CLB-08590; No. of pages: 7; 4C: Clinical Biochemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
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Weixian Chen a, Jun Zhang b, Gang Lu b, Zuowei Yuan c, Qian Wu a, Jingjing Li d, Guiping Xu d, An He c, Jian Zheng c, Juan Zhang d,⁎
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Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa
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The Clinical Laboratory Center, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Artron BioResearch Inc., Burnaby, British Columbia, Canada The Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China d The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China b
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Objectives: Cholera is an acute malignant infectious disease caused by the bacteria Vibrio cholerae leading to severe dehydrating diarrhea and vomiting, even high rates of mortality in some cases. However, the prevention of the epidemic disease is achievable if proper sanitation practices are followed, provided the accurate and prompt diagnosis of each prevalent serotype in cholera epidemic. The current gold standard of bacterial culture is inadequate for rapid diagnosis. Our aim is to develop an immunochromatographic test format for O1 serotype Ogawa diagnosis and provide the need for better epidemic prevention and early response. Design and methods: The monoclonal antibodies were raised in conventional method and subsequently screened for a match pair. A variety of related and unrelated bacteria strains recruited were employed to test their sensitivity, specificity etc. by indirect ELISA. The human fecal samples were used to test the final lateralflow device product to satisfy the measurement requirement. Results: A new monoclonal antibody (McAb) pair, named IXiao3G6 and IXiao1D9, was generated, which is specifically against V. cholerae O1 serotype Ogawa. Additionally, we developed an immunochromatographic lateral flow device (LFD) using this McAb pair for the highly specific and rapid (5 min) detection of Ogawa. Conclusions: Our product has advantages of simplicity and precision, and can benefit the scene and elementary medical institutions. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc.
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Article history: Received 22 August 2013 Received in revised form 3 December 2013 Accepted 23 December 2013 Available online xxxx
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Keywords: Cholera Ogawa Monoclonal antibody Rapid flow test Lateral flow device
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Introduction
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Historically, seven great pandemics (worldwide cholera epidemics) have been recorded. Epidemics as well as pandemics of cholera are the major health threat to human [1]. Vibrio cholerae strains can be subclassified into over 200 strains; however, only those belonging to the O1 serogroup, including two serotypes: Ogawa and Inaba; and O139 serogroup can lead to epidemic and pandemic cholera [2]. Each serogroup produces similar cholera toxin, causing the similar clinic symptoms [3,4]. In many countries, V. cholerae O1 serotype Ogawa has been the strain most frequently isolated and associated with cholera outbreaks [5–7]. A different prevalent serotype in cholera epidemic will invariantly occur during each episode [8,9]. Therefore, it is imperative to set up a rapid responsive mechanism and strategy for the rapid and accurate diagnosis [10,11], which may mitigate the scale of the outbreak and achieve better epidemic prevention.
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⁎ Corresponding author at: The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, #74 Linjiang Road, Yuzhong District, Chongqing 400010, China. E-mail address: [email protected] (J. Zhang).
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Currently, the gold standard to detect V. cholerae is still the bacterial culture method, which is laborious, time-consuming and lack of sensitivity. Several modern diagnostic methods have been developed, such as PCR [12–15], ELISA [16], indirect immunofluorescent assay (IFA) [17] etc. They are faster than the traditional culture method and have much higher sensitivity, yet remain to be heavily dependent on sophisticated equipment to analyze the samples and trained personnel to interpret the results. Many cholera prevalence areas are poor and usually not well equipped with modern machines and occupied with experienced laboratory staffs, seriously confining these applications in the field [18,19]. Rapid diagnostic tests (RDTs) are qualitative immunoassays intended for use as a point-of-care test to aid in the diagnosis of cholera infection, and are more affordable than laboratory-based tests and require no laboratory infrastructure to support scale-up. The simplicity of the LFD format allows the detection to be performed with minimal training and makes it irreplaceable for the rapid diagnosis of clinic cholera cases in field conditions for investigation of epidemiology and sanitation quarantine. Since 1990, nearly two dozens of RDTs have been developed for detection of V. cholerae in human fecal samples [20–30]. Many of them are capable of distinguishing serogroup O1 from O139. Nevertheless, there is no single product that can further distinguish
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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V. cholerae O1 serotype Ogawa and Inaba, and V. cholerae O139 were kindly donated by the Sichuan Provincial Center for Disease Control and Prevention. The following bacteria included the standard strain (shown by ATCC number) and its clinical-separated strains (not listed), e.g. Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 13076), Pseudomonas Aeruginosa (ATCC 27853), Shigella sonnei (ATCC 25931), and Staphylococcus Aureus (ATCC 25923). These strains and other clinical-separated stains such as Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio metschnihovii, Aeromonas hydrophila, Aeromonas caviae and Proteus mirabilis were kindly provided by the clinical laboratory of the Second Affiliated Hospital of Chongqing University of Medical Sciences. Bacteria were inactivated at 56 °C for 30 min and the protein content of all preparations was determined. Each bacteria strain was biochemically characterized and evaluated. The extraction of endotoxic lipopolysaccharide (LPS) from experimental bacteria was followed according to standard phenol–water method described by Westphal and Jann [31] and modified by Carlson et al. [32] and Carrion et al. [33], etc. The extracted LPS from different bacteria strains was analyzed by SDS–PAGE followed by silver staining analysis.
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Animal immunization
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The 6 to 8-week-old BALB/c female mice were given four intraperitoneal injection of killed Ogawa at 2-week intervals. The blood from mice inner canthus before immunization was used as negative control. On day 1, each mouse was immunized with 1:1 mixture (v/v) of bacterial antigens and complete Freund's adjuvant (Sigma-Aldrich, Beijing, China). The booster injections were given with the same proportion of immunogen emulsified with incomplete Freund's adjuvant (Sigma-Aldrich, Beijing, China) for three times at a 2-week interval. Two weeks after the third immunization, the immune response was assessed by measuring the titer of polyclonal antibody in mice sera using indirect ELISA. The immunized mice with the highest titer were used as a spleen cells donor in hybridoma production. The booster injection via tail vein was performed 3 days before fusion. The other sera were pooled and used as positive control.
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Hybridoma cell lines preparation
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Three days after the intravenous booster, the immunized mice were eye-bled and sacrificed by cervical dislocation. The spleen cells were fused with SP2/0 myeloma cells at a ratio of 5:1 using 50% (w/v) polyethylene glycol 4000 (BioUltra; Sigma-Aldrich, Beijing, China) according to a standard protocol. The hybridoma cells were suspended in RPMI 1640 (Gibco, Grand Island, NY), supplemented with penicillin streptomycin (Sigma-Aldrich, Beijing, China), sodium pyruvate (Sigma-Aldrich, Beijing, China), and 20% fetal bovine serum (FBS) (Hyclone, Tauranga, New Zealand). The cells were centrifuged (500 g, 10 min, RT) and the pellet was resuspended in hypoxanthine–aminopterin–thymidine (HAT) (Sigma-Aldrich, Beijing, China) medium. Cells were seeded in 96-well tissue culture plates (Costar, Corning, NY) and incubated in a humidified 37 °C, 5% CO2 incubator for 2 weeks. Clones were kept in hypoxanthine thymidine (HT) medium diluted from HyClone™ (HT)
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Indirect ELISA
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The 96-well microplates were coated with 100 μL 10 μg/mL different bacteria antigens including positive and negative controls, diluted in 0.1 M NaHCO3, and incubated overnight at 4 °C. Plates were blocked for 2 h with 200 μL blocking buffer (PBS/1% BSA) at RT and washed three times in PBST (PBS/0.5% Tween-20). One hundred microliters of hybridoma culture supernatants, positive control (immunized mouse serum), and negative control (SP2/0) were accordingly added to the plate and incubated at 37 °C for 1 h. Plates were washed five times with PBST and incubated with 100 μL horseradish peroxidase conjugated goat anti-mouse immunoglobulin (IgG-HRP) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China) in blocking buffer (1:1,000) for 30 min at 37 °C. Finally, plates were washed five times with PBST and developed with 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma-Aldrich, Beijing, China) in the dark for 15 min at RT. The reaction was terminated by supplementing 50 μL of 1 M H2SO4 and absorbance values were determined at 450 nm with BioTek™ ELx808™ Absorbance Microplate Readers (ThermoFisher Scientific Inc., Beijing, China) against the blank. The titer of the antibody preparation was defined as the highest dilution that could give reading 0.05. One indirect ELISA unit was defined as the smallest amount of the antibody which gave a positive detection of antigen.
