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Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus
Journal of Microbiological Methods 131 (2016) 78–84
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus Xinfeng Liu a,1, Yuyao Guan b,1, Shiliang Cheng a, Yidan Huang c, Qin Yan c, Jun Zhang c,d, Guanjun Huang e, Jian Zheng d,⁎, Tianqiang Liu e,⁎ a
Clinical Laboratory, Shandong Jiaotong Hospital, Ji'nan, Shandong Province 250031, PR China Pharmacy department, Shandong Jiaotong Hospital, Ji'nan, Shandong Province 250031, PR China Artron BioResearch Inc., 3938 North Fraser Way, Burnaby, British Columbia V5J 5H6, Canada d Jinan Kangbo Biotechnology, 2766 Ying Xiu Road, Ji'nan, Shandong Province 250101, PR China e National and Local Joint Engineering Laboratory for Aquaculture Animal Disease Prevention and Control Technology, Chengdu, Sichuan Province 610081, PR China b c
a r t i c l e
i n f o
Article history: Received 23 August 2016 Received in revised form 13 October 2016 Accepted 14 October 2016 Available online 15 October 2016 Keywords: Immunochromatographic assay Lateral flow device Monoclonal antibody Rapid diagnosis Vibrio parahaemolyticus
a b s t r a c t Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2 × 103 CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. © 2016 Published by Elsevier B.V.
1. Introduction Vibrio parahaemolyticus is a Gram-negative, curved, rod-shaped bacterium widely present in brackish water. Many aquatic animals, including Penaeus orientalis, tiger prawns, abalones and Iberian toothcarp, can be infected by V. parahaemolyticus (Alcaide et al., 1999; Liu et al., 2000). V. parahaemolyticus has been reported as a common cause of foodborne diseases worldwide (McLaughlin et al., 2005; Wang et al., 2007). In China, V. parahaemolyticus has been the leading cause of foodborne outbreaks and bacterial infectious diarrhea in coastal regions (Lin et al., 2011). In Japan, it has been found to account for 20–30% of all food poisoning cases (Alam et al., 2002). The typical symptom of V. parahaemolyticus infection is watery diarrhea, which usually occurs within 24 h (Newton et al., 2014). However, the etiology of diarrhea can be difficult to determine based on observing clinical symptoms
⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zheng), [email protected] (T. Liu). 1 Co-first authors.
http://dx.doi.org/10.1016/j.mimet.2016.10.007 0167-7012/© 2016 Published by Elsevier B.V.
alone. Besides V. parahaemolyticus, diarrhea can be caused by a variety of bacteria, viruses, and parasitic organisms. Hence, laboratory testing is a necessity for confirmation purposes. The most common method for diagnosis of V. parahaemolyticus infection is conventional bacteria isolation and identification, which is currently still considered as the “gold standard”. Another microbiological method widely used for detecting V. parahaemolyticus is the International Organization for Standardization (ISO) cultural method, where two selective media [thiosulfate–citrate–bile salts–sucrose agar (TCBS) and triphenyltetrazolium chloride soya tryptone agar (TSAT)] are employed (International Organization for Standardization, ISO, 1990; Hara-Kudo et al., 2001). However, the conventional microbiological methods are very time-consuming and usually take longer than one week. In addition, the test results can be affected by many factors, such as bacterial load of specimens and the experience of laboratory technicians. Also, when the culture of the target bacteria is contaminated, the results may be confusing and misleading. Recently, molecular approaches for detection of V. parahaemolyticus have been developed significantly. Many PCR-based methods have been introduced by targeting specific genes of V. parahaemolyticus (Kim et al.,
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1999; Di Pinto et al., 2005). They are generally less time-consuming and more sensitive than conventional microbiological methods. But these molecular methods require professional personnel and equipment, and still need several hours to give a test result. Lateral flow assay (LFA), on the other hand, is a newly developed technique that can obtain the results within only a few minutes. Moreover, it needs neither trained technicians nor expensive equipment. LFA, an immunoreaction-based assay, is developed by targeting specific epitopes of protein antigens. So far, several rapid tests have been developed for the detection of pathogens in aquatic products (Sithigorngula et al., 2007; Sheng et al., 2012; Liu et al., 2015). However, due to their low testing sensitivities, the process of bacterial culturing is usually required before those immunoassays. The report on LFA for direct detection of V. parahaemolyticus without requirement of culture enrichment has not been seen yet. In this study, two monoclonal antibody (MAb) pairs targeting V. parahaemolyticus were successfully prepared. The colloidal gold test strips were developed to establish a rapid and accurate diagnostic method for V. parahaemolyticus infection both in aquatic products and in human feces of diarrheal patients. 2. Materials and methods 2.1. Microbes V. parahaemolyticus, V. alginolyticus, V. cholerae, V. vulnificus, V. harveyi, V. fortis, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella typhimurium, Edwardsiella ictaluri, Bacillus subtilis, Shewanella putrefaciens, Enterobacter cloacae, Streptococcus iniae were provided by the Pathogen Center of National and Local Joint Engineering Laboratory for Disease Control and Prevention Technology in Aquaculture Animals (Sichuan, China). Other microbes, including Staphylococcus aureus, Helicobacter pylori, Aeromonas caviae, Stenotrophomonas maltophilia, Candida tropicalis, Aeromonas hydrophila, Citrobacter freundii, Candida albicans, Staphylococcus epidermidis, Salmonella paratyphi B, Salmonella typhi, Serratia marcescens, Proteus mirabilis, Escherichia coli, Staphylococcus haemolyticus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii were preserved in the laboratory of Artron BioResearch Inc. (Burnaby, Canada). All the microbes were cultured according to the standard methods. 2.2. Animal immunization The V. parahaemolyticus cells were inactivated at 56 °C for 30 min, followed by determination of protein concentration using bicinchoninic acid kit (Sigma-Aldrich, USA). Female BALB/c mice (8 weeks old), provided by Laboratory Animal Center of Shandong University, were immunized intraperitoneally with 50 μg (approximately 106 CFU), 100 μg, 150 μg and 200 μg, respectively, of whole V. parahaemolyticus cells emulsified in Freund's complete adjuvant (Sigma-Aldrich, USA). After four and eight weeks, the mice were injected intraperitoneally with the same amount of antigen emulsified in Freund's incomplete adjuvant (Sigma-Aldrich, USA). At ten weeks, the bloods of immunized mice were collected individually by tail vein puncture method, followed by centrifuging at 1500g to prepare sera samples. The antibody titers of sera were determined by indirect enzyme-linked immunosorbent assay (ELISA). The mouse with highest titer was selected as the spleen cell donor and received a final intraperitoneal immunization with V. parahaemolyticus cells alone (without any adjuvant). 2.3. Hybridoma production Mouse myeloma Sp2/0 cells, used as fusion partners, were cultured and propagated in RPMI-1640 culture medium (Gibco, ThermoFisher, USA) containing 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare, USA). Three days after the final immunization, the mice were
