The objective of the present study was to assess the ability of single-domain antibody clone 26 (dAb Cl 26) selected from phage display library to reduce Pasteurella multocida
(vaccine strain p52) LPS -induced effects on buffalo leukocytes in vitro
. The dAb Cl26 was expressed to an optimum level and later purified under denaturing condition using Ni-NTA chromatography. The protein concentration of purified dAb Cl-26 was estimated by BCA method and found to be 280 µg/ml. LPS of vaccine strain (p52) of P. multocida
was extracted and concentration was found to be 38 µg/ml. The purity of extracted LPS was assessed for nucleic acid and protein contamination and was found to be negative. The purified dAb Cl-26 showed reactivity with Pasteurella multocida
(p52) LPS by Indirect ELISA as well as Dot-Immunoblotting.
For in vitro testing of purified dAb Cl-26 on P. multocida (p52) LPS-induced activation of bovine peripheral leukocytes, Buffalo peripheral blood plastic adherent mononuclear cells were exposed to LPS alone and in combination with different concentration of dAb Cl-26 and the expression level of various genes namely TNFα, IL-1β and IL-6 were studied by qRT-PCR. The dAb Cl-26, in a dose dependent manner, reduced the LPS-induced TNFα, IL-1β and IL-6 transcripts in buffalo peripheral leukocytes in vitro.