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Bacteria strains and antigen preparation
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Materials and methods
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Solution (50×) (ThermoFisher Scientific Inc., Beijing, China) for further 2 weeks. After selection by indirect ELISA, the desired cell lines were recloned three times by limiting dilution using spleen cells from a nonimmune BALB/c mouse as feeder cells to achieve monoclonality and stability. Supernatants from these clones were retested and positive candidates were expanded in large scale in a nonselective medium, and stored in liquid nitrogen.
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the different serotype of O1 serogroup. The present study selected a pair of highly sensitive and specific McAbs targeting V. cholerae O1 serotype Ogawa through a novel screening technique; and further developed the rapid diagnostic strips using this McAb pair to diagnose Ogawa infection. The product can detect pathogen rapidly with high specificity, sensitivity and reliability.
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Monoclonal antibody production and purification
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McAbs against V. cholerae O1 serotype Ogawa were produced in traditional ascetic fluid method. The 8-week old female BALB/c mice, primed with liquid paraffin, were injected intraperitoneally with 1.0 × 106 hybridoma cells per mouse. One to two weeks later, the ascetic fluid was drained with a 12-gauge needle. The ascites were centrifuged at 4000 rpm at 4 °C for 10 min to remove cells. The collected supernatants were precipitated in 50% saturated ammonium sulfate (pH 7.4), followed with extensive dialysis against 0.02 M phosphate buffer (pH 7.4) at 4 °C. The solution was purified by protein A affinity chromatography to obtain high quality McAbs. First, the column was prepared with 5 mL Affi-Gel Protein A (Bio-Rad Laboratories (Shanghai) Co., Ltd.; Shanghai, China) agarose beads soaked in binding buffer (pH 8.2) for 15 min. The dialyzed solution was loaded to the column and extensively washed with binding buffer. The flow through was collected in the fractions of 4–5 mL/tube. The OD280 of all the fractions were monitored until the reading was below 0.05 to ensure that there was no more unbound protein in the solution. The elution was performed by sequentially loading 1 mL elution buffer (pH 3.0) five times. Three hundred microliters of 1 M Tris–HCl buffer (pH 9.0) was introduced to each collecting tube in advance to neutralize the pH. Fractions of 1 mL of the eluant were collected in test tubes until the OD280 of the eluant was below 0.05. Finally, the column was washed three times with wash buffer for storage.
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McAb characterization
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The purity of the products was assessed by 10% SDS–PAGE. The specificity of the McAbs was evaluated by western blotting analysis against killed related and unrelated bacteria strains, including V. cholerae O1 Ogawa, Inaba, V. cholerae O139, V. parahaemolyticus, V. fluvialis, S. sonnei, S. typhi, A. hydrophila, P. aeruginosa, E. coli, S. aureus, P. mirabilis, etc. The titer of McAbs was determined by indirect
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Preparation of colloidal gold and colloidal gold–MAb conjugate
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Colloidal gold was prepared as previously reported [36]. Briefly, 100 mL of 0.01% (w/v) HAuCl4 in a 250-mL siliconized flask was heated to boiling in a microwave oven, and then 1.4 mL 1% trisodium citrate was added to the solution. After the colloidal gold solution was allowed to cool gradually and stored at 4 °C in a dark-colored glass bottle. The pH of the colloidal gold was adjusted to 8.4 with 1% potassium carbonate (w/v). The McAb IXiao3G6 (30 μg) was added dropwise into 10 mL of colloidal gold solution on a magnetic stirring apparatus for 30 min. After the solution was stabilized at 4 °C for 30 min, 1 mL 10% (w/v) BSA was added to block excess reactivity of the gold colloid. The mixture was then stirred for an additional 30 min and stored at 4 °C for 2 h. After the mixture was centrifuged at 3000 g at 4 °C for 30 min, the supernatant was further centrifuged at 14,000 g at 4 °C for 45 min, and the resulting conjugate pellet was suspended in 10 mM borax buffer (pH 8.0) containing 2% (w/v) BSA and 0.05% NaN3.
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Manufacture and evaluation of colloidal gold conjugated strips
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The Ogawa LFD consisted of a sample binding region called an analyte absorption pad, a result showing region including a conjugate pad and a nitrocellulose membrane, and a tag terminal called wicking pad. First, the detection antibody IXiao1D9 (the test line antibody) and goat anti-mouse IgG (the procedural control line antibody) were diluted to the working concentration and sprayed onto a piece of nitrocellulose membrane then dried at 37 °C for 24 h. The strips were subsequently blocked and dried at 37 °C. The capture antibody IXiao3G6 conjugated with colloidal gold (40 nm diameter) was sprayed twice to the fiberglass (0.5–1.5 cm × 25 cm) and dried at 37 °C. Finally, the components were assembled as a package and sliced into 4-mm wide test strips. To perform a test, after fully contacted with sample solution, the strips were kept at RT for 5 min. Two red bands that appeared at both the
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The epitope of purified McAbs was characterized by a competent ELISA and the additivity index (AI) described by Friguet et al. [35]. Briefly, the 96-well plates were first coated with 100 μL 2 μg/mL deactivated Ogawa and incubated overnight at 4 °C. One hundred microliters of IXiao3G6 or IXiao1D9 or combination (50 μL of each) was separately incubated as the primary antibody overnight at 4 °C. The secondary antibody used 100 μL goat anti-mouse IgG-HRP (1:1,000) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China). Plates were developed and analyzed as described. The index AI was calculated for each pair of antibodies by the equation (2A1 + 2 / (A1 + A2) − 1) × 100%. A1, A2 and A1 + 2 were the absorbances obtained in the ELISA using IXiao3G6, IXiao1D9 and the combination, respectively. If the two antibodies are directed against different epitopes (no competition), A1 + 2 should be equal to the sum of A1 and A2 and the AI should approach 100%. Otherwise, if the two antibodies are directed against the same epitope (competition), A1 + 2 should be equal to the mean value for A1 and A2 and the AI should tend to be 0%. The threshold in this study was determined by AI ≥ 40%. The specific epitope of McAbs was further examined by western blotting analysis against a wide range of related and unrelated LPS antigens (V. cholerae O1 Ogawa and Inaba, V. cholerae O139, V. fluvialis, A. hydrophila, S. sonnei, E. coli, S. typhi). The primary antibody was 50 μL either IXiao3G6 or IXiao1D9, and the secondary antibody was 100 μL goat anti-mouse IgG-HRP (1:1000) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China).
Results
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Balb/c mice immunization and McAb production
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Three experimental mice, XiaoA1, XiaoB1 and XiaoB3, producing high titer antibody (≥ 1:100,000) were selected as spleen cell donors. The fusion rate was 90.2%. For selection of specific McAbs, two rounds of selection were performed using indirect ELISA and McAb pairs screening, by employing both homologous antigens and heterologous antigens. A total of 602 positive hybridoma cell lines generating McAb against Ogawa were selected. By repetitive indirect ELISA and across responsive screening, 15 cell lines were obtained. After pair assembly screening, the two best candidates, IXiao3G6 and IXiao1D9, were finally achieved.