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euthanized by cervical dislocation and the splenocytes were isolated. The freshly harvested splenocytes were then fused with the myeloma Sp2/0 cells at a ratio of 5:1 by 50% (w/v) polyethyleneglycol 4000 (Sigma-Aldrich, USA). Subsequently, the cells were re-suspended in Hypoxanthine-Aminopterin-Thymidine (HAT) medium (Sigma-Aldrich, USA), seeded in 96-well tissue culture plates and incubated in a humidified incubator with 5% CO2 at 37 °C for 2 weeks. Clones were kept in Hypoxanthine Thymidine (HT) medium (ThermoFisher, USA) for 2 more weeks. After selection by indirect ELISA, the desired cell lines were cloned three times by limiting dilution to stable monoclones. 2.4. Indirect ELISA The 96-well microplates were coated with 100 μl 5 μg/ml V. parahaemolyticus cells and incubated at 4 °C overnight. Plates were blocked for 2 h with blocking buffer, phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), at 37 °C and washed three times in PBST (PBS/0.05% Tween-20). 100 μl of hybridoma culture supernatants, the positive control (serum of immunized mice), and the negative control (Sp2/0 culture), were accordingly added to the plates and incubated at 37 °C for 1 h. Plates were washed four times with PBST and incubated with 100 μl horseradish peroxidase conjugated goat anti-mouse immunoglobulin (IgG-HRP) (Santa Cruz Biotechnology, USA) for 30 min at 37 °C. Finally, plates were washed five times with PBST and incubated with 100 μl 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, USA) in dark for 15 min at room temperature. The reaction was terminated by supplementing 50 μl H2SO4 solution (1 M) and the absorbance values were determined at 450 nm by BioTek™ ELx808™ Absorbance Microplate Readers (ThermoFisher, USA). 2.5. MAb production MAbs against V. parahaemolyticus were produced by traditional ascitic fluid method. Cells of desired monoclones in density of 1 × 106 cells/0.5 ml PBS were injected intraperitoneally into each mouse, which had been previously injected with 0.5 ml Pristane (Sigma-Aldrich, USA) two weeks earlier. Their ascites were harvested about ten days later when their abdomens were completely enlarged and their skins were extended. The supernatants were collected after centrifugation of the ascites at 2900g for 30 min. The antibodies were precipitated by 50% saturation of ammonium sulfate solution, followed by purification by protein G column (GE Healthcare, USA) affinity column chromatography according to the manufacturer's protocol. 2.6. MAb characterization The purity of prepared MAbs was analyzed by SDS-PAGE. The titers of MAbs were determined by indirect ELISA. The titer of an antibody solution was defined as the highest dilution that could give a positive reaction against the antigen. The immunoglobulin subclass was determined by a commercial Mouse Typer Sub-isotyping Kit (Bio-Rad, USA) according to the manufacturer's protocol. The affinity constant (Kaff) of MAbs were determined by indirect ELISA according to the method described previously (Beatty et al., 1987). 2.7. Preparation of colloidal gold and colloidal gold-MAb conjugate Colloidal gold was prepared according to a previous report (Grabar et al., 1995). Briefly, 100 ml of 0.01% (w/v) HAuCl4 (Sigma-Aldrich, USA) in a 250 ml siliconized flask was heated to boiling in a microwave oven, and 1.4 ml 1% trisodium citrate (Sigma-Aldrich, USA) was added. The solution was allowed to gradually cool down and then stored at 4 °C in a dark-colored glass bottle. The pH of the colloidal gold was adjusted to 8.4 with 1% (w/v) potassium carbonate. The capture MAb (30 μg) was added dropwise into 10 ml colloidal gold solution on a magnetic stirrer
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for 30 min. After the solution was stabilized at 4 °C for 30 min, 1 ml 10% (w/v) BSA (Sigma-Aldrich, USA) was added to block the excess gold colloid. The mixture was then stirred for another 30 min and stored at 4 °C for 2 h, followed by centrifuging at 3000g at 4 °C for 30 min. The resultant supernatant was further centrifuged at 14,000g at 4 °C for 45 min to obtain the pellets of colloidal gold-MAb conjugate, which were resuspended in 10 mM borax (Sigma-Aldrich, USA) buffer (pH 8.0) containing 2% (w/v) BSA and 0.05% (w/v) NaN3 (Sigma-Aldrich, USA) for further use.
buffer (pH 7.8) and was tested by using the strips. The lowest concentration that gave a positive signal was defined as the testing limit. To verify the specificity, a total of 32 microbial species, including 6 Vibrio species, were diluted to 106 CFU/ml and used for the LFA strip test. To study the testing repeatability, three V. parahaemolyticus samples (106, 105 and 104 CFU/ml) and three negative control samples (106, 105 and 104 CFU/ml of V. alginolyticus) were tested repeatedly by the prepared LFA strips with ten replicates per sample. The stability was evaluated by storing the strips at room temperature for one year and testing relative samples similarly.
2.8. Manufacture of LFA strips 2.11. Test of shrimp samples The LFA 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 (Fig. 1). First, the detection antibody (the test line antibody) and goat antimouse IgG (the procedural control line antibody) were diluted to the working concentration and sprayed onto a piece of nitrocellulose membrane, which was subsequently dried at 37 °C for 24 h. The strips were then blocked and dried at 37 °C. The capture antibody conjugated with colloidal gold (40 nm diameter) was sprayed twice onto the fiberglass (0.5–1.5 × 25 cm) and dried at 37 °C. Finally, the components were assembled as a package and sliced into 4 mm wide testing strips. To perform a test, the strip was dipped fully into the sample solution and kept at room temperature for 5 min. When there are two red bands at both test and control lines, this shows a positive testing result. With only one red band at control line, this represents a negative result. The absence of the band at the control line tells the invalidity of the test. 2.9. MAb pairing The LFA strips with all possible MAb pairs were prepared. The cell suspensions of V. parahaemolyticus and those of other Vibrio species (50 μg/ml) were used as test samples. The MAb pairs that gave the strongest signals for V. parahaemolyticus, but negative to all other Vibrio species were selected for further study. 2.10. Characterization of LFA strips The testing limit of LFA strips was determined by the method of serial dilution. The V. parahaemolyticus suspension was serially diluted at 10-fold from 1.2 × 108 CFU/ml to 1.2 × 101 CFU/ml in 20 mM Tris-HCl
Whiteleg shrimp (Litopenaeus vannamei, formerly Penaeus vannamei) samples were collected from a shrimp farm located in Taishan, Guangzhou province, China. The sick shrimps were weak and swam close to the water surface. Their shells were pale red with white spots. To identify the V. parahaemolyticus infection, the shrimp was cleaned with 70% ethanol, followed by aseptic isolation of hepatopancreas samples and inoculation in Alkaline Peptone Water (APW). After being incubated at 37 °C overnight, the APW cultures were sub-cultured onto Vibrio Chromogenic Medium (CHROMagar, France). The appearance of mauve colonies after overnight incubation at 37 °C indicated the V. parahaemolyticus infection. In this study, six healthy and seven V. parahaemolyticus infected shrimps, which had been confirmed by clinical symptoms and microbiological method, were used for strip tests. The hepatopancreases from all the shrimps were homogenized individually with mortar and pestle. One milliliter of 20 mM Tris-HCl buffer (pH 7.8) was added and mixed with the hepatopancreas homogenates. The suspensions were detected by the LFA strips. White feces from healthy and sick shrimps were collected separately, followed by supplement of 0.2 ml of Tris-HCl buffer (pH 7.8) and complete mix by pipette. The fecal suspensions were tested by the LFA strips as well. 2.12. Test of human fecal samples from diarrheal patients To verify the detection limit of V. parahaemolyticus in human fecal samples, feces from three healthy persons were collected and suspended completely in 20 mM Tris-HCl buffer (pH 7.8) by the ratio of 5 ml of buffer per 1 g of feces. Five milliliters of each suspension were collected and mixed together to prepare the negative control and diluting agent. The V. parahaemolyticus culture was then serially
Fig. 1. Description of LFA architecture. LFA consists of a sample binding region called a sample absorption pad, a result showing region including a conjugate pad and a nitrocellulose membrane, and a tag terminal called wicking pad. When a sample is applied to the sample pad, it flows towards the terminal wicking pad. A positive signal is visualized by the gold nano-particles when the sample, captured by gold-conjugated antibody, binds the antibody on the test line. Meanwhile, a test strip also contains a control line, where anti-IgG antibody is coated, to assure accuracy of the assay.
