Production and characterization of anti-Salmonella polyclonal antibodies as bio-recognition element for developing a microbial monitoring method

All the foodborne pathogens listed in Table 1, except for bacteria ending with ATCC, were obtained from the Food Microbiology Laboratory at Auburn University Auburn, AL, USA. The other foodborne pathogens with ATCC label were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Each bacterial strain was cultivated in 20 mL of Tryptic Soy Broth (TSB, Difco Laboratories, Sparks, MD, USA) while L. monocytogenes was cultivated in TSB with 0.6% yeast extract. After incubation at 37℃ at 200 rpm for 16 h, each culture was washed three times with phosphate buffered saline (PBS, pH 7.2, Sigma-Aldrich Co., St. Louis, MO, USA) by centrifugation at 5,000 ×g for 5 min. The collected bacterial cells were re-suspended in PBS and the bacterial concentration was adjusted to 10⁸ CFU/mL using a pre-constructed standard curve, determined by optical density at 640 nm. Salmonella mixture was prepared by mixing equal amounts of S. Typhimurium and S. Enteritidis.

A New Zealand white rabbit (approximately 3 kg) was used for the production of pAbs against Salmonella. The purification of pAbs was performed following a modified Kohler and Milstein’s method (1975). For the immunization of the rabbit, Salmonella mixture (10⁸ cells), inactivated with 1% formalin, was emulsified with Ribi’s adjuvant system at 285 g/mL. The rabbit was immunized with 1 mL of the adjuvant emulsion through an intradermal injection of 50 μL into each of eight sites on the back, close to the spinal cord, and 300 μL aliquot intramuscular into each hind leg. The same immunization procedure with the adjuvant emulsion was performed to boost the antibody production in the rabbit at 4 week intervals after the initial immunization. Seven days later, the rabbit blood containing anti-Salmonella pAbs was collected from the central ear artery and centrifuged at 5,000 ×g for 20 min at 4℃. Solid ammonium sulfate was added slowly into 1 mL of the collected anti-Salmonella rabbit serum until a final concentration of 0.5 g/mL. After placing on ice for 5 min, the serum was centrifuged at 5,000 ×g for 5 min at 4℃ and the precipitate was washed twice with 1 mL of saturated ammonium sulfate. The precipitate was then suspended in PBS and dialyzed in a 20 mM sodium phosphate buffer (pH 7.0) at 4℃ overnight. The suspension containing anti-Salmonella pAbs was applied to protein A affinity column (Sigma-Aldrich Co., St. Louis, MO, USA) and eluted with 0.1 M citric acid (pH 3.45). After adjusting pH of the eluates with 2 M Tris (pH 9), the protein content of each fraction was estimated by measuring adsorption at 280 nm. Finally, all the eluates were dialyzed with PBS at 4℃ overnight, and the protein content of purified anti-Salmonella pAbs was determined by Bradford method.

Salmonella mixture (S. Typhimurium and S. Enteritidis) and forty foodborne pathogens (20 Salmonella serotypes and 20 non-Salmonella strains) were used for the investigation of the reactivity and specificity of anti-Salmonella pAbs, respectively. A 96-well polystyrene assay plate (Costar, Cambridge, MA, USA) was coated with 100 μL of Salmonella mixture for the reactivity test and each bacterial suspension (10⁸ CFU/mL) for specificity test at 37℃ for 1 h. After washing three times with 200 mL PBS containing 0.1% Tween-20 and 0.02% sodium azide (PBST), the plate was blocked with 200 μL of 1% bovine serum albumin (BSA, Equitech-Bio Inc., Kerrville, TX, USA) for 1 h at 22℃, followed by washing with 200 mL PBST. An aliquot of 100 μL of purified anti-Salmonella pAbs was added and the plate was incubated at 22℃ for 2 h, prior to washing with PBST. Commercial anti-Salmonella pAbs (Abcam Co., Cambridge, MA, USA) were used for the comparison of reactivity with that of purified anti-Salmonella pAbs. An aliquot of 100 μL of alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma-Aldrich Co.) was added to each well for incubation at 22℃ for 1 h. Finally, 100 μL of p-nitrophenyl phosphate (p-npp, Sigma-Aldrich Co.) in 10 mM diethanolamine buffer (pH 9.5) was added to the wells for color development. Finally, the absorbance at 405 nm was measured at 0 min and 15 min using a microplate reader (Thermo Labsystems Inc., Helsinki, Finland).

A glass sensor platform was prepared by cutting a microscopic cover glass (0.17 mm thickness) into 5×5 mm with micro-dicing saw (MPE Inc., Grass Valley, CA, USA). After cutting, it was ultrasonically cleaned with an acetone solution, and rinsed with alcohol and deionized (DI) water. The cleaned sensor was sputtered with 140 nm gold by using a Pelco SC-6 sputter coater (Ted Pella Inc., Redding, CA, USA). For antibody immobilization, 100 μL of purified anti-Salmonella pAbs were added on the gold sensor surface for incubation at 37℃ for 1 h. After washing three times with PBS, the pAbs-immobilized gold sensor was blocked with 100 μL of 1% BSA at 22℃ for 1 h. Finally, the sensor was incubated with Salmonella mixture at 37℃ for 1 h and washed four times with DI water, prior to SEM observation (S-4800, Hitachi Ltd., Tokyo, Japan).

All experiments were performed at least in triplicates and the data are expressed as mean±SD. Student’s paired t-test, for two groups, and one-way analysis of variance (ANOVA), for more than two groups, were used to compare the means by using GraphPad and InStat V.3 software (GraphPad Co., San Diego, CA, USA). Differences were considered to be statistically significant at p<0.05.