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Purification and characterization of McAb pair
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IXiao3G6 and IXiao1D9 were produced in large scale through ascitic fluid, and then purified by 50% saturated ammonium sulfate precipitation and protein A affinity chromatography. SDS–PAGE demonstrated that the purity of final McAb products was over 95% (Fig. 1A). Western blot analysis demonstrated clearly that both IXiao3G6 and IXiao1D9 could specifically detect Ogawa and did not cross-react to other experimental bacteria stains (Fig. 1B). The titer of the two McAbs in the supernatant culture of hybridomas and ascites indicated high activity (both N10− 6). The affinity constant was 1.39 × 10 9 (IXiao 3G 6) and 3.27 × 109 (IXiao1 D9 ). The subtypes of the two McAbs were IgG2b (IXiao3G6) and IgG3 (IXiao1D9). The epitope characterization study showed that the absorbance values for IXiao3 G6 , IXiao 1D 9 and the combination were 0.722, 0.744 and 1.244 respectively, which gives an AI value of 66.3%, indicating that IXiao3 G6 and IXiao1 D9 are directed against different epitopes. In order to identify the antigen epitopes of these two McAb targeted, the LPS of a range of related and unrelated bacteria were extracted, shown by silver-staining (Fig. 1C) and simultaneously detected against both McAbs. It is shown in Fig. 1D that only the extracted LPS from Ogawa could be specifically recognized by IXiao3G6 but not IXiao1D9 (data not shown), suggesting the two McAbs should target different epitopes of Ogawa: IXiao3G6 for LPS sites, and IXiao1D9 for non-LPS sites.
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Systematic characterization of LFD strips
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The sensitivity of the LFD was demonstrated by testing against the serial dilution of Ogawa pure cultures, and it was determined that the detection threshold was 1.0 × 104 cfu/mL. In 961 clinicalseparated strains, 112 of them, which were pre-identified as positive Ogawa by bacterial culture method, were equally confirmed to be positive by strips, shown by the representative test image with two solid red lines, i.e. control line and test line, in Table 1; and the rest
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test and control sites represent a positive test result. Only one red band at control location represents a negative test result. The absence of a line at the control site means the test is invalid. To determine the sensitivity of the strips for detecting Ogawa, the V. cholerae pure cultures were serially diluted at 10-fold from 109 to 103 cells/mL by saline and tested. To determine the specificity of the strips, 961 clinical-separated strains were prepared (Table 1). A total of 726 diarrhea patients feces samples (Jul, 2012–Sep 2012) provided by the Second Affiliated Hospital of Chongqing Medical University were examined by our strips, commercial strips from Zhuangdi Haohe Biological Medicine Co., Ltd. and standard bacterial culture method in parallel for comparison. The repeatability of our strips was statistically tested against 100 Ogawa cultures and 100 control cultures with 10 repeats for each culture. The stability was determined by strips kept in RT for 1 year and tested similarly.
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ELISA. The immunoglobulin subclass was determined with a commercial mouse McAb Isotyping kit following manufacturers' instructions (SigmaAldrich, Beijing, China). The affinity constant of McAb was determined by indirect ELISA according to the method described by Beatty et al. [34].
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Bacteria culture identification Ogawa positive
Aeromonas caviae
26
0
26
t1:6
Aeromonas hydrophila
21
0
21
t1:7
Escherichia coli
110
0
110
t1:8
Proteus mirabilis
96
0
96
t1:9
Pseudomonas aeruginosa
105
0
105
t1:10
Salmonella typhi
100
0
100
t1:11
Shigella sonnei
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0
15
t1:12
Staphylococcus aureus
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0
102
t1:13
V. cholerae O1 serotype Inaba
108
0
108
t1:14
V. cholerae O1 serotype Ogawa
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112
0
t1:15
V. cholerae O139
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0
t1:16
Vibrio fluvialis
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0
t1:17
Vibrio metschnihovii
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0
t1:18
Vibrio parahaemolyticus
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93
16
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of the strains were simultaneously substantiated to be negative, shown by the representative test images with only one solid red line, i.e. control line, in Table 1. In addition, 726 diarrhea patients' feces samples tested by strips and standard bacterial culture method also presented the same outcomes, which all of the fecal samples were tested to be Ogawa negative. These results collectively suggested that our LFD strip has diagnostic sensitivity of 100% and diagnostic specificity of 100%. The parallel examination of V. cholerae O1 serotype Ogawa and Inaba, and V. cholerae O139 samples (Table 1) of our strips and strips from Zhuangdi Haohe Biological Medicine Co., Ltd. demonstrated better sensitivity (Fig. 2A) of our LFD strip, with detection limit of 1.0 × 104 cfu/mL. Furthermore, our product is capable not only of distinguishing V. cholerae O1 from O139 (Fig. 2B), but also further distinguishing V. cholerae O1 serotype Ogawa from Inaba, because it is clearly demonstrated in Fig. 2C that strips from Zhuangdi Haohe continued to report positive results when Inaba samples were examined. Table 2 statistically presented all the test outcomes of comparison with strips from Zhuangdi Haohe. The repeatability of the colloidal gold strip was 100% for pure cultures both positive and negative samples, and the strips were confirmed to be stable when kept in RT for 1 year.
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Representative result
Ogawa negative
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Number of each strain
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Table 1 Bacteria strain list.
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Discussion
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Cholera continues to be considered as a serious world health issue and causes an adverse impact on economic development in many countries, especially the third world. McAb can specifically recognize and bind to the single antigenic determinants from diverse bioactive molecules such as protein, polysaccharides, and nucleic acid, which makes it an ideal candidate to V. cholerae diagnosis and prevention. Colloidal gold is one of the four pillar technologies of immuno-label. Twenty years has witnessed its rapid improvement and ever-increasing application in biomedical research, especially in medical diagnosis [37]. The combination of these two technologies delivers the promising practice of the rapid diagnosis of V. cholerae. Antibodies recognize that the similar or shared antigens among these bacteria can lower their specificity. To screen out the nonspecific McAbs, two significant biopanning processes were conducted. In the first panning process, all available vibrio bacteria and other common intestinal bacteria were engaged to absorb the nonspecific antibodies, in order to screen out the cross-reactive hybridomas. The second panning process was developed based on industrial procedures to improve the preparation and screening efficacy of McAb cell lines by combining
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Dick et al. reviewed the last two decades of peer-reviewed and gray literature on rapid diagnostic tests for V. cholerae [38]. Research showed that twenty four diagnostic tests have been developed for the detection of V. cholerae in human fecal samples. Compared to the shortcomings of
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conjugating McAbs to colloid gold (as capture antibody) and coating McAbs onto cellulose nitrate membrane (as detection antibody) to circumvent the laborious traditional screening methods, e.g. ELISA, and considerably expedited the selection.
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Fig. 1. Purification of McAb and specificity determination. A: SDS–PAGE results of the purification result of IXiao3G6 (line 1) and IXiao1D9 (line 2) from protein A affinity chromatograph. B: Western blotting analysis of IXiao 3 G 6 (upper panel) and IXiao 1 D 9 (lower panel) against different bacteria (1. V. cholerae O1 serotype Inaba; 2. V. cholerae O139; 3. V. parahaemolyticus; 4. V. fluvialis; 5. S. sonnei; 6. S. typhi; 7. A. hydrophila; 8. P. aeruginosa; 9. E. coli; 10. S. aureus; 11. P. mirabilis; 12. V. cholerae O1 serotype Ogawa). C: Silver staining of LPS extracted from different bacteria; D: western blotting analysis of these LPS samples against IXiao3G6. 1. V. cholerae O1 serotype Ogawa; 2. V. cholerae O1 serotype Inaba; 3. V. cholerae O139; 4. V. fluvialis; 5. A. caviae; 6. S. sonnei; 7. E. coli; 8. S. typhi.
