Goat anti rabbit igg horseradish peroxidase hrp conjugate

AFB₁–BSA, rabbit anti-AFB₁, goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, carbonate–bicarbonate buffer (capsule), multi-walled carbon nanotubes (MWCNTs), medium molecular weight chitosan (CS) and potassium ferricyanide, K₃[Fe(CN)₆], were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Aflatoxin B₁ (AFB₁) standard solution was purchased from Supelco Analytical (Bellefonte, PA, USA). Skimmed milk powder was obtained from Nacalai Tesque (Kyoto, Japan). Other reagents were of analytical grade, and all aqueous solutions were prepared using deionized water. Phosphate buffer saline (PBS) was prepared at 10× stock concentration and diluted to 1×. Tween 20 (0.05%) was added to 1 L of 1× PBS to make PBST as a washing buffer in ELISA. For supporting electrolyte, 0.17 g of potassium ferricyanide, K₃[Fe(CN)₆], was added into 100 mL of 1× PBS to make a 5 mM solution, to study the reversible electrochemical behavior in cyclic voltammetry analysis.

To assess immune reactivity of ClpB_Li, SDS-PAGE electrophoresis was performed according to [16 (link)] using 10 % polyacrylamide gels and Western blotting was performed as described [17 ]. The blots were blocked with 0.1 % Tween 20 in Tris-buffered saline (TBS) for 1 h at room temperature and then incubated overnight at 4 °C with anti-ClpB_Li158–334 serum (1: 2000 dilution) [8 (link)] or polyclonal rabbit and bovine sera (1:100 dilution) against Leptospira strains. After primary antibody incubation, the blots were washed three times with TBS containing 0.05 % Tween 20 and incubated for 1 h at room temperature with the goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Sigma) diluted 1: 3000 or the polyclonal rabbit anti-cow Ig/HRP conjugate (DakoCytomation) diluted 1: 1000. The blots were then washed three times as described above and were developed using 3,3′-diaminobenzidine (Sigma), and H₂O₂ as substrates.

AFB₁-BSA, rabbit anti-AFB₁, goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, carbonate-bicarbonate buffer (capsule), multi-walled carbon nanotubes (MWCNTs), medium molecular weight chitosan (CS) and potassium ferricyanide, K₃[Fe(CN)₆], were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aflatoxin B₁ (AFB₁) standard solution (20 µg/mL in methanol) was purchased from Supelco Analytical (Bellefonte, PA, USA). Skimmed-milk powder was obtained from Nacalai Tesque (Kyoto, Japan). Other reagents were of analytical grade, and all aqueous solutions were prepared using deionized water. Phosphate buffer saline (PBS) was prepared at 10× stock concentration and diluted to 1×. Tween 20 (0.05%) was added to 1 L of 1× PBS to make PBST as a washing buffer in ELISA. For supporting electrolyte, 0.17 g K₃[Fe(CN)₆] was added into 100 mL of 1× PBS to make a 5 mM solution.