Journal of Microbiological Methods 108 (2015) 1–3
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Monoclonal antibody-based competitive enzyme-linked immunosorbent assay to detect antibodies to O:4 Salmonella in the sera of livestock and poultry Swarmistha Devi Aribam a, Yohsuke Ogawa a, Hidenori Matsui b, Jiro Hirota a, Masashi Okamura c, Masato Akiba a, Yoshihiro Shimoji a, Masahiro Eguchi a,⁎ a b c
Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, NARO, Tsukuba, Ibaraki 305-0856, Japan Kitasato Institute for Life Sciences and Graduate School of Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan Laboratory of Zoonoses, Kitasato University School of Veterinary Medicine, Towada, Aomori 034-8628, Japan
a r t i c l e
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Article history: Received 26 October 2014 Received in revised form 4 November 2014 Accepted 4 November 2014 Available online 13 November 2014
a b s t r a c t Serotyping is an important element for surveillance of Salmonella. In this study, an anti-O:4 Salmonella monoclonal antibody-based competitive enzyme-linked immunosorbent assay that could identify Salmonella infection in cow, pig, horse, and chicken was developed. This detection system can therefore be useful for a wide range of animals and for humans. © 2014 Elsevier B.V. All rights reserved.
Keywords: Animal salmonellosis Competitive ELISA Group O:4 Salmonella Monoclonal antibody
Salmonella, an important zoonotic pathogen, is one of the major causative agents of food-borne infectious diseases worldwide. Many of the human salmonellosis cases reported to date were caused by consumption of contaminated egg, chicken, pork, beef, and milk products (Geimba et al., 2004, Meemken et al., 2014, Vo et al., 2014). The loss in animal production and public health issues associated with salmonellosis has a substantial impact on the economy of several countries (Hendriksen et al., 2009, Hoelzer et al., 2011, Mather et al., 2013, Schrader et al., 2008). Therefore, to reduce the prevalence of the bacteria and the risk of transmission to humans, efﬁcient surveillance has become increasingly important. A critical component of current surveillance programs involves serotyping using serum/antibodies to the O-antigen of Salmonella lipopolysaccharide (LPS) (Ronholm et al., 2011). Routine serotyping helps in monitoring the public health response to the global challenge of salmonellosis and the effectiveness of control measures (Wahlström et al., 2014, Herikstad et al., 2002; Wattiau et al., 2011). However, shortage of reagents and lack of capital required for mass-scale laboratory diagnosis may be a hindrance to such surveillance techniques. Therefore, the development of readily available detection systems of Salmonella antibody in livestock is important. In this study, we developed an anti-O:4 Salmonella monoclonal
⁎ Corresponding author. Tel./fax: +81 29 838 7790. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.mimet.2014.11.002 0167-7012/© 2014 Elsevier B.V. All rights reserved.
antibody (mAb)-based competitive enzyme-linked immunosorbent assay (ELISA) by using a novel mAb, 414B. One of the most commonly isolated Salmonella serotypes with a broad host range is the O:4 serotype of Salmonella enterica serovar Typhimurium (S. Typhimurium, O:1,4,,12) (Singh, 2013). We therefore used the S. Typhimurium strain χ3306 for our study (Eguchi and Kikuchi, 2010). The hybridoma-secreting mAb 414B (subclass IgG2a) was developed by fusing P3-X63-Ag8-U1 myeloma cells with lymph node cells from the spleens of 8-week-old BALB/c mice infected with aroA- S. Typhimurium strain UF20 (Gulig and Doyle, 1993) by using 50% polyethylene glycol (Yokoyama, 1991). Hybridoma supernatants were screened on S. Typhimurium LPS-coated wells in an indirect ELISA (Fig. 1A) (Ko et al., 2009). LPS was puriﬁed by the hot phenol–water method (Westphal and Jann, 1965). For indirect ELISA, a 96-well plate (Nunc; Roskilde, Denmark) was coated with 50 μl of 5 μg/ml S. Typhimurium LPS diluted in carbonate–bicarbonate-buffered saline (pH 9.4). All incubations were carried out at 37 °C for 60 min and every incubation step was followed by three washes with phosphate-buffered saline supplemented with 0.05% Tween-20 (PBS-T). After washing, LPS-coated plates were incubated with 2 μg/ml mAb 414B or control IgG2a in serial two-fold dilutions (2–0 μg/ml) (Fig. 1A). Bound antibodies were detected by using rabbit anti-mouse IgG2a conjugated to horseradish peroxidase (HRP; Invitrogen; Carlsbad, CA USA) diluted 1:2000 in PBS-T and developed using 3,3′,5,5′-tetramethylbenzinine substrate (Thermo Scientiﬁc Pierce; Rockford, IL, USA). Indirect ELISA using LPS from four Salmonella
S.D. Aribam et al. / Journal of Microbiological Methods 108 (2015) 1–3
Fig. 1. Development of mAb 414B and establishment of a competitive ELISA using mAb 414B for detection of O:4 LPS. (A) Indirect ELISA to determine the reactivity of mAb 414B with S. Typhimurium LPS. The negative control (IgG2a) demonstrated no reaction with LPS. (B) Analysis of serotype speciﬁcity of mAb 414B by using LPS puriﬁed from S. Typhimurium (ST), S. Choleraesuis (SC), S. Dublin (SD), and S. Enteritidis (SE) in an indirect ELISA. IgG2a was used as a negative control. (C) Bacterial agglutination of S. Typhimurium (1 × 107 CFU) by using rabbit O:4 antiserum and control serum. The circle and triangles represent antibody titer of aggregation reaction for the rabbit O:4 antiserum, and the control serum respectively. Data is shown for three independent experiments performed in triplicate and the line in each panel represents the average of the resultant serum dilutions obtained. (D) Competitive ELISA to analyze the effect of O:4 antiserum and control serum at 24- to 768-times dilutions on blocking the binding of mAb 414B with LPS. The Student's t-test showed that the difference in the blocking ability of O:4 and control serum were signiﬁcant at 24- to 192-times dilution for all concentration of mAb 414B tested (*, p b 0.05). All ELISA data shown here (A, B, and D) represent mean ± standard deviation from three independent experiments performed in triplicate.