Fig. 2. Specificity comparison between our RDT strips (generated from IXiao3G6 and IXiao1D9) and Zhuangdi Haohe RDT devices. The green strips are products derived from IXiao3G6 and IXiao1D9. The white cassettes are RDT devices from Zhuangdi Haohe. For each strip or cassette, the upper line is control line, and the lower line (if presented) is test line. Panel A shows the comparison results tested against of V. cholerae Ogawa (from left to right: negative control, 109, 108, 107, 106, 105, 104, and 103 cfu/mL). Panel B showed the comparison results tested against V. cholerae O139 (from left to right: 109, 108, 107, and 106 cfu/mL). Panel C showed the comparison results tested against V. cholerae Inaba (from left to right: 109, 108, 107, 106, 105, 104, and 103 cfu/mL).
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Table 2 Results of comparison with Zhuangdihaohe strips.
t2:3
Strip source
112 strains of Ogawa (106–109 cfu/mL)
108 strains of Inaba (106–109 cfu/mL)
93 strains of O139 (106–109 cfu/mL)
t2:4 t2:5 t2:6
Zhuangdihaohe strips (V. cholerae O1) Zhuangdihaohe strips (V. cholerae O139) Homemade strips (V. cholerae O1 Ogawa)
Positive (+) Negative (−) Positive (+)
Positive (+) Negative (−) Negative (−)
Negative (−) Positive (+) Negative (−)
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402
C
384 385
E
The authors have no financial interests to disclose. Acknowledgements
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This study was supported by Artron BioResearch Inc.; State HighTech Development Plan (863 program) (code: 2002AA215015); and Chongqing Medical scientific research project (code: 2009-2-200).
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[7] Siddique AK, Nair GB, Alam M, Sack DA, Huq A, Nizam A, et al. El Tor cholera with severe disease: a new threat to Asia and beyond. Epidemiol Infect Mar 2010;138(3):347–52. [8] Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, et al. Molecular analysis of V. cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates. Appl Environ Microbiol Feb 2001;67(2):910–21. [9] LaRocque RC, Harris JB, Ryan ET, Qadri F, Calderwood SB. Postgenomic approaches to cholera vaccine development. Expert Rev Vaccines 2006;5(3):337–46. [10] World Health Organization. Cholera (2010). Wkly Epidemiol Rec 2011;86:325–39. [11] Preston NW. Prevention of cholera. Lancet 2004;363(9412):898. [12] Le Roux WJ, Masoabi D, de Wet CM, Venter SN. Evaluation of a rapid polymerase chain reaction based identification technique for V. cholerae isolates. Water Sci Technol 2004;50(1):229–32. [13] Fykse EM, Skogan G, Davies W, Olsen JS, Blatny JM. Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl Environ Microbiol 2007;73(5):1457–66. [14] Lalitha P, Siti Suraiya MN, Lim KL, Lee SY, Nur Haslindawaty AR, Chan YY, et al. Analysis of lolB gene sequence and its use in the development of a PCR assay for the detection of Vibrio cholerae. J Microbiol Methods Sep 2008;75(1):142–4. [15] Low KF, Karimah A, Yean CY. A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae. Biosens Bioelectron Sep 15 2013;47:38–44. [16] Ramamurthy T, Bhattacharya SK, Uesaka Y, Horigome K, Paul M, Sen D, et al. Evaluation of the bead enzyme-linked immunosorbent assay for detection of cholera toxin directly from stool specimens. J Clin Microbiol 1992;30(7):1783–6. [17] Hasan JA, Bernstein D, Huq A, Loomis L, Tamplin ML, Colwell RR. Cholera DFA: an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of V. cholerae O1. FEMS Microbiol Lett 1994;120:143–8. [18] Dodin A, Fournier JM. Diagnosis of the cholera vibrio. In: Institut Pasteur Paris (France), editor. Laboratory methods for the diagnosis of cholera vibrio and other vibrios; 1992. p. 59–82. [19] Alam M, Hasan NA, Sultana M, Nair GB, Sadique A, Faruque AS, et al. Diagnostic limitations to accurate diagnosis of cholera. J Clin Microbiol 2010;48(11):3918–22. [20] Hasan JA, Huq A, Tamplin ML, Siebeling RJ, Colwell RR. A novel kit for rapid detection of Vibrio cholerae O1. J Clin Microbiol Jan 1994;32(1):249–52. [21] Hasan JA, Huq A, Nair GB, Garg S, Mukhopadhyay AK, Loomis L, et al. Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal. J Clin Microbiol 1995;33(11):2935–9. [22] Qadri F, Hasan JA, Hossain J, Chowdhury A, Begum YA, Azim T, et al. Evaluation of the monoclonal antibody-based kit Bengal SMART for rapid detection of Vibrio cholerae O139 synonym Bengal in stool samples. J Clin Microbiol Mar 1995;33(3):732–4. [23] Nato F, Boutonnier A, Rajerison M, Grosjean P, Dartevelle S, Guénolé A, et al. Onestep immunochromatographic dipstick tests for rapid detection of Vibrio cholerae O1 and O139 in stool samples. Clin Diagn Lab Immunol May 2003;10(3):476–8. [24] Bhuiyan NA, Qadri F, Faruque AS, Malek MA, Salam MA, et al. Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs. J Clin Microbiol 2003;41:3939–41. [25] Wang XY, Ansaruzzaman M, Vaz R, Mondlane C, Lucas ME, von Seidlein L, et al. Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population. BMC Infect Dis Feb 1 2006;6:17. [26] Kalluri P, Naheed A, Rahman S, Ansaruzzaman M, Faruque AS, Bird M, et al. Evaluation of three rapid diagnostic tests for cholera: does the skill level of the technician matter? Trop Med Int Health Jan 2006;11(1):49–55. [27] Harris JR, Cavallaro EC, de Nóbrega AA, Dos S, Barrado JC, Bopp C, et al. Field evaluation of Crystal VC Rapid Dipstick test for cholera during a cholera outbreak in Guinea-Bissau. Trop Med Int Health Sep 2009;14(9):1117–21. [28] Mukherjee P, Ghosh S, Ramamurthy T, Bhattacharya MK, Nandy RK, Takeda Y, et al. Evaluation of a rapid immunochromatographic dipstick kit for diagnosis of cholera emphasizes its outbreak utility. Jpn J Infect Dis Jul 2010;63(4):234–8. [29] Pengsuk C, Chaivisuthangkura P, Longyant S, Sithigorngul P. Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples. Biosens Bioelectron Apr 15 2013;42:229–35. [30] Banoo S, Bell D, Bossuyt P, Herring A, et al. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol Dec 2010;8(12 Suppl.): S17–29. [31] Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. In: Whistler RL, Wolfan ML, editors. Methods in carbohydrate chemistry. New York: Academic press; 1965. p. 83–91. [32] Carlson RW, Kalembasa S, Turowski D, Pachori P, Noel KD. Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development. J Bacteriol Nov 1987;169(11):4923–8.