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diluted at 10-fold from 0.8 × 106 CFU/ml to 0.8 × 102 CFU/ml in the prepared negative control and was tested by the strips. Fecal samples from 146 diarrheal patients, whose infectious bacteria were determined by traditional microbiological method, were collected from Shandong Jiaotong Hospital, Shandong province, China, during July–September 2015. One milliliter of 20 mM Tris-HCl buffer (pH 7.8) was added to 0.2 g of fresh fecal sample and mixed completely. The suspensions were directly tested by the LFA strips.
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to be strongly positive, those of all other microbial species were negative (Fig. 3). These results indicated that both types of LFA strip were highly sensitive and specific to V. parahaemolyticus culture. The reproducibility of the LFA strips was also examined. The testing results were 100% consistent for both positive and negative samples. The strips were confirmed to be stable at room temperature for 1 year (data not shown). 3.4. Detection of V. parahaemolyticus infection in Whiteleg shrimps
3. Results 3.1. MAb production and pairing Eight BALB/c mice were used for immunization at different dosages of V. parahaemolyticus. The one with the highest antibody titer (1:105) was chosen as the spleen cell donor. After fusion of splenocytes and freshly grown Sp2/0 cells, positive hybridomas were observed in cell culture plates. The fusion rate was 84.7%. The supernatants of growing hybridoma cells were screened based on reactivity against V. parahaemolyticus by indirect ELISA. A total of 1278 positive hybridoma clones were selected. The hybridomas were subcloned 3 times and consequently 79 stable positive clones, whose antibodies specifically recognized V. parahaemolyticus, were obtained. Based on the isolated positive clones, 79 ascites containing relevant MAbs were prepared. Subsequently, the MAbs were purified with a combination of 50% saturated ammonium sulfate precipitation and Protein G affinity chromatography. After cross-pairing all the 79 MAbs, the antibody pair GA1a-IC9 and IC9-KB4c, which showed strong signal to V. parahaemolyticus and negative to all other microbes, were selected for further study. 3.2. MAb characterization SDS-PAGE demonstrated the purities of selected MAb products, GA1a, IC9 and KB4c, were all over 95% (data not shown). The antibody titers of cell culture supernatants and mouse ascites were determined to be 4.3 × 10−3 and 1.3 × 10−6 (GA1a), 7.2 × 10−3 and 2.1 × 10−6 (IC9), and 2.7 × 10− 3 and 8.3 × 10−5 (KB4c), respectively. The Kaff was calculated to be 2.15 × 109 l/mol (GA1a), 3.61 × 109 l/mol (IC9) and 1.82 × 109 l/mol (KB4c), respectively (Table 1). Therefore, the GA1a, IC9 and KB4c were believed to be satisfying MAbs with high specificities, titers and affinities. The isotype test indicated that the GA1a MAb was of IgG2b isotype, IC9 was of IgG1 and KB4c was of IgA (Table 1). 3.3. Characterization of LFA strips Two types of LFA strips were developed. The type I applied GA1a as the capture antibody (gold-conjugated antibody) and IC9 as the detection antibody (coating antibody); while the type II used IC9 as the capture antibody and KB4c as the detection antibody. The testing limits of the LFA strips were examined by testing the serial dilutions of V. parahaemolyticus culture. The Tris-HCl buffer (pH 7.8) was used as negative control. Both types of LFA strip were found to be of the same testing limit, which was 1.2 × 103 CFU/ml (Fig. 2). To test the specificity, a total of 32 microbial species, including 6 Vibrio species, were tested by the LFA strips. While the culture of V. parahaemolyticus was confirmed Table 1 Characterization of MAbs. Clone ID
GA1a IC9 KB4c
Antibody titer Culture supernatant
Ascites
4.3 × 10−3 7.2 × 10−3 2.7 × 10−3
1.3 × 10−6 2.1 × 10−6 8.3 × 10−5
Kaff(l/mol)
Isotype
2.15 × 109 3.61 × 109 1.82 × 109
IgG2b IgG1 IgA
The hepatopancreas samples of healthy and V. parahaemolyticus infected shrimps were tested by the LFA strips. Both types of LFA strip showed exactly the same results. All seven samples from sick shrimps were confirmed to be positive, while the six healthy samples were all found to be negative (Fig. 4). Similar results were also observed when the fecal samples were tested (Fig. 5). Thus, the diagnostic sensitivity and specificity of the homemade LFA strips for shrimp hepatopancreas and fecal samples in this study were both 100%. 3.5. Detection of V. parahaemolyticus infection in human diarrheal patients The detection limit of V. parahaemolyticus in human fecal samples was studied (Fig. 6). At the concentration of 0.8 × 104 CFU/ml, strips of both types exhibited clearly positive. When the concentration decreased to 0.8 × 103 CFU/ml, while the T line of type II strip was still visible, that of type I strip became difficult to see. Thus, the testing limit of type II strip for fecal samples, which could reach as low as 0.8 × 103 CFU/ ml, was slightly lower than that of type I strip. To evaluate the clinical application in V. parahaemolyticus detection in humans, a total of 146 fecal samples from diarrheal patients were freshly collected and immediately tested by the two types of LFA strips. The testing data are summarized in Table 2. Of the 27 fecal samples from V. parahaemolyticus infected patients, 26 were detected positive by type I strip and all positive by type II strip. The diagnostic sensitivities were 96.3% and 100%, respectively. When it comes to specificity, both types of LFA strips showed no cross-activity to any of the other bacteria associated with diarrhea. Therefore, the type II LFA, with IC9 as the capture antibody and KB4c as the detection antibody, was considered as a better product, whose sensitivity and specificity for V. parahaemolyticus detection in fecal samples from diarrheal patients were both 100% in the study. 4. Discussion Globally, V. parahaemolyticus is a leading cause of seafood-associated gastroenteritis (Hara-Kudo et al., 2012; Haendiges et al., 2014). Detection of V. parahaemolyticus infection is of great significance not only for aquaculture disease detection and food safety control, but also for accurate diagnosis and proper treatment for human diarrheal patients. The traditional microbiological culture method, though considered as the gold-standard for V. parahaemolyticus detection, is labor-intensive and time-consuming with complicated procedures and low sensitivity (Chitkara, 2005; DeBoer et al., 2010). Currently, many commercial PCR-based detection kits, such as BioFire FilmArray Gastrointestinal Panel and Luminex xTAG Gastrointestinal Pathogen Panel (Khare et al., 2014; Buss et al., 2015), are available. Comparing to traditional biological method, these commercial kits can finish the tests within hours with much higher sensitivity and specificity. However, due to the high costs, their application is highly restricted in primary public healthcare laboratories, especially in developing regions. In contract, LFA is an immunological method that requires neither expensive equipment nor highly trained personnel, and that can be accomplished within a few minutes. In this study, using the inactivated V. parahaemolyticus as immunogen, two pairs of murine anti-V. parahaemolyticus MAbs, GA1a-IC9 and IC9-KB4c, were prepared for LFA development. Based on the two MAb
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Fig. 2. Testing limit of V. parahaemolyticus LFA strips. C Line, Control line; T Line, Test line. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); 1–8, serial dilutions of V. parahaemolyticus culture: 1, 1.2 × 108 CFU/ml; 2, 1.2 × 107 CFU/ml; 3, 1.2 × 106 CFU/ml; 4, 1.2 × 105 CFU/ml; 5, 1.2 × 104 CFU/ml; 6, 1.2 × 103 CFU/ml; 7, 1.2 × 102 CFU/ml; and 8, 1.2 × 101 CFU/ml. The detection threshold for both types of LFA strips was 1.2 × 103 CFU/ml. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Specificity analysis of V. parahaemolyticus strips. C Line, Control line; T Line, Test line. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); 1–32: 106 CFU/ml of microbial culture diluted in 20 mM Tris-HCl buffer (pH 7.8). 1, V. alginolyticus; 2, V. cholerae; 3, V. vulnificus; 4, V. harveyi; 5, V. fortis; 6, Enterococcus faecalis; 7, Klebsiella pneumoniae; 8, Enterobacter cloacae; 9, Staphylococcus aureus; 10, Streptococcus iniae; 11, Helicobacter pylori; 12, Salmonella typhimurium; 13, Aeromonas caviae; 14, Edwardsiella ictaluri; 15, Stenotrophomonas maltophilia; 16, Bacillus subtilis; 17, Candida tropicalis; 18, Shewanella putrefaciens; 19, Aeromonas hydrophila; 20, Citrobacter freundii; 21, Candida albicans; 22, Staphylococcus epidermidis; 23, Salmonella paratyphi B; 24, Salmonella typhi; 25, Serratia marcescens; 26, Proteus mirabilis; 27, Escherichia coli; 28, Staphylococcus haemolyticus; 29, Neisseria gonorrhoeae; 30, Pseudomonas aeruginosa; 31, Acinetobacter baumannii; 32, V. parahaemolyticus. Both types of LFA strips were highly specific to V. parahaemolyticus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Test of hepatopancreas samples from Whiteleg shrimps. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); Pos, positive control, suspension of V. parahaemolyticus cells; H1–H6, hepatopancreas samples from six healthy shrimps; S1-S7, hepatopancreas samples from seven V. parahaemolyticus infected shrimps. All the seven samples from sick shrimps were confirmed to be positive, while the six healthy samples were all found negative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pairs, two types of LFA strips were prepared. The testing limit for V. parahaemolyticus culture was both 1.2 × 103 CFU/ml, which is close to those reported for PCR-base methods (Panicker et al., 2004; Hanabara and Ueda, 2016), but much lower than a previously reported V.