Unlike the preparation of monoclonal antibodies (mAbs), the preparation of pAbs is less complicated since it does not require an immortal hybridoma (18-19). Anti-Salmonella pAbs were produced from a rabbit and purified by saturated ammonium sulfate precipitation and protein A affinity column. The final antibody concentration was determined to be 2.0 mg/mL, which was greater than that of commercial anti-Salmonella pAbs (1.0 mg/mL).

Reactivity of purified anti-Salmonella pAbs was tested and compared with that of commercial ones by indirect ELISA (Fig. 1). Since the original concentration of purified anti-Salmonella pAbs was two-fold greater than that of commercial anti-Salmonella pAbs, its concentration was adjusted with that of commercial ones to enable the direct comparison between both antibodies. The reactivity of purified anti-Salmonella pAbs was greater than those of commercial anti-Salmonella pAbs over the entire range of pAbs concentration, although there were no significant differences between the two groups at the same dilution of anti-Salmonella pAbs (p>0.05). Higher the antibody concentration, less differences of absorbance was observed for both anti-Salmonella pAbs. However, the purified anti-Salmonella pAbs obviously had greater binding efficiency against Salmonella mixture at relatively lower concentration of pAbs, ranging from 1/3,200 to 1/6,400 dilution of the original concentration of pAbs (1.0 mg/mL). The greater binding efficiency of purified anti-Salmonella pAbs over commercial pAbs can presumably be attributed to targeting S. Typhimurium and S. Enteritidis and relatively delicate purification procedures. Based on our results, the concentration of purified anti-Salmonella pAbs was determined to be 0.00125 mg/mL (1,600 times dilution of original concentration of purified anti-Salmonella pAbs) considering both reactivity and economic aspects. Finally, this study confirmed that the purified anti-Salmonella pAbs had a competitive reactivity over commercial anti-Salmonella pAbs.

Fig. 1. Comparative reactivity of purified anti-Salmonella pAbs and commercial anti-Salmonella pAbs. The x-axis numbers, corresponding dilutions are as follows: 1, 200 times dilution; 2, 400 times dilution; 3, 800 times dilution; 4, 1,600 times dilution; 5, 3,200 times dilutions; 6, 6,400 times dilution; 7, 12,800 times dilution; 8, 25,600 times dilution; 9, 51,200 times dilution of anti-Salmonella pAbs (1.0 mg/mL). Vertical bars represent SD and paired t-test was performed at p<0.05 (N=3).

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Besides reactivity, the other desired property, for the antibody to be useful as a bio-recognition element, is its specificity against all pathogenic Salmonella serotypes. So far, 20 most frequent serotypes have been held responsible for approximately 70% of Salmonella outbreaks (approximately 13% unknown serotypes and 15% other serotypes) (7). As shown in Table 1, all Salmonella serotypes, except S. Diarizonae, showed significantly higher binding efficiencies with purified anti-Salmonella pAbs (p<0.05). Among the 20 Salmonella serotypes, purified anti-Salmonella pAbs showed the best binding reactivity with S. Typhimurium and S. Enteritidis, which exhibited maximum color change during incubation. However, the purified anti-Salmonella pAbs showed no cross-reactivity against other exclusive foodborne pathogens including major strains of Bacillus, Campylobacter, E. coli/E. coli O157:H7, Listeria, Shigella, Staphylococcus, Vibrio, and Yersinia. Their absorbance changes were obviously lower than those of Salmonella serotypes, except for S. Diarizonae.

Based on the recent annual report of Salmonella outbreaks, there were 45,735 cases recognized by CDC in 2013 (7). Among them, S. Typhimurium and S. Enteritidis accounted for 12.8% and 15.1%, respectively. Our purified antibodies showed the best reactivity with these two major serotypes of Salmonella. Furthermore, our pAbs also showed binding efficiency with some of the recent top 20 major serotypes of Salmonella including S. Newport, S. Javiana, S. Heidelberg, S. Montevideo, and S. Typhi. Overall, the purified anti-Salmonella pAbs showed an excellent specificity against pathogenic Salmonella.

For the efficient performance of purified anti-Salmonella pAbs, the antibodies need to bind with Salmonella after their immobilization on the gold sensor surface (15). If the purified antibodies are immobilized on gold sensor properly, their binding with Salmonella will be increased so that numerous Salmonella can bind on the antibody-immobilized sensor surface. As seen in Fig. 2B and 2C, there were numerous bindings of Salmonella observed, corresponding with the increase in Salmonella concentration. Meanwhile, there are a few non-specific binding of Salmonella on the sensor surface devoid of antibody, but they are very weak (p<0.05). Capture capacity of the pAbs against Salmonella could be enhanced by using various immobilization methods, such as a covalent attachment with cross-linker and the application of protein A, G, and other derivatives on the sensor platforms (13,14,20,21). However, such studies will be reserved for the future, and our focus for this report will remain on developing new bio-recognition element for Salmonella detection with excellent reactivity and specificity.

Fig. 2. SEM images of Salmonella captured on the surface of (A) antibody-devoid gold biosensor after exposure to Salmonella mixture with the concentration of 10⁸ CFU/mL, and of antibody-immobilized gold biosensor after exposure to Salmonella mixture with the concentrations of (B) 106 CFU/mL and (C) 10⁸ CFU/mL.

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In summary, anti-Salmonella pAbs were successfully isolated and purified from a rabbit and demonstrated competitive reactivity over commercial anti-Salmonella pAbs. In addition, it showed excellent specificity against pathogenic Salmonella serotypes and sufficient binding capability with Salmonella on the gold sensor. Therefore, the purified anti-Salmonella pAbs could be a novel bio-recognition element for application in a gold biosensor combined with LMIS for Salmonella monitoring.