests, chapter 12 in Rasooly A. and Herold K. E. (eds.), Methods in Molecular Biology: Biosensors and Biodetection, Vol. 504, © Humana Press. NGHIÊN CỨU PHÁT TRIỂN QUE THỬ CHẨN ĐOÁN NHANH VIRUS ROTA TRONG MẪU PHÂN Đỗ Thị Thu Hà1, Ngô Thu Hường2, Nguyễn Đăng Hiền2, Lê Thị Luân2, Lương Trịnh Thùy Linh1, Trương Quốc Phong1 1Trường Đại học Bách khoa Hà Nội, Bộ Giáo dục và Đào tạo 2Trung tâm Nghiên cứu sản xuất vắc-xin và sinh phẩm y tế, Bộ Y tế TÓM TẮT Virus rota là nguyên nhân phổ biến nhất gây tiêu chảy ở trẻ em trên thế giới, chiếm 50% trường hợp trẻ tiêu chảy phải nhập viện. Nếu không được điều trị, việc nhiễm virus có thể gây huy hiểm cho trẻ nhỏ do mất nước và mất cân bằng điện giải. Chẩn đoán nhanh tác nhân gây bệnh này sẽ giúp bác sĩ đưa ra phác đồ điều trị hợp lý và hiệu quả. Trong nghiên cứu này, chúng tôi trình bày kết quả nghiên cứu tạo que thử nhanh dùng để phát hiện virus rota trong mẫu phân. Kháng thể kháng virus rota được nghiên cứu sản xuất và tinh sạch từ huyết thanh thỏ, chuột lang và được sử dụng để nghiên cứu tạo que thử. Một số điều kiện đã được nghiên cứu và tối ưu bao gồm nồng độ kháng thể thỏ kháng virus rota cố định lên hạt nano vàng (0,25 µg/miếng thấm), nồng độ kháng thể chuột lang cố định lên màng (0,1 µg/vạch). Điều kiện cố định kháng thể, ngưỡng phát hiện của que thử và thử nghiệm que thử với mẫu phân bệnh phẩm cũng đã được nghiên cứu và khảo sát. Từ khóa: Bệnh tiêu chảy, chẩn đoán nhanh, mẫu phân, que thử, virus rota. Ngày nhận bài: 22-10-2014