parahaemolyticus LFA (105 CFU/ml) (Guo et al., 2012). The concentrations of foodborne pathogenic bacteria are usually N106 CFU/g feces in acute phase of illness (Hanabara and Ueda, 2016). However, fecal samples are often collected in the non-acute phase, when the bacterial
Fig. 5. Test of fecal samples from Whiteleg shrimps. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); Pos, positive control, suspension of V. parahaemolyticus cells; H1–H6, fecal samples from six healthy shrimps; S1–S7, fecal samples from seven V. parahaemolyticus infected shrimps. The test results of fecal samples were highly correlated to those of hepatopancreas samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Testing limit of V. parahaemolyticus LFA strips for fecal samples. Neg, negative control (healthy feces suspension); 1–5, V. parahaemolyticus samples serially diluted in healthy feces sample: 1, 0.8 × 106 CFU/ml; 2, 0.8 × 105 CFU/ml; 3, 0.8 × 104 CFU/ml; 4, 0.8 × 103 CFU/ml; 5, 0.8 × 102 CFU/ml.
concentration may be much lower. Therefore, the low testing limits of our homemade strips guarantee a high diagnostic sensitivity for V. parahaemolyticus detection, and no requirement of culture enrichment in testing human fecal samples. Due to the high specificity and low testing limit, the strips successfully detected the V. parahaemolyticus infection in both hepatopancreas and fecal samples from 13 Whiteleg shrimps with 100% diagnostic specificity and sensitivity. During the test of human feces from diarrheal patients, the two types of strips showed a slight difference in diagnostic sensitivities. While type II strip still exhibited 100% sensitivity, type I failed to detect one of the total 27 positive samples. It might be caused by different testing limits of the two strips in fecal samples, as the sample that type I strip failed to identify showed very weak signal in type I strip as well. The fecal samples are more complex than those from bacterial culture. Thus, type II strip is believed to be a more promising product to diagnose the V. parahaemolyticus infection in diarrheal patients. To date, few studies on V. parahaemolyticus detection by immunological methods have been reported. Guo et al. developed an immunochromatographic strip for detection of V. parahaemolyticus in seafoods by employing a polyclonal antibody (PAb)–MAb pair (Guo et al., 2012). Our study used MAb pairs, which should be more specific than PAb-MAb pairs. In addition, the testing limit of Guo's strip was as high as 105 CFU/ml, which made it unsuitable to directly detect V. parahaemolyticus in human feces. Kawatsu et al. reported a test strip to detect thermostable direct hemolysin (TDH) produced by V. parahaemolyticus (Kawatsu et al., 2006). However, due to the low content of TDH in the bacteria, culture enrichment of the samples was required to get a signal for positive fecal specimens. In conclusion, we successfully developed two types of LFA strips for V. parahaemolyticus detection. Their testing limits are both 1.2 × 103 CFU/ml for bacterial culture. Both strips are able to directly detect V. parahaemolyticus in hepatopancreas and fecal samples from infected Whiteleg shrimps and, potentially, from other aquatic products as well. Importantly, type II strip, due to its higher sensitivity in human fecal specimens, can be used to rapidly diagnose V. parahaemolyticus in human feces without prior culture enrichment process, which allows the test to be finished within only a few minutes.
Table 2 Testing results of fecal samples from diarrheal patients. Infection causes
V. parahaemolyticus Diarrheagenic E. coli Salmonella spp. Shigella spp. Campylobacter Total
Sample numbers
27 44 31 26 18 146
Type I LFA
Type II LFA
Positive
Negative
Positive
Negative
26 0 0 0 0 26
1 44 31 26 18 120
27 0 0 0 0 27
0 44 31 26 18 119
Conflict of interest The authors declare that no conflict of interest exists. References Alam, M.J., Tomochika, K.I., Miyoshi, S.I., Shinoda, S., 2002. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol. Lett. 208, 83–87. Alcaide, E., Amaro, C., Todoli, R., Oltra, R., 1999. Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus. Dis. Aquat. Org. 35, 77–80. Beatty, J.D., Beatty, B.G., Vlahos, W.G., 1987. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immunol. Methods 100, 173–179. Buss, S.N., Leber, A., Chapin, K., Fey, P.D., Bankowski, M.J., Jones, M.K., Rogatcheva, M., Kanack, K.J., Bourzac, K.M., 2015. Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J. Clin. Microbiol. 53, 915–925. Chitkara, Y.K., 2005. Limited value of routine stool cultures in patients receiving antibiotic therapy. Am. J. Clin. Pathol. 123, 92–95. DeBoer, R.F., Ott, A., Kesztyüs, B., Kooistra-Smid, A.M., 2010. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J. Clin. Microbiol. 48, 4140–4146. Di Pinto, A., Ciccarese, G., Tantillo, G., Catalano, D., Forte, V.T., 2005. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J. Food Prot. 68, 150–153. Grabar, K.C., Freeman, R.G., Hommer, M.B., Natan, M.J., 1995. Preparation and characterization of Au colloid monolayers. Anal. Chem. 67, 735–743. Guo, A., Sheng, H., Zhang, M., Wu, R., Xie, J., 2012. Development and evaluation of a colloidal gold immunochromatography strip for rapid detection of Vibrio parahaemolyticus in food. J. Food Qual. 35, 366–371. Haendiges, J., Rock, M., Myers, R.A., Brown, E.W., Evans, P., Gonzalez-Escalona, N., 2014. Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012. Emerg. Infect. Dis. 20, 718–720. Hanabara, Y., Ueda, Y., 2016. A rapid and simple real-time polymerase chain reaction assay for detecting foodborne pathogenic bacteria in human feces. Japan J. Infect. Dis. (Epub ahead of print). Hara-Kudo, Y., Nishina, T., Nakagawa, H., Konuma, H., Hasegawa, J., Kumagai, S., 2001. Improved method for detection of Vibrio parahaemolyticus in seafood. Appl. Environ. Microbiol. 67, 5819–5823. Hara-Kudo, Y., Saito, S., Ohtsuka, K., Yamasaki, S., Yahiro, S., Nishio, T., Iwade, Y., Otomo, Y., Konuma, H., Tanaka, H., Nakagawa, H., Sugiyama, K., Sugita-Konishi, Y., Kumagai, S., 2012. Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan. Int. J. Food Microbiol. 157, 95–101. International Organization for Standardization (ISO), 1990. General guidance for the detection of Vibrio parahaemolyticus. ISO 8914:1990(E). Switzerland, Geneva. Kawatsu, K., Ishibashi, M., Tsukamoto, T., 2006. Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable in enrichment cultures of stool specimens. J. Clin. Microbiol. 44, 1821–1827. Khare, R., Espy, M.J., Cebelinski, E., Boxrud, D., Sloan, L.M., Cunningham, S.A., Pritt, B.S., Patel, R., Binnicker, M.J., 2014. Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stools pecimens. J. Clin. Microbiol. 52, 3667–3673. Kim, Y.B., Okuda, J., Matsumoto, C., Takahashi, N., Hashimoto, S., Nishibuchi, M., 1999. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR Gene. J. Clin. Microbiol. 37, 1173–1177. Lin, X., Ran, L., Ma, L., Wang, Z., Feng, Z., 2011. Analysis on the cases of infectious diarrhea (rather than cholera, dysentery, typhoid and paratyphoid) reported in China, 2010 [in Chinese]. China J. Food Hyg. 23, 385–389. Liu, P.C., Chen, Y.C., Huang, C.Y., Lee, K.K., 2000. Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome. Lett. Appl. Microbiol. 31, 433–437. Liu, H., Wang, Y., Xiao, J., Wang, Q., Liu, Q., Zhang, Y., 2015. An immunochromatographic test strip for rapid detection of fish pathogen Edwardsiella tarda. Bioresour. Bioprocessing 2, 20.
