Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae

Immunization of mice and rabbits by recombinant Shiga toxin B (rStxB)

In our previous study10, the stxB gene (289 bp from 967 to 1255, accession number EF685161) coding for B chain of Stx was amplified. After cloning, StxB was expressed and purified in the native conditions11. BALB/c mice and New Zealand female white rabbits were maintained in animal care facility of Defence Research & Development Establishment (DRDE), Gwalior, India, at temperature 25°C±2°C and humidity 30-70 per cent with 12:12-h light:dark period. Animals were provided food obtained from Amrut Ltd., Chandigarh. The study was approved by the Institutional Animal Ethics Committee of DRDE. Route of immunization for mice was intraperitoneal and that for rabbits was intramuscular. Immunization was carried out on 0, 7, 14 and 21 days in BALB/c mice. One set (12 mice) was immunized with cross-linked recombinant Shiga toxin B (rStxB) and another set of four mice was immunized with rStxB without glutaraldehyde. The antigen was emulsified in Freund's complete adjuvant for the first dose, and on days 7, 14 and 21, Freund's incomplete adjuvant- based emulsion was used. The antigen was injected (10, 10, 20 and 40 μg/mice), respectively, as on the days mentioned above. The rabbits (n=3) were also immunized with the cross-linked rStxB. The rabbits were primed with the dose of 50 μg followed by the booster doses of 100 μg.

Collection of antiserum: Pre- immunized mice and rabbits were used as negative control. Initially, the test bleed was collected on the 10^th day and final bleed on the 25^th day of immunization. Mice were bled through retro- orbital route by inserting a microcapillary into the orbital plexus at an angle of 45°. The blood was collected through the capillary and pooled from each of the mice. From rabbits the blood was collected from ear vein for the test and by cardiac puncture for the final bleed. The collected blood was incubated at 37°C for 30 min and at 4°C for one hour. After dislodging and centrifugation at 6000×g for 10 min, antiserum was collected and stored at −80°C till further use.

Specificity of mouse and rabbit antiserum: Specificity of rabbit and mouse antiserum was checked against different toxins for specific detection of rStxB. Various toxins, i.e. rStxB, Staphylococcus enterotoxin B (SEB), Escherichia coli and Vibrio cholerae with the concentration of 500 ng/well in coating buffer (50 mM sodium carbonate-bicarbonate buffer, pH 9.6) (Sigma-Aldrich, USA) were coated on 96-well MaxiSorp microtitre plate (Nunc-Nalgene, USA). A dilution of 1:1000 of rabbit and mouse antiserum was prepared in one per cent bovine serum albumin (BSA); 100 μl of each was added in respective wells and incubated for one hour at 37°C, and absorbance was read at 450 nm using the ELISA plate reader (BioTek Instruments, USA).

Rabbit and mouse antiserum titre: The final bleed antiserum samples of mice and rabbits were subjected to the Western blot to check the titre. Two-fold serial dilutions of antiserum were prepared starting with 1:1000 up to 1:512,000 in phosphate-buffered saline (PBS). Standard Western blot protocol was performed as described elsewhere11. ELISA was performed in which antigen solution (500 ng) was prepared in coating buffer. The dilutions of mouse and rabbit antiserum ranging from 1:1000 to 1:2,048,000 were prepared in one per cent BSA in PBS. Pre-immunized serum was used as negative control. Subsequently, 100 μl of HRP (horse raddish peroxidase) conjugated anti-mouse and anti- rabbit immunoglobulin (1:2000) (Sigma-Aldrich, USA) were added to the respective wells and incubated for one hour at 37°C. Finally, the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (100 μl/well) was added, and the reaction was stopped with 50 μl (2N H₂ SO₄). Absorbance was read at 450 nm using ELISA plate reader. The cut-off value was calculated by the formula described by Snyder et al9, cut-off value = (average OD+standard deviation of blank ×3).