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these diagnostic tests evaluated by authors [38], the present study has achieved several advantages. From the study design point of view, first, the large sample size employed in this study could avoid limited validity and reproducibility. Second, our study, which involved using stool samples instead of only using purified bacteria cultures, happened in many other studies, although certain limitation due to the fact of a paucity of positive cholera stool samples recruited led to the diffident interpretation. Third, this study applied a range of related and unrelated bacteria to specifically test against profound gut microflora and avoided limited data against common gut organism. All of these efforts greatly enhanced the specificity evaluation outcome. From test descriptors' point of view, this study provided real time stability and reproducibility tests results compared to others and confirmed the strips were valid in 1 year at RT. In conclusion, we achieved two McAbs against V. cholerae O1 Ogawa, IXiao3G6 and IXiao1D9, with high specificity and activity. They can specifically bind to different epitopes of Ogawa: IXiao3G6 for LPS sites, and IXiao1D9 for non-LPS sites. The RDT strip had been further successfully developed followed with conducting of a systematic evaluation to assess its specificity, sensitivity and reliability. The detection threshold for bacterial culture of the strip was 1.0 × 104 cfu/mL, which is higher than the current stipulated threshold (1.0 × 105 cfu/mL). More importantly, the product was capable of distinguishing the V. cholerae O1 Ogawa and V. cholerae O1 Inaba, which filled the gaps of the area in rapid cholera diagnosis. Overall, the outcome confirmed that this product has superior detective aspects than other parallel commercial products. Through the combination of our methods and other commercial available kits, the pathogens of cholera can be simultaneously and rapidly diagnosed, classified and differentially typed. Our perspective for future development is to design a combined detection device that can spontaneously detect and distinguish three different types of cholera all at once thereby providing more comprehensive information for better, faster monitoring and control of cholera pandemic.
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[1] World Health Organisation. Cholera (2011). Wkly Epidemiol Rec 2010;86:325–40. [2] Burgos JM, Perez JL, Garcia L, Gonzalez GS, Benitez JA, Galindo F, et al. Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo. Vaccine Feb 26 1999;17(7–8):949–56. [3] Qadri F, Wennerås C, Albert MJ, Hossain J, Mannoor K, Begum YA, et al. Comparison of immune responses in patients infected with Vibrio cholerae O139 and O1. Infect Immun Sep 1997;65(9):3571–6. [4] Smirnova NI. Cholera pathogens from the new serogroup O139: molecular-genetic features and origin. Mol Gen Mikrobiol Virusol 2002;3:23–33. [5] Nair GB, Qadri F, Holmgren J, Svennerholm AM, Safa A, Bhuiyan NA, et al. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J Clin Microbiol Nov 2006;44(11):4211–3. [6] Acosta CJ, Galindo CM, Kimario J, Senkoro K, Urassa H, Casals C, et al. Cholera outbreak in southern Tanzania: risk factors and patterns of transmission. Emerg Infect Dis 2001;7(3 Suppl.):583–7.
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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[33] Carrion M, Bhat UR, Reuhs B, Carlson RW. Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum. J Bacteriol 1990;172(4):1725–31. [34] Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods Jun 26 1987;100(1–2):173–9. [35] Friguet B, Djavadi-Ohaniance L, Pages J, Bussard A, Goldberg M. A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta
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2-subunit of Escherichia coli tryptophan synthase. J Immunol Methods Jun 10 1983;60(3):351–8. [36] Grabar KC, Freeman RG, Hommer MB, Natan MJ. Preparation and characterization of Au colloid monolayers. Anal Chem 1995;67:735–43. [37] Bendayan M. A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology. Biotech Histochem Sep 2000;75(5):203–42. [38] Dick MH, Guillerm M, Moussy F, Chaignat CL. Review of two decades of cholera diagnostics—how far have we really come? PLoS Negl Trop Dis 2012;6(10):e1845.
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
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Weixian Chen a, Jun Zhang b, Gang Lu b, Zuowei Yuan c, Qian Wu a, Jingjing Li d, Guiping Xu d, An He c, Jian Zheng c, Juan Zhang d,⁎
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Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa
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The Clinical Laboratory Center, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Artron BioResearch Inc., Burnaby, British Columbia, Canada The Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China d The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China b
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Objectives: Cholera is an acute malignant infectious disease caused by the bacteria Vibrio cholerae leading to severe dehydrating diarrhea and vomiting, even high rates of mortality in some cases. However, the prevention of the epidemic disease is achievable if proper sanitation practices are followed, provided the accurate and prompt diagnosis of each prevalent serotype in cholera epidemic. The current gold standard of bacterial culture is inadequate for rapid diagnosis. Our aim is to develop an immunochromatographic test format for O1 serotype Ogawa diagnosis and provide the need for better epidemic prevention and early response. Design and methods: The monoclonal antibodies were raised in conventional method and subsequently screened for a match pair. A variety of related and unrelated bacteria strains recruited were employed to test their sensitivity, specificity etc. by indirect ELISA. The human fecal samples were used to test the final lateralflow device product to satisfy the measurement requirement. Results: A new monoclonal antibody (McAb) pair, named IXiao3G6 and IXiao1D9, was generated, which is specifically against V. cholerae O1 serotype Ogawa. Additionally, we developed an immunochromatographic lateral flow device (LFD) using this McAb pair for the highly specific and rapid (5 min) detection of Ogawa. Conclusions: Our product has advantages of simplicity and precision, and can benefit the scene and elementary medical institutions. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc.
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Article history: Received 22 August 2013 Received in revised form 3 December 2013 Accepted 23 December 2013 Available online xxxx
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Keywords: Cholera Ogawa Monoclonal antibody Rapid flow test Lateral flow device
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Introduction
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Historically, seven great pandemics (worldwide cholera epidemics) have been recorded. Epidemics as well as pandemics of cholera are the major health threat to human [1]. Vibrio cholerae strains can be subclassified into over 200 strains; however, only those belonging to the O1 serogroup, including two serotypes: Ogawa and Inaba; and O139 serogroup can lead to epidemic and pandemic cholera [2]. Each serogroup produces similar cholera toxin, causing the similar clinic symptoms [3,4]. In many countries, V. cholerae O1 serotype Ogawa has been the strain most frequently isolated and associated with cholera outbreaks [5–7]. A different prevalent serotype in cholera epidemic will invariantly occur during each episode [8,9]. Therefore, it is imperative to set up a rapid responsive mechanism and strategy for the rapid and accurate diagnosis [10,11], which may mitigate the scale of the outbreak and achieve better epidemic prevention.
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⁎ Corresponding author at: The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, #74 Linjiang Road, Yuzhong District, Chongqing 400010, China. E-mail address: [email protected] (J. Zhang).
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Currently, the gold standard to detect V. cholerae is still the bacterial culture method, which is laborious, time-consuming and lack of sensitivity. Several modern diagnostic methods have been developed, such as PCR [12–15], ELISA [16], indirect immunofluorescent assay (IFA) [17] etc. They are faster than the traditional culture method and have much higher sensitivity, yet remain to be heavily dependent on sophisticated equipment to analyze the samples and trained personnel to interpret the results. Many cholera prevalence areas are poor and usually not well equipped with modern machines and occupied with experienced laboratory staffs, seriously confining these applications in the field [18,19]. Rapid diagnostic tests (RDTs) are qualitative immunoassays intended for use as a point-of-care test to aid in the diagnosis of cholera infection, and are more affordable than laboratory-based tests and require no laboratory infrastructure to support scale-up. The simplicity of the LFD format allows the detection to be performed with minimal training and makes it irreplaceable for the rapid diagnosis of clinic cholera cases in field conditions for investigation of epidemiology and sanitation quarantine. Since 1990, nearly two dozens of RDTs have been developed for detection of V. cholerae in human fecal samples [20–30]. Many of them are capable of distinguishing serogroup O1 from O139. Nevertheless, there is no single product that can further distinguish
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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V. cholerae O1 serotype Ogawa and Inaba, and V. cholerae O139 were kindly donated by the Sichuan Provincial Center for Disease Control and Prevention. The following bacteria included the standard strain (shown by ATCC number) and its clinical-separated strains (not listed), e.g. Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 13076), Pseudomonas Aeruginosa (ATCC 27853), Shigella sonnei (ATCC 25931), and Staphylococcus Aureus (ATCC 25923). These strains and other clinical-separated stains such as Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio metschnihovii, Aeromonas hydrophila, Aeromonas caviae and Proteus mirabilis were kindly provided by the clinical laboratory of the Second Affiliated Hospital of Chongqing University of Medical Sciences. Bacteria were inactivated at 56 °C for 30 min and the protein content of all preparations was determined. Each bacteria strain was biochemically characterized and evaluated. The extraction of endotoxic lipopolysaccharide (LPS) from experimental bacteria was followed according to standard phenol–water method described by Westphal and Jann [31] and modified by Carlson et al. [32] and Carrion et al. [33], etc. The extracted LPS from different bacteria strains was analyzed by SDS–PAGE followed by silver staining analysis.