serotypes, S. Typhimurium (group O:4), S. Choleraesuis (group O:7), S. Dublin (group O:9), and S. Enteritidis (group O:9), showed that mAb 414B (1 μg/ml) is speciﬁc to group O:4 LPS (Fig. 1B). To develop a competitive ELISA based on the ability of rabbit O:4 antiserum (Denka Seiken; Tokyo, Japan) to block the binding site targeted by mAb 414B, a bacterial agglutination reaction was carried out and the cutoff titer for O:4 antiserum was determined (Fig. 1C). To this end, 50 μl of two-fold serial dilutions of the O:4 antiserum or normal rabbit serum wAS dispensed in a 96-well microtiter plate with a starting dilution of 1:6, and 100 μl of S. Typhimurium adjusted to 1 × 107 CFU was emulsiﬁed in each well. After incubation at 4 °C overnight, agglutination was determined by observation and 192 to 768-times dilution was found to be the highest serum dilution with a positive limit for detection (Fig. 1C). The speciﬁcity of mAb 414B at 0.5, 1.0, and 2.0 μg/ml was evaluated in a competitive ELISA by using O:4 antiserum at 24 to 768-times dilutions. The LPS coating, incubation, and washing steps were carried out according to the protocol described for the indirect ELISA (Fig. 1A). After coating, the plate was washed three times with PBS-T and nonantigen binding sites were blocked with 0.5% bovine serum albumin in PBS-T. The wells were incubated with 50 μl of O:4 antiserum or normal rabbit serum at two-fold serial dilutions (24- to 768-times). This was followed by incubation with mAb 414B at 0.5, 1.0, and 2.0 μg/ml (Fig. 1A) and detection of bound antibody by using rabbit anti-mouse IgG2a conjugated to HRP (1:2000). The presence of O:4 antiserum blocked the reaction of mAb 414B with LPS, as indicated by a low optical density value, whereas in the absence of the O:4 antiserum, the antigens reacted freely with mAb 414B (Fig. 1D). The results also indicated that for all concentrations of mAb 414B tested, signiﬁcant blocking (p b 0.05) was observed even with a very high dilution (192-times) of the antiserum. To validate our assay, S. Typhimurium-infected animals belonging to four animal models (cow, pig, horse, and chicken) were selected as reference animals. Using sera from the infected and non-infected reference animals, bacterial agglutination was carried out as described above
(Fig. 1C) and the optimal titers that demonstrated Salmonella infection in the infected animals were determined. For the cow and horse sera, the lowest serum dilution that showed an agglutination reaction was 6, while for chicken and pig it was 48 (Fig. S1). At these dilutions, no agglutination was seen for the non-infected serum samples. To test the efﬁcacy of our method, we evaluated the mAb 414B-based competitive ELISA for detection of antibodies to Salmonella in the sera of the infected animals. For this assay, we used sera at the dilutions determined above (Fig. S1), and mAb 414B was used at 1 and 2 μg/ml (Fig. 1D). Noninfected sera were used as negative control in this assay. The addition of infected sera signiﬁcantly blocked the reaction of mAb 414B, whereas the addition of non-infected sera did not block the reaction between mAb 414B and Salmonella LPS, as indicated by increased absorbance (Fig. 2A). To evaluate the optimal cut-off for our assay, the average of the control readings were normalized to 100% and the relative percentages of all the infected sera were plotted on a continuous scale of 0 to 100% (Fig. 2B). Thus, sera with a cut-off value less than or equal to about 40% can be considered as seropositive (Fig. 2B). The competitive ELISA using the O:4 group speciﬁc mAb 414B was thus found to be speciﬁc and sensitive for the detection of cow, pig, horse, and chicken antibodies to O:4 Salmonella. We suggest that the mAb 414B will be useful in screening for salmonellosis in livestock and poultry as a supplement to other available tests. In addition, this method may also be effective in diagnosing Salmonella infection in reptiles, domestic pets, as well as zoo animals that are often found to be carriers of Salmonella (Hoelzer et al., 2011; Jang et al., 2008). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2014.11.002. Acknowledgments This research was supported in part by Grant-in-Aid for Scientiﬁc Research to ME (26660254) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.
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Fig. 2. mAb 414B-mediated detection of antibody to S. Typhimurium in infected reference animals and evaluation of the optimal cut-off value for the assay. (A) Competitive ELISA using sera from infected and non-infected cow, pig, horse, and chicken at 6-, 48-, 6-, and 48-times serum dilution, respectively. A Student's t-test was used to compare the difference in the blocking ability of the infected and non-infected serum, which showed signiﬁcant values (p b 0.01) for all sera tested. (B) Normalized data for the competitive ELISA to calculate the optimal cut-off value for the assay. Data shown here represents mean ± standard deviation from three independent experiments performed in triplicate.
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