Nunc elisa plates

For serum anti-commensal IgG analysis, colonic fecal contents were processed as described above. Bacteria were homogenized using BugBuster 10X protein extraction reagent (Novagen), centrifuged at 20,000 g for 10 min, and the supernatant recovered for a crude commensal bacterial antigen preparation. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 96-well Nunc ELISA plates (Thermo Fisher Scientific) were coated with 5 μg/mL commensal antigen preparation overnight at 4°C, washed extensively, and murine sera incubated in doubling dilutions for 4 h at room temperature. For small volumes of sera, samples were incubated at a 1:150 dilution. In the case of serum anti-flagellin IgG detection, 96-well Nunc plates were coated overnight with 200 ng/mL flagellin purified from Salmonella typhimurium (InvivoGen). Commensal antigen-specific IgG was detected using a goat anti-mouse IgG-horseradish peroxidase (HRP) conjugated antibody (Thermo Fisher Scientific, 1:10000 dilution), and TMB peroxidase substrate (BD biosciences). After 15-20 min, the reaction was quenched with 1 M Na₂SO₄ and the optical densities measured at 450 nm using a CLARIOstar spectrophotometer (BMG Labtech).

Gold chloride (HAuCl₄), Aflatoxin B1, OTA, trysulfonium hexafluorophosphate salt, propylene glycol monomethyl ether acetate, and negative photoresist were purchased from Merck, Delhi, India. Sodium citrate and sodium chloride were purchased from Sisco Research Laboratories Pvt. Ltd, Delhi, India. The 36-mer aptamer sequence of OTA was 5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′, and 21-mer aptamer sequence of aflatoxin B1 5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCG CTAGGCCCACA-(3′) was purchased from GCC Biotech (India) Pvt. Ltd, Kolkata, India. Nunc Elisa plates were purchased from ThermoFisher Scientific, Delhi, India. Corn and groundnut samples were purchased from a local supermarket in Hyderabad, India. For HPLC analysis, acetonitrile, water, and acetic acid were purchased at a high purity grade from Merck, Delhi, India. All reagents used for the experiments were of analytical grade.

Endpoint titres of serum antibodies were determined using an in-house indirect ELISA as previously described [41 (link)]. Briefly, 96-well Nunc ELISA plates (Thermo Scientific, Waltham, MA, USA) coated with 50 μL/well of 1 μg/mL of recombinant S1 protein (SinoBiological, Beijing, China) overnight, were washed and blocked for 1 h with 10% skimmed milk in phosphate-buffered saline (PBS) with 0.05% tween-20 (PBS-T). Then, 50 μL/well from each serum sample was added in duplicates in a 3-fold serial dilution prepared in PBS-T starting from a 1:100 dilution. After 1 h of incubation, alkaline phosphatase-conjugated goat anti-human IgG secondary antibodies (Thermo Scientific) were added at 1:3000. Finally, p-nitrophenyl phosphate substrate (pNPP) (Sigma Aldrich, St. Louis, MO, USA) was used for colour development, and the OD was measured at 405 nm. The procedure steps were all performed at room temperature. The endpoint titre for each tested serum was determined as the reciprocal value of the serum dilution that gave the OD signal converging with the cut-off determined as the average OD of a seronegative mouse or camel serum plus 3 SD, as described by previous studies [42 (link)]. For MVA whole-virus ELISA, the total protein of purified MVA-wt was quantified in Bradford Assay, 10 µg/mL was used to coat ELISA plates.