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Animal immunization
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The 6 to 8-week-old BALB/c female mice were given four intraperitoneal injection of killed Ogawa at 2-week intervals. The blood from mice inner canthus before immunization was used as negative control. On day 1, each mouse was immunized with 1:1 mixture (v/v) of bacterial antigens and complete Freund's adjuvant (Sigma-Aldrich, Beijing, China). The booster injections were given with the same proportion of immunogen emulsified with incomplete Freund's adjuvant (Sigma-Aldrich, Beijing, China) for three times at a 2-week interval. Two weeks after the third immunization, the immune response was assessed by measuring the titer of polyclonal antibody in mice sera using indirect ELISA. The immunized mice with the highest titer were used as a spleen cells donor in hybridoma production. The booster injection via tail vein was performed 3 days before fusion. The other sera were pooled and used as positive control.
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Hybridoma cell lines preparation
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Three days after the intravenous booster, the immunized mice were eye-bled and sacrificed by cervical dislocation. The spleen cells were fused with SP2/0 myeloma cells at a ratio of 5:1 using 50% (w/v) polyethylene glycol 4000 (BioUltra; Sigma-Aldrich, Beijing, China) according to a standard protocol. The hybridoma cells were suspended in RPMI 1640 (Gibco, Grand Island, NY), supplemented with penicillin streptomycin (Sigma-Aldrich, Beijing, China), sodium pyruvate (Sigma-Aldrich, Beijing, China), and 20% fetal bovine serum (FBS) (Hyclone, Tauranga, New Zealand). The cells were centrifuged (500 g, 10 min, RT) and the pellet was resuspended in hypoxanthine–aminopterin–thymidine (HAT) (Sigma-Aldrich, Beijing, China) medium. Cells were seeded in 96-well tissue culture plates (Costar, Corning, NY) and incubated in a humidified 37 °C, 5% CO2 incubator for 2 weeks. Clones were kept in hypoxanthine thymidine (HT) medium diluted from HyClone™ (HT)
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Indirect ELISA
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The 96-well microplates were coated with 100 μL 10 μg/mL different bacteria antigens including positive and negative controls, diluted in 0.1 M NaHCO3, and incubated overnight at 4 °C. Plates were blocked for 2 h with 200 μL blocking buffer (PBS/1% BSA) at RT and washed three times in PBST (PBS/0.5% Tween-20). One hundred microliters of hybridoma culture supernatants, positive control (immunized mouse serum), and negative control (SP2/0) were accordingly added to the plate and incubated at 37 °C for 1 h. Plates were washed five times with PBST and incubated with 100 μL horseradish peroxidase conjugated goat anti-mouse immunoglobulin (IgG-HRP) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China) in blocking buffer (1:1,000) for 30 min at 37 °C. Finally, plates were washed five times with PBST and developed with 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma-Aldrich, Beijing, China) in the dark for 15 min at RT. The reaction was terminated by supplementing 50 μL of 1 M H2SO4 and absorbance values were determined at 450 nm with BioTek™ ELx808™ Absorbance Microplate Readers (ThermoFisher Scientific Inc., Beijing, China) against the blank. The titer of the antibody preparation was defined as the highest dilution that could give reading 0.05. One indirect ELISA unit was defined as the smallest amount of the antibody which gave a positive detection of antigen.
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Solution (50×) (ThermoFisher Scientific Inc., Beijing, China) for further 2 weeks. After selection by indirect ELISA, the desired cell lines were recloned three times by limiting dilution using spleen cells from a nonimmune BALB/c mouse as feeder cells to achieve monoclonality and stability. Supernatants from these clones were retested and positive candidates were expanded in large scale in a nonselective medium, and stored in liquid nitrogen.
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the different serotype of O1 serogroup. The present study selected a pair of highly sensitive and specific McAbs targeting V. cholerae O1 serotype Ogawa through a novel screening technique; and further developed the rapid diagnostic strips using this McAb pair to diagnose Ogawa infection. The product can detect pathogen rapidly with high specificity, sensitivity and reliability.
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Monoclonal antibody production and purification
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McAbs against V. cholerae O1 serotype Ogawa were produced in traditional ascetic fluid method. The 8-week old female BALB/c mice, primed with liquid paraffin, were injected intraperitoneally with 1.0 × 106 hybridoma cells per mouse. One to two weeks later, the ascetic fluid was drained with a 12-gauge needle. The ascites were centrifuged at 4000 rpm at 4 °C for 10 min to remove cells. The collected supernatants were precipitated in 50% saturated ammonium sulfate (pH 7.4), followed with extensive dialysis against 0.02 M phosphate buffer (pH 7.4) at 4 °C. The solution was purified by protein A affinity chromatography to obtain high quality McAbs. First, the column was prepared with 5 mL Affi-Gel Protein A (Bio-Rad Laboratories (Shanghai) Co., Ltd.; Shanghai, China) agarose beads soaked in binding buffer (pH 8.2) for 15 min. The dialyzed solution was loaded to the column and extensively washed with binding buffer. The flow through was collected in the fractions of 4–5 mL/tube. The OD280 of all the fractions were monitored until the reading was below 0.05 to ensure that there was no more unbound protein in the solution. The elution was performed by sequentially loading 1 mL elution buffer (pH 3.0) five times. Three hundred microliters of 1 M Tris–HCl buffer (pH 9.0) was introduced to each collecting tube in advance to neutralize the pH. Fractions of 1 mL of the eluant were collected in test tubes until the OD280 of the eluant was below 0.05. Finally, the column was washed three times with wash buffer for storage.
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McAb characterization
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The purity of the products was assessed by 10% SDS–PAGE. The specificity of the McAbs was evaluated by western blotting analysis against killed related and unrelated bacteria strains, including V. cholerae O1 Ogawa, Inaba, V. cholerae O139, V. parahaemolyticus, V. fluvialis, S. sonnei, S. typhi, A. hydrophila, P. aeruginosa, E. coli, S. aureus, P. mirabilis, etc. The titer of McAbs was determined by indirect
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Preparation of colloidal gold and colloidal gold–MAb conjugate
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Colloidal gold was prepared as previously reported [36]. Briefly, 100 mL of 0.01% (w/v) HAuCl4 in a 250-mL siliconized flask was heated to boiling in a microwave oven, and then 1.4 mL 1% trisodium citrate was added to the solution. After the colloidal gold solution was allowed to cool gradually and stored at 4 °C in a dark-colored glass bottle. The pH of the colloidal gold was adjusted to 8.4 with 1% potassium carbonate (w/v). The McAb IXiao3G6 (30 μg) was added dropwise into 10 mL of colloidal gold solution on a magnetic stirring apparatus for 30 min. After the solution was stabilized at 4 °C for 30 min, 1 mL 10% (w/v) BSA was added to block excess reactivity of the gold colloid. The mixture was then stirred for an additional 30 min and stored at 4 °C for 2 h. After the mixture was centrifuged at 3000 g at 4 °C for 30 min, the supernatant was further centrifuged at 14,000 g at 4 °C for 45 min, and the resulting conjugate pellet was suspended in 10 mM borax buffer (pH 8.0) containing 2% (w/v) BSA and 0.05% NaN3.