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McLaughlin, J.B., DePaola, A., Bopp, C.A., Martinek, K.A., Napolilli, N.P., Allison, C.G., Murray, S.L., Thompson, E.C., Bird, M.M., Middaugh, J.P., 2005. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. N. Engl. J. Med. 253, 1463–1470. Newton, A.E., Garrett, N., Stroika, S.G., Halpin, J.L., Turnsek, M., Mody, R.K., Centers for Disease Control and Prevention (CDC), 2014. Increase in Vibrio parahaemolyticus infections associated with consumption of Atlantic Coast shellfish-2013. MMWR Morb. Mortal. Wkly Rep. 63, 335–336. Panicker, G., Call, D.R., Krug, M.J., Bej, A.K., 2004. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays. Appl. Environ. Microbiol. 70, 7436–7444.
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus Xinfeng Liu a,1, Yuyao Guan b,1, Shiliang Cheng a, Yidan Huang c, Qin Yan c, Jun Zhang c,d, Guanjun Huang e, Jian Zheng d,⁎, Tianqiang Liu e,⁎ a
Clinical Laboratory, Shandong Jiaotong Hospital, Ji'nan, Shandong Province 250031, PR China Pharmacy department, Shandong Jiaotong Hospital, Ji'nan, Shandong Province 250031, PR China Artron BioResearch Inc., 3938 North Fraser Way, Burnaby, British Columbia V5J 5H6, Canada d Jinan Kangbo Biotechnology, 2766 Ying Xiu Road, Ji'nan, Shandong Province 250101, PR China e National and Local Joint Engineering Laboratory for Aquaculture Animal Disease Prevention and Control Technology, Chengdu, Sichuan Province 610081, PR China b c
a r t i c l e
i n f o
Article history: Received 23 August 2016 Received in revised form 13 October 2016 Accepted 14 October 2016 Available online 15 October 2016 Keywords: Immunochromatographic assay Lateral flow device Monoclonal antibody Rapid diagnosis Vibrio parahaemolyticus
a b s t r a c t Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2 × 103 CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. © 2016 Published by Elsevier B.V.
1. Introduction Vibrio parahaemolyticus is a Gram-negative, curved, rod-shaped bacterium widely present in brackish water. Many aquatic animals, including Penaeus orientalis, tiger prawns, abalones and Iberian toothcarp, can be infected by V. parahaemolyticus (Alcaide et al., 1999; Liu et al., 2000). V. parahaemolyticus has been reported as a common cause of foodborne diseases worldwide (McLaughlin et al., 2005; Wang et al., 2007). In China, V. parahaemolyticus has been the leading cause of foodborne outbreaks and bacterial infectious diarrhea in coastal regions (Lin et al., 2011). In Japan, it has been found to account for 20–30% of all food poisoning cases (Alam et al., 2002). The typical symptom of V. parahaemolyticus infection is watery diarrhea, which usually occurs within 24 h (Newton et al., 2014). However, the etiology of diarrhea can be difficult to determine based on observing clinical symptoms
⁎ Corresponding authors. E-mail addresses: [email protected] (J. Zheng), [email protected] (T. Liu). 1 Co-first authors.
http://dx.doi.org/10.1016/j.mimet.2016.10.007 0167-7012/© 2016 Published by Elsevier B.V.
alone. Besides V. parahaemolyticus, diarrhea can be caused by a variety of bacteria, viruses, and parasitic organisms. Hence, laboratory testing is a necessity for confirmation purposes. The most common method for diagnosis of V. parahaemolyticus infection is conventional bacteria isolation and identification, which is currently still considered as the “gold standard”. Another microbiological method widely used for detecting V. parahaemolyticus is the International Organization for Standardization (ISO) cultural method, where two selective media [thiosulfate–citrate–bile salts–sucrose agar (TCBS) and triphenyltetrazolium chloride soya tryptone agar (TSAT)] are employed (International Organization for Standardization, ISO, 1990; Hara-Kudo et al., 2001). However, the conventional microbiological methods are very time-consuming and usually take longer than one week. In addition, the test results can be affected by many factors, such as bacterial load of specimens and the experience of laboratory technicians. Also, when the culture of the target bacteria is contaminated, the results may be confusing and misleading. Recently, molecular approaches for detection of V. parahaemolyticus have been developed significantly. Many PCR-based methods have been introduced by targeting specific genes of V. parahaemolyticus (Kim et al.,
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1999; Di Pinto et al., 2005). They are generally less time-consuming and more sensitive than conventional microbiological methods. But these molecular methods require professional personnel and equipment, and still need several hours to give a test result. Lateral flow assay (LFA), on the other hand, is a newly developed technique that can obtain the results within only a few minutes. Moreover, it needs neither trained technicians nor expensive equipment. LFA, an immunoreaction-based assay, is developed by targeting specific epitopes of protein antigens. So far, several rapid tests have been developed for the detection of pathogens in aquatic products (Sithigorngula et al., 2007; Sheng et al., 2012; Liu et al., 2015). However, due to their low testing sensitivities, the process of bacterial culturing is usually required before those immunoassays. The report on LFA for direct detection of V. parahaemolyticus without requirement of culture enrichment has not been seen yet. In this study, two monoclonal antibody (MAb) pairs targeting V. parahaemolyticus were successfully prepared. The colloidal gold test strips were developed to establish a rapid and accurate diagnostic method for V. parahaemolyticus infection both in aquatic products and in human feces of diarrheal patients. 2. Materials and methods 2.1. Microbes V. parahaemolyticus, V. alginolyticus, V. cholerae, V. vulnificus, V. harveyi, V. fortis, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella typhimurium, Edwardsiella ictaluri, Bacillus subtilis, Shewanella putrefaciens, Enterobacter cloacae, Streptococcus iniae were provided by the Pathogen Center of National and Local Joint Engineering Laboratory for Disease Control and Prevention Technology in Aquaculture Animals (Sichuan, China). Other microbes, including Staphylococcus aureus, Helicobacter pylori, Aeromonas caviae, Stenotrophomonas maltophilia, Candida tropicalis, Aeromonas hydrophila, Citrobacter freundii, Candida albicans, Staphylococcus epidermidis, Salmonella paratyphi B, Salmonella typhi, Serratia marcescens, Proteus mirabilis, Escherichia coli, Staphylococcus haemolyticus, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii were preserved in the laboratory of Artron BioResearch Inc. (Burnaby, Canada). All the microbes were cultured according to the standard methods. 