Plasticware and Chemicals
Plasticware and chemicals used in the study were procured from reputed manufacturers, 96 well ELISA plates (Nunc, Denmark), conical centrifuge tubes, microcentrifuge tubes and micropipette tips (Tarsons, India). All chemicals used were of analytical or molecular biology grade, skimmed milk powder (HiMedia, India), bovine serum albumin (SRL, India), tween-20 (Amresco, USA), citric acid (SRL, India), di-sodium hydrogen phosphate (SRL, India), 30% H2O2 (Merck, India), H2SO4 (Merck, India). Antibody and conjugates were procured from reputed suppliers, rabbit anti-SEA polyclonal antibody (Sigma-Aldrich-St Louis, MO, USA), mouse anti-SEA monoclonal antibody (Santa Cruz Biotechnology Inc., USA), goat anti-mouse IgG-HRP conjugate and goat anti-rabbit IgG-HRP conjugate (Merck-GeNei, India), o-Phenylenediamine dihydrochloride (Sigma-Aldrich-St Louis, MO, USA).
Microorganisms and their culture
Five reference strains of S. aureus with known enterotoxigenic potential and seven non-Staphylococcus bacterial genera were included in the study as mentioned in Table 1. A total of 86 (MS-1 to MS-86) strains of Staphylococci isolated from food and clinical sources tested by standardized sandwich ELISA to evaluate its applicability in detecting SEA (Table 2) (unpublished data). All the bacterial strains were procured form Food Microbiology Laboratory of Division of Livestock Products Technology, ICAR-IVRI Izatnagar (UP) India and revived using recommended selective broth and selective agar. The cultures were maintained on enriched media in refrigerated conditions for further use.
Table1: Reference strains of Staphylococci and non-Staphylococci used in the study
Strain
|
Number
|
Characteristics
|
S. aureus
|
ATCC 700699
|
SEA positive
|
S. aureus
|
ATCC 29213
|
SEA positive
|
S. aureus
|
ATCC 43300
|
SEA positive
|
S. aureus
|
CFTRI 722
|
SEB positive
|
S. aureus
|
MTCC 96
|
SEA negative
|
B. cereus
|
ATCC 10876
|
Specificity
|
C. perfringens
|
ATCC 13124
|
Specificity
|
V. parahaemolyticus
|
ATCC 17802
|
Specificity
|
E. coli O157
|
ATCC 43888
|
Specificity
|
Salmonella spp.
|
09/epi
|
Specificity
|
Y. enterocolitica
|
ATCC 23715
|
Specificity
|
L. monocytogenes
|
ATCC 19115
|
Specificity
|
Preparation of Buffers
The buffers used for sandwich ELISA include (a) coating buffer, 50 mM carbonate buffer (pH 9.6): 1.59 g Na2CO3, 2.93 g NaHCO3 in 1 L distilled water, (b) blocking buffer, 10 mM PBS (pH 7.4): 2.9 g Na2HPO4·12H2O, 8 g NaCl, 0.2 g KCl, and 0.2 g KH2PO4 in 1 L distilled water containing 3% (w/v) BSA, 5% (w/v) skim milk powder and 0.1% (v/v) tween-20, (c) washing buffer (PBS-T), 10 mM PBS (pH 7.4) containing 0.05% (v/v) tween-20, (d) dilution buffer, blocking buffer was used as diluent, (e) OPD solution, 15 mL sodium citrate buffer (pH 3.6) containing 6 mg OPD and 15 μL 30% (v/v) H2O2, and (f) stop solution: 2 M H2SO4.