Sandwich ELISA for detection of Shiga toxin (Stx): Limit of detection (LOD) of rStxB was checked by sandwich ELISA using rabbit antiserum as capture antibody and mouse antiserum as revealing. Rabbit antiserum (1:1600) was coated (100 μl/well) on 96-well MaxiSorp microtitre plate in coating buffer (pH 9.6). The plate was incubated overnight at 4°C, after decanting blocking was done with three per cent BSA (2 h at 37°C). Washing was done thrice with PBS and tween-20 (PBST) and once with PBS. Two- fold serial dilutions from 500 ng/well of rStxB were prepared in one per cent BSA in PBS. One hundred microlitres/well was added and was incubated for one hour at 37°C. For the detection of Stx in S. dysenteriae type 1 (SD-1) and STEC (Shiga-like toxin (SLT)-producing E. coli), StxB was replaced with culture lysates. The wells containing one per cent BSA and pre-immune mouse serum served as the negative controls. BoNT/B, BoNT/E and ETX were used for specificity determination. Mouse anti-StxB antiserum (1:1600) was added and plate was incubated as above. After washing, 100 μl anti- mouse HRP immunoglobulin (1:5000) was added and incubated for one hour at 37°C. The substrate, TMB (100 μl/well), was added, the reaction was stopped and absorbance was read at described above. Food samples such as milk, paneer and apple juice were spiked with purified rStxB (1 μg/ml) and detected by sandwich ELISA as described above.

Dot-ELISA for detection of Shiga toxin B (StxB): Dot-ELISA was performed to determine LOD of StxB using nitrocellulose membrane (Micro Devices, India). Five microlitres of rabbit antiserum (1:250 in coating buffer) was placed on membrane of all eight strips of comb and left for drying at room temperature for 10 min. The membrane was incubated for one hour at 37°C in moist chamber. Skimmed milk powder (200 μl 5% in PBS) was used to block active sites. The membrane was washed thrice with PBST and once with PBS (5 min each). Two-fold serial dilutions of rStxB starting with 125 to 1.95 ng/well were prepared and 200 μl was added in the plate and the membrane was dipped. The last well containing one per cent BSA served as negative control. The membrane was washed as above and kept in plate containing mouse anti-StxB serum (1:500) for one hour at 37°C. After washing, membrane was incubated in anti-mouse HRP conjugate (1:2000). Substrate 3,3’-diaminobenzidine (DAB) + H₂O₂ was added after the washing and kept until the appearance of brown dots.

Flow-through assay for detection of Shiga toxin (Stx): Nitrocellulose membrane and absorbent pads were placed and fitted into small-sized cassettes (Micro Devices, India) used for performing flow-through assay in sandwich format. Mouse anti-StxB serum was mixed (1:1 v/v) in coating buffer and 2 μl was added on membrane. Pre-immunized rabbit serum (1:100) was added on the top of the membrane which served as positive control and incubated at 37°C for one hour. Non-specific sites were blocked by incubating at 37°C for one hour in three per cent BSA. The membrane was washed by soaking it in PBST followed by PBS. Two-fold serially diluted rStxB was added in different cassettes with concentrations of 15 to 0.46 μg/cassette and kept for two minutes. BSA (1%) was used as negative control. Subsequently, the membrane was washed by soaking in PBST and PBS. Purified rabbit immunoglobulin G (IgG) (500 ng/cassette) was added and kept for one minute, and after washing, rabbit HRP conjugate (1:5000) was added, kept for one minute and detected by adding DAB and H₂O₂.

Flow-through assay for detection of Shigella dysenteriae type 1: Cassettes were prepared as described above and mouse anti-StxB serum was mixed (500 ng) in coating buffer, and 2 μl was added on the membrane and incubated at 37°C for one hour. Pre-immunized rabbit serum (1:100) served as positive control. Non-specific sites were blocked by incubating with three per cent BSA. After washing SD-1 lysates of National Institute of Cholera and Enteric Diseases (NICED), Kolkata, and Christian Medical College (CMC), Vellore, cultures (1:10 in 1% BSA) were added in the respective cassettes and kept for two minutes. E. coli SG13009 lysate was used as negative control. After washing, purified rabbit IgG (500 ng/cassette) was added and kept for one minute, after washing, rabbit HRP conjugate (1:5000) was added, kept for one minute and substrate used for detection.