2.2. Animal immunization The V. parahaemolyticus cells were inactivated at 56 °C for 30 min, followed by determination of protein concentration using bicinchoninic acid kit (Sigma-Aldrich, USA). Female BALB/c mice (8 weeks old), provided by Laboratory Animal Center of Shandong University, were immunized intraperitoneally with 50 μg (approximately 106 CFU), 100 μg, 150 μg and 200 μg, respectively, of whole V. parahaemolyticus cells emulsified in Freund's complete adjuvant (Sigma-Aldrich, USA). After four and eight weeks, the mice were injected intraperitoneally with the same amount of antigen emulsified in Freund's incomplete adjuvant (Sigma-Aldrich, USA). At ten weeks, the bloods of immunized mice were collected individually by tail vein puncture method, followed by centrifuging at 1500g to prepare sera samples. The antibody titers of sera were determined by indirect enzyme-linked immunosorbent assay (ELISA). The mouse with highest titer was selected as the spleen cell donor and received a final intraperitoneal immunization with V. parahaemolyticus cells alone (without any adjuvant). 2.3. Hybridoma production Mouse myeloma Sp2/0 cells, used as fusion partners, were cultured and propagated in RPMI-1640 culture medium (Gibco, ThermoFisher, USA) containing 10% fetal bovine serum (FBS) (Hyclone, GE Healthcare, USA). Three days after the final immunization, the mice were
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euthanized by cervical dislocation and the splenocytes were isolated. The freshly harvested splenocytes were then fused with the myeloma Sp2/0 cells at a ratio of 5:1 by 50% (w/v) polyethyleneglycol 4000 (Sigma-Aldrich, USA). Subsequently, the cells were re-suspended in Hypoxanthine-Aminopterin-Thymidine (HAT) medium (Sigma-Aldrich, USA), seeded in 96-well tissue culture plates and incubated in a humidified incubator with 5% CO2 at 37 °C for 2 weeks. Clones were kept in Hypoxanthine Thymidine (HT) medium (ThermoFisher, USA) for 2 more weeks. After selection by indirect ELISA, the desired cell lines were cloned three times by limiting dilution to stable monoclones. 2.4. Indirect ELISA The 96-well microplates were coated with 100 μl 5 μg/ml V. parahaemolyticus cells and incubated at 4 °C overnight. Plates were blocked for 2 h with blocking buffer, phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), at 37 °C and washed three times in PBST (PBS/0.05% Tween-20). 100 μl of hybridoma culture supernatants, the positive control (serum of immunized mice), and the negative control (Sp2/0 culture), were accordingly added to the plates and incubated at 37 °C for 1 h. Plates were washed four times with PBST and incubated with 100 μl horseradish peroxidase conjugated goat anti-mouse immunoglobulin (IgG-HRP) (Santa Cruz Biotechnology, USA) for 30 min at 37 °C. Finally, plates were washed five times with PBST and incubated with 100 μl 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich, USA) in dark for 15 min at room temperature. The reaction was terminated by supplementing 50 μl H2SO4 solution (1 M) and the absorbance values were determined at 450 nm by BioTek™ ELx808™ Absorbance Microplate Readers (ThermoFisher, USA). 2.5. MAb production MAbs against V. parahaemolyticus were produced by traditional ascitic fluid method. Cells of desired monoclones in density of 1 × 106 cells/0.5 ml PBS were injected intraperitoneally into each mouse, which had been previously injected with 0.5 ml Pristane (Sigma-Aldrich, USA) two weeks earlier. Their ascites were harvested about ten days later when their abdomens were completely enlarged and their skins were extended. The supernatants were collected after centrifugation of the ascites at 2900g for 30 min. The antibodies were precipitated by 50% saturation of ammonium sulfate solution, followed by purification by protein G column (GE Healthcare, USA) affinity column chromatography according to the manufacturer's protocol. 2.6. MAb characterization The purity of prepared MAbs was analyzed by SDS-PAGE. The titers of MAbs were determined by indirect ELISA. The titer of an antibody solution was defined as the highest dilution that could give a positive reaction against the antigen. The immunoglobulin subclass was determined by a commercial Mouse Typer Sub-isotyping Kit (Bio-Rad, USA) according to the manufacturer's protocol. The affinity constant (Kaff) of MAbs were determined by indirect ELISA according to the method described previously (Beatty et al., 1987). 2.7. Preparation of colloidal gold and colloidal gold-MAb conjugate Colloidal gold was prepared according to a previous report (Grabar et al., 1995). Briefly, 100 ml of 0.01% (w/v) HAuCl4 (Sigma-Aldrich, USA) in a 250 ml siliconized flask was heated to boiling in a microwave oven, and 1.4 ml 1% trisodium citrate (Sigma-Aldrich, USA) was added. The solution was allowed to gradually cool down and then stored at 4 °C in a dark-colored glass bottle. The pH of the colloidal gold was adjusted to 8.4 with 1% (w/v) potassium carbonate. The capture MAb (30 μg) was added dropwise into 10 ml colloidal gold solution on a magnetic stirrer
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for 30 min. After the solution was stabilized at 4 °C for 30 min, 1 ml 10% (w/v) BSA (Sigma-Aldrich, USA) was added to block the excess gold colloid. The mixture was then stirred for another 30 min and stored at 4 °C for 2 h, followed by centrifuging at 3000g at 4 °C for 30 min. The resultant supernatant was further centrifuged at 14,000g at 4 °C for 45 min to obtain the pellets of colloidal gold-MAb conjugate, which were resuspended in 10 mM borax (Sigma-Aldrich, USA) buffer (pH 8.0) containing 2% (w/v) BSA and 0.05% (w/v) NaN3 (Sigma-Aldrich, USA) for further use.
buffer (pH 7.8) and was tested by using the strips. The lowest concentration that gave a positive signal was defined as the testing limit. To verify the specificity, a total of 32 microbial species, including 6 Vibrio species, were diluted to 106 CFU/ml and used for the LFA strip test. To study the testing repeatability, three V. parahaemolyticus samples (106, 105 and 104 CFU/ml) and three negative control samples (106, 105 and 104 CFU/ml of V. alginolyticus) were tested repeatedly by the prepared LFA strips with ten replicates per sample. The stability was evaluated by storing the strips at room temperature for one year and testing relative samples similarly.