Preparation of Antigen
Recombinant SEA was expressed and purified using SEA clone (SEA-Tru15-pProEXHT/1) available at Food Microbiology Laboratory of the Division of Livestock Products Technology, ICAR-IVRI Izatnagar (UP) India (unpublished data). Briefly, SEA clone was sub-cultures in 10 ml Luria-Bertani (LB) broth containing ampicillin (100 µg ml-1). Confirmation of the clone was done by restriction digestion of the isolated plasmid and visualizing specific size insert. After confirmation, the clone was inoculated into 200 ml LB broth containing ampicillin (100 µg ml-1) and incubated at 37 °C under shaking (200 rpm) for 3-4 h till OD reached ~0.5-0.6. The culture was induced by 0.6 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated further 6 h under similar conditions. Thereafter, bacterial cells were pelleted by centrifugation at 10,000 g for 10 min. Recombinant SEA was purified by Ni-NTA affinity chromatography. Eluted protein was dialyzed against 10 mM PBS and confirmed by SDS-PAGE and western blotting. The concentration of purified protein was determined by Nanodrop spectrophotometer (Thermo, USA).
Optimization of antigen concentration
Optimization of antigen concentration for sandwich ELISA was carried out by indirect ELISA. 96 wells ELISA plates were coated with 100 μl of five-fold serially diluted recombinant SEA (1:5-1:24414625) and 1 X PBS (negative control) and incubated at 4 °C for overnight. Three washes were performed with wash buffer (PBS-T) to remove unbound antigen and each well was blocked with 300 μl of blocking buffer and incubated at 37 °C for 120 min. The washing step was repeated thrice and 100 μl of two-fold serially diluted (1:500-1:64000) rabbit anti-SEA polyclonal antibody in prepared in diluent was added to each well of plates and incubated at 37 °C for 60 min. The washing step was repeated thrice and 100 μl goat anti-rabbit IgG-HRP conjugate (1:1000 prepared in diluent) was added to each well of plates and incubated at 37 °C for 60 min. The plates were washed four times with wash buffer and 100 μl of OPD solution was added to each well and incubated at 37 °C for 15 min. The reaction was terminated by the addition of 100 μl of stop solution (2 M H2SO4) and absorbance was measured at 492 nm using microplate reader (Multiskan GO, Thermo Scientific, USA).
Optimization of Sandwich ELISA
Optimization of sandwich ELISA was attempted in two formats. In first, rabbit anti-SEA polyclonal was used as capture antibody and mouse anti-SEA monoclonal was used as detector antibody, while in second, mouse anti-SEA monoclonal was used as capture antibody and rabbit anti-SEA polyclonal was used as detector antibody. ELISA plates were coated with 100 μl of 2-fold serially diluted (1:200-1:409600 in coating buffer) mouse anti-SEA monoclonal (1 μg ml-1) or rabbit anti-SEA polyclonal as a capture antibody and incubated at 4 °C for overnight. Washing with wash buffer (PBS-T) was performed thrice to remove unbound antibodies and each well was blocked with 300 μl of blocking buffer and incubated at 37 °C for 120 min. The washing step was repeated thrice and 100 μl of 1:125 diluted recombinant SEA protein in diluent (as standardized by indirect ELISA) and 1X PBS was added (negative control) to each well of plates and incubated at 37 °C for 120 min. After three times washing with wash buffer (PBS-T), 100 μl of two-fold serially diluted (1:500-1:64000) rabbit anti-SEA polyclonal or mouse anti-SEA monoclonal antibody prepared in diluent was added to each well of the respective plates and incubated at 37 °C for 60 min. Washing was repeated three times with wash buffer (PBS-T) and 100 μl (1:1000 prepared in diluent) goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP conjugate was added to each well of the respective plates and incubated at 37 °C for 60 min. Plates were washed four times with wash buffer (PBS-T) and 100 μl of OPD solution was added to each well for 15 min. The reaction was terminated by the addition of 100 μl of stop solution (2 M H2SO4) and the absorbance was measured at 492 nm as mentioned above.