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Manufacture and evaluation of colloidal gold conjugated strips
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The Ogawa LFD consisted of a sample binding region called an analyte absorption pad, a result showing region including a conjugate pad and a nitrocellulose membrane, and a tag terminal called wicking pad. First, the detection antibody IXiao1D9 (the test line antibody) and goat anti-mouse IgG (the procedural control line antibody) were diluted to the working concentration and sprayed onto a piece of nitrocellulose membrane then dried at 37 °C for 24 h. The strips were subsequently blocked and dried at 37 °C. The capture antibody IXiao3G6 conjugated with colloidal gold (40 nm diameter) was sprayed twice to the fiberglass (0.5–1.5 cm × 25 cm) and dried at 37 °C. Finally, the components were assembled as a package and sliced into 4-mm wide test strips. To perform a test, after fully contacted with sample solution, the strips were kept at RT for 5 min. Two red bands that appeared at both the
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The epitope of purified McAbs was characterized by a competent ELISA and the additivity index (AI) described by Friguet et al. [35]. Briefly, the 96-well plates were first coated with 100 μL 2 μg/mL deactivated Ogawa and incubated overnight at 4 °C. One hundred microliters of IXiao3G6 or IXiao1D9 or combination (50 μL of each) was separately incubated as the primary antibody overnight at 4 °C. The secondary antibody used 100 μL goat anti-mouse IgG-HRP (1:1,000) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China). Plates were developed and analyzed as described. The index AI was calculated for each pair of antibodies by the equation (2A1 + 2 / (A1 + A2) − 1) × 100%. A1, A2 and A1 + 2 were the absorbances obtained in the ELISA using IXiao3G6, IXiao1D9 and the combination, respectively. If the two antibodies are directed against different epitopes (no competition), A1 + 2 should be equal to the sum of A1 and A2 and the AI should approach 100%. Otherwise, if the two antibodies are directed against the same epitope (competition), A1 + 2 should be equal to the mean value for A1 and A2 and the AI should tend to be 0%. The threshold in this study was determined by AI ≥ 40%. The specific epitope of McAbs was further examined by western blotting analysis against a wide range of related and unrelated LPS antigens (V. cholerae O1 Ogawa and Inaba, V. cholerae O139, V. fluvialis, A. hydrophila, S. sonnei, E. coli, S. typhi). The primary antibody was 50 μL either IXiao3G6 or IXiao1D9, and the secondary antibody was 100 μL goat anti-mouse IgG-HRP (1:1000) (Santa Cruz Biotechnology (Shanghai) Co., Ltd.; Shanghai, China).
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Balb/c mice immunization and McAb production
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Three experimental mice, XiaoA1, XiaoB1 and XiaoB3, producing high titer antibody (≥ 1:100,000) were selected as spleen cell donors. The fusion rate was 90.2%. For selection of specific McAbs, two rounds of selection were performed using indirect ELISA and McAb pairs screening, by employing both homologous antigens and heterologous antigens. A total of 602 positive hybridoma cell lines generating McAb against Ogawa were selected. By repetitive indirect ELISA and across responsive screening, 15 cell lines were obtained. After pair assembly screening, the two best candidates, IXiao3G6 and IXiao1D9, were finally achieved.
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Purification and characterization of McAb pair
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IXiao3G6 and IXiao1D9 were produced in large scale through ascitic fluid, and then purified by 50% saturated ammonium sulfate precipitation and protein A affinity chromatography. SDS–PAGE demonstrated that the purity of final McAb products was over 95% (Fig. 1A). Western blot analysis demonstrated clearly that both IXiao3G6 and IXiao1D9 could specifically detect Ogawa and did not cross-react to other experimental bacteria stains (Fig. 1B). The titer of the two McAbs in the supernatant culture of hybridomas and ascites indicated high activity (both N10− 6). The affinity constant was 1.39 × 10 9 (IXiao 3G 6) and 3.27 × 109 (IXiao1 D9 ). The subtypes of the two McAbs were IgG2b (IXiao3G6) and IgG3 (IXiao1D9). The epitope characterization study showed that the absorbance values for IXiao3 G6 , IXiao 1D 9 and the combination were 0.722, 0.744 and 1.244 respectively, which gives an AI value of 66.3%, indicating that IXiao3 G6 and IXiao1 D9 are directed against different epitopes. In order to identify the antigen epitopes of these two McAb targeted, the LPS of a range of related and unrelated bacteria were extracted, shown by silver-staining (Fig. 1C) and simultaneously detected against both McAbs. It is shown in Fig. 1D that only the extracted LPS from Ogawa could be specifically recognized by IXiao3G6 but not IXiao1D9 (data not shown), suggesting the two McAbs should target different epitopes of Ogawa: IXiao3G6 for LPS sites, and IXiao1D9 for non-LPS sites.
288
Systematic characterization of LFD strips
311
The sensitivity of the LFD was demonstrated by testing against the serial dilution of Ogawa pure cultures, and it was determined that the detection threshold was 1.0 × 104 cfu/mL. In 961 clinicalseparated strains, 112 of them, which were pre-identified as positive Ogawa by bacterial culture method, were equally confirmed to be positive by strips, shown by the representative test image with two solid red lines, i.e. control line and test line, in Table 1; and the rest
312 313
R O
205 206
P
Epitope characterization
D
204
test and control sites represent a positive test result. Only one red band at control location represents a negative test result. The absence of a line at the control site means the test is invalid. To determine the sensitivity of the strips for detecting Ogawa, the V. cholerae pure cultures were serially diluted at 10-fold from 109 to 103 cells/mL by saline and tested. To determine the specificity of the strips, 961 clinical-separated strains were prepared (Table 1). A total of 726 diarrhea patients feces samples (Jul, 2012–Sep 2012) provided by the Second Affiliated Hospital of Chongqing Medical University were examined by our strips, commercial strips from Zhuangdi Haohe Biological Medicine Co., Ltd. and standard bacterial culture method in parallel for comparison. The repeatability of our strips was statistically tested against 100 Ogawa cultures and 100 control cultures with 10 repeats for each culture. The stability was determined by strips kept in RT for 1 year and tested similarly.
T
202 203
ELISA. The immunoglobulin subclass was determined with a commercial mouse McAb Isotyping kit following manufacturers' instructions (SigmaAldrich, Beijing, China). The affinity constant of McAb was determined by indirect ELISA according to the method described by Beatty et al. [34].
E
200 201
3
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
278 279 280 281 282 283 284 285 286
289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
314 315 316 317 318
4
Bacteria culture identification Ogawa positive
Aeromonas caviae
26
0
26
t1:6
Aeromonas hydrophila
21
0
21
t1:7
Escherichia coli
110
0
110
t1:8
Proteus mirabilis
96
0
96
t1:9
Pseudomonas aeruginosa
105
0
105
t1:10
Salmonella typhi
100
0
100
t1:11
Shigella sonnei
15
0
15
t1:12
Staphylococcus aureus
102
0
102
t1:13
V. cholerae O1 serotype Inaba
108
0
108
t1:14
V. cholerae O1 serotype Ogawa
112
112
0
t1:15
V. cholerae O139
93
0
t1:16
Vibrio fluvialis
16
0
t1:17
Vibrio metschnihovii
9
0
t1:18
Vibrio parahaemolyticus
E
C
T
E
D
t1:5
0
93
16
9
48
324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
C
322 323
of the strains were simultaneously substantiated to be negative, shown by the representative test images with only one solid red line, i.e. control line, in Table 1. In addition, 726 diarrhea patients' feces samples tested by strips and standard bacterial culture method also presented the same outcomes, which all of the fecal samples were tested to be Ogawa negative. These results collectively suggested that our LFD strip has diagnostic sensitivity of 100% and diagnostic specificity of 100%. The parallel examination of V. cholerae O1 serotype Ogawa and Inaba, and V. cholerae O139 samples (Table 1) of our strips and strips from Zhuangdi Haohe Biological Medicine Co., Ltd. demonstrated better sensitivity (Fig. 2A) of our LFD strip, with detection limit of 1.0 × 104 cfu/mL. Furthermore, our product is capable not only of distinguishing V. cholerae O1 from O139 (Fig. 2B), but also further distinguishing V. cholerae O1 serotype Ogawa from Inaba, because it is clearly demonstrated in Fig. 2C that strips from Zhuangdi Haohe continued to report positive results when Inaba samples were examined. Table 2 statistically presented all the test outcomes of comparison with strips from Zhuangdi Haohe. The repeatability of the colloidal gold strip was 100% for pure cultures both positive and negative samples, and the strips were confirmed to be stable when kept in RT for 1 year.