2.8. Manufacture of LFA strips 2.11. Test of shrimp samples The LFA 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 (Fig. 1). First, the detection antibody (the test line antibody) and goat antimouse IgG (the procedural control line antibody) were diluted to the working concentration and sprayed onto a piece of nitrocellulose membrane, which was subsequently dried at 37 °C for 24 h. The strips were then blocked and dried at 37 °C. The capture antibody conjugated with colloidal gold (40 nm diameter) was sprayed twice onto the fiberglass (0.5–1.5 × 25 cm) and dried at 37 °C. Finally, the components were assembled as a package and sliced into 4 mm wide testing strips. To perform a test, the strip was dipped fully into the sample solution and kept at room temperature for 5 min. When there are two red bands at both test and control lines, this shows a positive testing result. With only one red band at control line, this represents a negative result. The absence of the band at the control line tells the invalidity of the test. 2.9. MAb pairing The LFA strips with all possible MAb pairs were prepared. The cell suspensions of V. parahaemolyticus and those of other Vibrio species (50 μg/ml) were used as test samples. The MAb pairs that gave the strongest signals for V. parahaemolyticus, but negative to all other Vibrio species were selected for further study. 2.10. Characterization of LFA strips The testing limit of LFA strips was determined by the method of serial dilution. The V. parahaemolyticus suspension was serially diluted at 10-fold from 1.2 × 108 CFU/ml to 1.2 × 101 CFU/ml in 20 mM Tris-HCl
Whiteleg shrimp (Litopenaeus vannamei, formerly Penaeus vannamei) samples were collected from a shrimp farm located in Taishan, Guangzhou province, China. The sick shrimps were weak and swam close to the water surface. Their shells were pale red with white spots. To identify the V. parahaemolyticus infection, the shrimp was cleaned with 70% ethanol, followed by aseptic isolation of hepatopancreas samples and inoculation in Alkaline Peptone Water (APW). After being incubated at 37 °C overnight, the APW cultures were sub-cultured onto Vibrio Chromogenic Medium (CHROMagar, France). The appearance of mauve colonies after overnight incubation at 37 °C indicated the V. parahaemolyticus infection. In this study, six healthy and seven V. parahaemolyticus infected shrimps, which had been confirmed by clinical symptoms and microbiological method, were used for strip tests. The hepatopancreases from all the shrimps were homogenized individually with mortar and pestle. One milliliter of 20 mM Tris-HCl buffer (pH 7.8) was added and mixed with the hepatopancreas homogenates. The suspensions were detected by the LFA strips. White feces from healthy and sick shrimps were collected separately, followed by supplement of 0.2 ml of Tris-HCl buffer (pH 7.8) and complete mix by pipette. The fecal suspensions were tested by the LFA strips as well. 2.12. Test of human fecal samples from diarrheal patients To verify the detection limit of V. parahaemolyticus in human fecal samples, feces from three healthy persons were collected and suspended completely in 20 mM Tris-HCl buffer (pH 7.8) by the ratio of 5 ml of buffer per 1 g of feces. Five milliliters of each suspension were collected and mixed together to prepare the negative control and diluting agent. The V. parahaemolyticus culture was then serially
Fig. 1. Description of LFA architecture. LFA consists of a sample binding region called a sample absorption pad, a result showing region including a conjugate pad and a nitrocellulose membrane, and a tag terminal called wicking pad. When a sample is applied to the sample pad, it flows towards the terminal wicking pad. A positive signal is visualized by the gold nano-particles when the sample, captured by gold-conjugated antibody, binds the antibody on the test line. Meanwhile, a test strip also contains a control line, where anti-IgG antibody is coated, to assure accuracy of the assay.
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diluted at 10-fold from 0.8 × 106 CFU/ml to 0.8 × 102 CFU/ml in the prepared negative control and was tested by the strips. Fecal samples from 146 diarrheal patients, whose infectious bacteria were determined by traditional microbiological method, were collected from Shandong Jiaotong Hospital, Shandong province, China, during July–September 2015. One milliliter of 20 mM Tris-HCl buffer (pH 7.8) was added to 0.2 g of fresh fecal sample and mixed completely. The suspensions were directly tested by the LFA strips.
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to be strongly positive, those of all other microbial species were negative (Fig. 3). These results indicated that both types of LFA strip were highly sensitive and specific to V. parahaemolyticus culture. The reproducibility of the LFA strips was also examined. The testing results were 100% consistent for both positive and negative samples. The strips were confirmed to be stable at room temperature for 1 year (data not shown). 3.4. Detection of V. parahaemolyticus infection in Whiteleg shrimps
3. Results 3.1. MAb production and pairing Eight BALB/c mice were used for immunization at different dosages of V. parahaemolyticus. The one with the highest antibody titer (1:105) was chosen as the spleen cell donor. After fusion of splenocytes and freshly grown Sp2/0 cells, positive hybridomas were observed in cell culture plates. The fusion rate was 84.7%. The supernatants of growing hybridoma cells were screened based on reactivity against V. parahaemolyticus by indirect ELISA. A total of 1278 positive hybridoma clones were selected. The hybridomas were subcloned 3 times and consequently 79 stable positive clones, whose antibodies specifically recognized V. parahaemolyticus, were obtained. Based on the isolated positive clones, 79 ascites containing relevant MAbs were prepared. Subsequently, the MAbs were purified with a combination of 50% saturated ammonium sulfate precipitation and Protein G affinity chromatography. After cross-pairing all the 79 MAbs, the antibody pair GA1a-IC9 and IC9-KB4c, which showed strong signal to V. parahaemolyticus and negative to all other microbes, were selected for further study. 3.2. MAb characterization SDS-PAGE demonstrated the purities of selected MAb products, GA1a, IC9 and KB4c, were all over 95% (data not shown). The antibody titers of cell culture supernatants and mouse ascites were determined to be 4.3 × 10−3 and 1.3 × 10−6 (GA1a), 7.2 × 10−3 and 2.1 × 10−6 (IC9), and 2.7 × 10− 3 and 8.3 × 10−5 (KB4c), respectively. The Kaff was calculated to be 2.15 × 109 l/mol (GA1a), 3.61 × 109 l/mol (IC9) and 1.82 × 109 l/mol (KB4c), respectively (Table 1). Therefore, the GA1a, IC9 and KB4c were believed to be satisfying MAbs with high specificities, titers and affinities. The isotype test indicated that the GA1a MAb was of IgG2b isotype, IC9 was of IgG1 and KB4c was of IgA (Table 1). 3.3. Characterization of LFA strips Two types of LFA strips were developed. The type I applied GA1a as the capture antibody (gold-conjugated antibody) and IC9 as the detection antibody (coating antibody); while the type II used IC9 as the capture antibody and KB4c as the detection antibody. The testing limits of the LFA strips were examined by testing the serial dilutions of V. parahaemolyticus culture. The Tris-HCl buffer (pH 7.8) was used as negative control. Both types of LFA strip were found to be of the same testing limit, which was 1.2 × 103 CFU/ml (Fig. 2). To test the specificity, a total of 32 microbial species, including 6 Vibrio species, were tested by the LFA strips. While the culture of V. parahaemolyticus was confirmed Table 1 Characterization of MAbs. Clone ID
GA1a IC9 KB4c
Antibody titer Culture supernatant
Ascites
4.3 × 10−3 7.2 × 10−3 2.7 × 10−3
1.3 × 10−6 2.1 × 10−6 8.3 × 10−5
Kaff(l/mol)
Isotype
2.15 × 109 3.61 × 109 1.82 × 109
IgG2b IgG1 IgA