Pair-wise interaction analysis of sandwich ELISA
To select the best pair of capture and detector antibodies yielding maximum sensitivity with recombinant SEA antigen (100ng, 10 ng, 1 ng, 0.5 ng, 0.25 ng), pair-wise interaction analysis was carried out. Pairs of different concentrations of capture (mouse anti-SEA monoclonal) and detector antibodies (rabbit anti-SEA polyclonal) were made viz. (1) 1:200 mouse monoclonal anti-SEA as capture with 1:500 rabbit polyclonal anti-SEA as detector, (2) 1:200 mouse monoclonal anti-SEA as capture with 1:1000 rabbit polyclonal anti-SEA as detector, (3) 1:400 mouse monoclonal anti-SEA as capture with 1:500 rabbit polyclonal anti-SEA as detector, (4) 1:400 mouse monoclonal anti-SEA as capture with 1:1000 rabbit polyclonal anti-SEA as detector, (5) 1:800 mouse monoclonal anti-SEA as capture with 1:1000 rabbit polyclonal anti-SEA as detector. Other steps followed for performing sandwich ELISA were similar as described in the previous section. All the reactions were put in triplicates.
Specificity of developed sandwich ELISA
For evaluating the specificity of developed sandwich ELISA, standard or field isolates of important toxigenic bacteria or foodborne pathogens (Table 1) were cultured in 20 mL of brain heart infusion (BHI) broth. Culture supernatants were collected by centrifugation at 10,000 g for 10 min and used as a source of bacterial toxins.
Calibration curve of developed sandwich ELISA
Calibration curve (standard curve) was plotted utilizing selected pair of capture (1:200 mouse anti-SEA monoclonal) and detector (1:500 rabbit anti-SEA polyclonal) antibodies which yielded maximum sensitivity in detecting SEA. Different concentrations (100 ng, 10 ng, 1 ng, 0.5 ng, 0.25 ng) of recombinant SEA antigen were used for plotting calibration curve either directly and after inoculation into food matrix (meat). The calibration curve was used in detecting concentration of SEA in food samples.
Evaluation of the developed sandwich ELISA by spiking studies in meat
Raw meat was procured from local market of Bareilly (UP), India and was confirmed to be free from SEA producing Staphylococci by SEA gene specific PCR (Johnson et al. 1991). Meat sample was inoculated with reference strain of SEA producing S. aureus (ATCC 29213). For this, S. aureus (ATCC 29213) was inoculated in brain heart infusion (BHI) broth and incubated at 37º C for overnight. Bacterial cells were collected by centrifuging at 10,000 g for 10 min, washed with 1 X PBS and re-dissolved. ̴ 2×10-8 cfu were inoculated in to one gram of meat sample followed by incubation at 37 °C for 18-20 h for production of SEA. Inoculated meat was homogenized using homogenizer (Ultra Turrax, IKA, Germany) and centrifuged at 10,000 g for 10 min. Supernatant was collected and used as a source of SEA for its detection by developed sandwich ELISA. Similar steps were followed for performing sandwich ELISA as described in the previous section.
Screening of Staphylococcal strains using developed sandwich ELISA
A total of 86 strains of Staphylococci isolated from food and clinical sources were screened for SEA production potential by developed sandwich ELISA (Table 2).
Comparative evaluation of the developed sandwich ELISA with commercially ELISA kit
The developed sandwich ELISA was evaluated against the commercially available RIDASCREEN staphylococcal enterotoxin detection kit (R. Biopharm, GMBH, Germany). Interpretation of results of RIDASCREEN kit was carried out as per interpretation chart provided by the manufacturer. A sample was considered positive for SEA if OD of the sample was more than the threshold value, which was calculated by using formula –
Threshold value = Mean negative OD + 0.15
Whereas, in case of developed sandwich ELISA, a sample was considered positive for SEA production, if OD value of the sample was more than three times of mean negative OD value.
Statistical Analysis
Student t- test was used for the comparison between developed sandwich ELISA and RIDASCREEN Staphylococcus enterotoxin detection kit (R. Biopharm, GMBH, Germany). The data was analyzed by using SAS (v 9.3) software. Data was expressed as mean ± SEM (standard error mean). Significance was accepted at p < 0.05.