N
320 321
U
319
O
R
R
48
Representative result
Ogawa negative
F
Number of each strain
O
Strain type
t1:4
R O
t1:3
Table 1 Bacteria strain list.
P
t1:1 t1:2
W. Chen et al. / Clinical Biochemistry xxx (2013) xxx–xxx
Discussion
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Cholera continues to be considered as a serious world health issue and causes an adverse impact on economic development in many countries, especially the third world. McAb can specifically recognize and bind to the single antigenic determinants from diverse bioactive molecules such as protein, polysaccharides, and nucleic acid, which makes it an ideal candidate to V. cholerae diagnosis and prevention. Colloidal gold is one of the four pillar technologies of immuno-label. Twenty years has witnessed its rapid improvement and ever-increasing application in biomedical research, especially in medical diagnosis [37]. The combination of these two technologies delivers the promising practice of the rapid diagnosis of V. cholerae. Antibodies recognize that the similar or shared antigens among these bacteria can lower their specificity. To screen out the nonspecific McAbs, two significant biopanning processes were conducted. In the first panning process, all available vibrio bacteria and other common intestinal bacteria were engaged to absorb the nonspecific antibodies, in order to screen out the cross-reactive hybridomas. The second panning process was developed based on industrial procedures to improve the preparation and screening efficacy of McAb cell lines by combining
341
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359
5
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F
W. Chen et al. / Clinical Biochemistry xxx (2013) xxx–xxx
T
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Dick et al. reviewed the last two decades of peer-reviewed and gray literature on rapid diagnostic tests for V. cholerae [38]. Research showed that twenty four diagnostic tests have been developed for the detection of V. cholerae in human fecal samples. Compared to the shortcomings of
R
363
conjugating McAbs to colloid gold (as capture antibody) and coating McAbs onto cellulose nitrate membrane (as detection antibody) to circumvent the laborious traditional screening methods, e.g. ELISA, and considerably expedited the selection.
N C O
361 362
U
360
E
D
Fig. 1. Purification of McAb and specificity determination. A: SDS–PAGE results of the purification result of IXiao3G6 (line 1) and IXiao1D9 (line 2) from protein A affinity chromatograph. B: Western blotting analysis of IXiao 3 G 6 (upper panel) and IXiao 1 D 9 (lower panel) against different bacteria (1. V. cholerae O1 serotype Inaba; 2. V. cholerae O139; 3. V. parahaemolyticus; 4. V. fluvialis; 5. S. sonnei; 6. S. typhi; 7. A. hydrophila; 8. P. aeruginosa; 9. E. coli; 10. S. aureus; 11. P. mirabilis; 12. V. cholerae O1 serotype Ogawa). C: Silver staining of LPS extracted from different bacteria; D: western blotting analysis of these LPS samples against IXiao3G6. 1. V. cholerae O1 serotype Ogawa; 2. V. cholerae O1 serotype Inaba; 3. V. cholerae O139; 4. V. fluvialis; 5. A. caviae; 6. S. sonnei; 7. E. coli; 8. S. typhi.
Fig. 2. Specificity comparison between our RDT strips (generated from IXiao3G6 and IXiao1D9) and Zhuangdi Haohe RDT devices. The green strips are products derived from IXiao3G6 and IXiao1D9. The white cassettes are RDT devices from Zhuangdi Haohe. For each strip or cassette, the upper line is control line, and the lower line (if presented) is test line. Panel A shows the comparison results tested against of V. cholerae Ogawa (from left to right: negative control, 109, 108, 107, 106, 105, 104, and 103 cfu/mL). Panel B showed the comparison results tested against V. cholerae O139 (from left to right: 109, 108, 107, and 106 cfu/mL). Panel C showed the comparison results tested against V. cholerae Inaba (from left to right: 109, 108, 107, 106, 105, 104, and 103 cfu/mL).
Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
364 365 366 367
6 t2:1 t2:2
W. Chen et al. / Clinical Biochemistry xxx (2013) xxx–xxx
Table 2 Results of comparison with Zhuangdihaohe strips.
t2:3
Strip source
112 strains of Ogawa (106–109 cfu/mL)
108 strains of Inaba (106–109 cfu/mL)
93 strains of O139 (106–109 cfu/mL)
t2:4 t2:5 t2:6
Zhuangdihaohe strips (V. cholerae O1) Zhuangdihaohe strips (V. cholerae O139) Homemade strips (V. cholerae O1 Ogawa)
Positive (+) Negative (−) Positive (+)
Positive (+) Negative (−) Negative (−)
Negative (−) Positive (+) Negative (−)
386 387 388 389 390 391 392 393 394 395 396 397 398
402
C
384 385
E
The authors have no financial interests to disclose. Acknowledgements
404 405 406
This study was supported by Artron BioResearch Inc.; State HighTech Development Plan (863 program) (code: 2002AA215015); and Chongqing Medical scientific research project (code: 2009-2-200).
407
References
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F
O
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382 383
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374 375
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Disclosure
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these diagnostic tests evaluated by authors [38], the present study has achieved several advantages. From the study design point of view, first, the large sample size employed in this study could avoid limited validity and reproducibility. Second, our study, which involved using stool samples instead of only using purified bacteria cultures, happened in many other studies, although certain limitation due to the fact of a paucity of positive cholera stool samples recruited led to the diffident interpretation. Third, this study applied a range of related and unrelated bacteria to specifically test against profound gut microflora and avoided limited data against common gut organism. All of these efforts greatly enhanced the specificity evaluation outcome. From test descriptors' point of view, this study provided real time stability and reproducibility tests results compared to others and confirmed the strips were valid in 1 year at RT. In conclusion, we achieved two McAbs against V. cholerae O1 Ogawa, IXiao3G6 and IXiao1D9, with high specificity and activity. They can specifically bind to different epitopes of Ogawa: IXiao3G6 for LPS sites, and IXiao1D9 for non-LPS sites. The RDT strip had been further successfully developed followed with conducting of a systematic evaluation to assess its specificity, sensitivity and reliability. The detection threshold for bacterial culture of the strip was 1.0 × 104 cfu/mL, which is higher than the current stipulated threshold (1.0 × 105 cfu/mL). More importantly, the product was capable of distinguishing the V. cholerae O1 Ogawa and V. cholerae O1 Inaba, which filled the gaps of the area in rapid cholera diagnosis. Overall, the outcome confirmed that this product has superior detective aspects than other parallel commercial products. Through the combination of our methods and other commercial available kits, the pathogens of cholera can be simultaneously and rapidly diagnosed, classified and differentially typed. Our perspective for future development is to design a combined detection device that can spontaneously detect and distinguish three different types of cholera all at once thereby providing more comprehensive information for better, faster monitoring and control of cholera pandemic.
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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Please cite this article as: Chen W, et al, Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa, Clin Biochem (2013), http://dx.doi.org/10.1016/j.clinbiochem.2013.12.022
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