The hepatopancreas samples of healthy and V. parahaemolyticus infected shrimps were tested by the LFA strips. Both types of LFA strip showed exactly the same results. All seven samples from sick shrimps were confirmed to be positive, while the six healthy samples were all found to be negative (Fig. 4). Similar results were also observed when the fecal samples were tested (Fig. 5). Thus, the diagnostic sensitivity and specificity of the homemade LFA strips for shrimp hepatopancreas and fecal samples in this study were both 100%. 3.5. Detection of V. parahaemolyticus infection in human diarrheal patients The detection limit of V. parahaemolyticus in human fecal samples was studied (Fig. 6). At the concentration of 0.8 × 104 CFU/ml, strips of both types exhibited clearly positive. When the concentration decreased to 0.8 × 103 CFU/ml, while the T line of type II strip was still visible, that of type I strip became difficult to see. Thus, the testing limit of type II strip for fecal samples, which could reach as low as 0.8 × 103 CFU/ ml, was slightly lower than that of type I strip. To evaluate the clinical application in V. parahaemolyticus detection in humans, a total of 146 fecal samples from diarrheal patients were freshly collected and immediately tested by the two types of LFA strips. The testing data are summarized in Table 2. Of the 27 fecal samples from V. parahaemolyticus infected patients, 26 were detected positive by type I strip and all positive by type II strip. The diagnostic sensitivities were 96.3% and 100%, respectively. When it comes to specificity, both types of LFA strips showed no cross-activity to any of the other bacteria associated with diarrhea. Therefore, the type II LFA, with IC9 as the capture antibody and KB4c as the detection antibody, was considered as a better product, whose sensitivity and specificity for V. parahaemolyticus detection in fecal samples from diarrheal patients were both 100% in the study. 4. Discussion Globally, V. parahaemolyticus is a leading cause of seafood-associated gastroenteritis (Hara-Kudo et al., 2012; Haendiges et al., 2014). Detection of V. parahaemolyticus infection is of great significance not only for aquaculture disease detection and food safety control, but also for accurate diagnosis and proper treatment for human diarrheal patients. The traditional microbiological culture method, though considered as the gold-standard for V. parahaemolyticus detection, is labor-intensive and time-consuming with complicated procedures and low sensitivity (Chitkara, 2005; DeBoer et al., 2010). Currently, many commercial PCR-based detection kits, such as BioFire FilmArray Gastrointestinal Panel and Luminex xTAG Gastrointestinal Pathogen Panel (Khare et al., 2014; Buss et al., 2015), are available. Comparing to traditional biological method, these commercial kits can finish the tests within hours with much higher sensitivity and specificity. However, due to the high costs, their application is highly restricted in primary public healthcare laboratories, especially in developing regions. In contract, LFA is an immunological method that requires neither expensive equipment nor highly trained personnel, and that can be accomplished within a few minutes. In this study, using the inactivated V. parahaemolyticus as immunogen, two pairs of murine anti-V. parahaemolyticus MAbs, GA1a-IC9 and IC9-KB4c, were prepared for LFA development. Based on the two MAb
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Fig. 2. Testing limit of V. parahaemolyticus LFA strips. C Line, Control line; T Line, Test line. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); 1–8, serial dilutions of V. parahaemolyticus culture: 1, 1.2 × 108 CFU/ml; 2, 1.2 × 107 CFU/ml; 3, 1.2 × 106 CFU/ml; 4, 1.2 × 105 CFU/ml; 5, 1.2 × 104 CFU/ml; 6, 1.2 × 103 CFU/ml; 7, 1.2 × 102 CFU/ml; and 8, 1.2 × 101 CFU/ml. The detection threshold for both types of LFA strips was 1.2 × 103 CFU/ml. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Specificity analysis of V. parahaemolyticus strips. C Line, Control line; T Line, Test line. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); 1–32: 106 CFU/ml of microbial culture diluted in 20 mM Tris-HCl buffer (pH 7.8). 1, V. alginolyticus; 2, V. cholerae; 3, V. vulnificus; 4, V. harveyi; 5, V. fortis; 6, Enterococcus faecalis; 7, Klebsiella pneumoniae; 8, Enterobacter cloacae; 9, Staphylococcus aureus; 10, Streptococcus iniae; 11, Helicobacter pylori; 12, Salmonella typhimurium; 13, Aeromonas caviae; 14, Edwardsiella ictaluri; 15, Stenotrophomonas maltophilia; 16, Bacillus subtilis; 17, Candida tropicalis; 18, Shewanella putrefaciens; 19, Aeromonas hydrophila; 20, Citrobacter freundii; 21, Candida albicans; 22, Staphylococcus epidermidis; 23, Salmonella paratyphi B; 24, Salmonella typhi; 25, Serratia marcescens; 26, Proteus mirabilis; 27, Escherichia coli; 28, Staphylococcus haemolyticus; 29, Neisseria gonorrhoeae; 30, Pseudomonas aeruginosa; 31, Acinetobacter baumannii; 32, V. parahaemolyticus. Both types of LFA strips were highly specific to V. parahaemolyticus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Test of hepatopancreas samples from Whiteleg shrimps. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); Pos, positive control, suspension of V. parahaemolyticus cells; H1–H6, hepatopancreas samples from six healthy shrimps; S1-S7, hepatopancreas samples from seven V. parahaemolyticus infected shrimps. All the seven samples from sick shrimps were confirmed to be positive, while the six healthy samples were all found negative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pairs, two types of LFA strips were prepared. The testing limit for V. parahaemolyticus culture was both 1.2 × 103 CFU/ml, which is close to those reported for PCR-base methods (Panicker et al., 2004; Hanabara and Ueda, 2016), but much lower than a previously reported V.
parahaemolyticus LFA (105 CFU/ml) (Guo et al., 2012). The concentrations of foodborne pathogenic bacteria are usually N106 CFU/g feces in acute phase of illness (Hanabara and Ueda, 2016). However, fecal samples are often collected in the non-acute phase, when the bacterial
Fig. 5. Test of fecal samples from Whiteleg shrimps. The blue (dark color) strips were of type I and the yellow (light color) ones were of type II. Neg, negative control, 20 mM Tris-HCl buffer (pH 7.8); Pos, positive control, suspension of V. parahaemolyticus cells; H1–H6, fecal samples from six healthy shrimps; S1–S7, fecal samples from seven V. parahaemolyticus infected shrimps. The test results of fecal samples were highly correlated to those of hepatopancreas samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Testing limit of V. parahaemolyticus LFA strips for fecal samples. Neg, negative control (healthy feces suspension); 1–5, V. parahaemolyticus samples serially diluted in healthy feces sample: 1, 0.8 × 106 CFU/ml; 2, 0.8 × 105 CFU/ml; 3, 0.8 × 104 CFU/ml; 4, 0.8 × 103 CFU/ml; 5, 0.8 × 102 CFU/ml.
concentration may be much lower. Therefore, the low testing limits of our homemade strips guarantee a high diagnostic sensitivity for V. parahaemolyticus detection, and no requirement of culture enrichment in testing human fecal samples. Due to the high specificity and low testing limit, the strips successfully detected the V. parahaemolyticus infection in both hepatopancreas and fecal samples from 13 Whiteleg shrimps with 100% diagnostic specificity and sensitivity. During the test of human feces from diarrheal patients, the two types of strips showed a slight difference in diagnostic sensitivities. While type II strip still exhibited 100% sensitivity, type I failed to detect one of the total 27 positive samples. It might be caused by different testing limits of the two strips in fecal samples, as the sample that type I strip failed to identify showed very weak signal in type I strip as well. The fecal samples are more complex than those from bacterial culture. Thus, type II strip is believed to be a more promising product to diagnose the V. parahaemolyticus infection in diarrheal patients. To date, few studies on V. parahaemolyticus detection by immunological methods have been reported. Guo et al. developed an immunochromatographic strip for detection of V. parahaemolyticus in seafoods by employing a polyclonal antibody (PAb)–MAb pair (Guo et al., 2012). Our study used MAb pairs, which should be more specific than PAb-MAb pairs. In addition, the testing limit of Guo's strip was as high as 105 CFU/ml, which made it unsuitable to directly detect V. parahaemolyticus in human feces. Kawatsu et al. reported a test strip to detect thermostable direct hemolysin (TDH) produced by V. parahaemolyticus (Kawatsu et al., 2006). However, due to the low content of TDH in the bacteria, culture enrichment of the samples was required to get a signal for positive fecal specimens. In conclusion, we successfully developed two types of LFA strips for V. parahaemolyticus detection. Their testing limits are both 1.2 × 103 CFU/ml for bacterial culture. Both strips are able to directly detect V. parahaemolyticus in hepatopancreas and fecal samples from infected Whiteleg shrimps and, potentially, from other aquatic products as well. Importantly, type II strip, due to its higher sensitivity in human fecal specimens, can be used to rapidly diagnose V. parahaemolyticus in human feces without prior culture enrichment process, which allows the test to be finished within only a few minutes.
Table 2 Testing results of fecal samples from diarrheal patients. Infection causes
V. parahaemolyticus Diarrheagenic E. coli Salmonella spp. Shigella spp. Campylobacter Total
Sample numbers
27 44 31 26 18 146
Type I LFA
Type II LFA
Positive
Negative
Positive
Negative
26 0 0 0 0 26
1 44 31 26 18 120
27 0 0 0 0 27
0 44 31 26 18 119
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