Application of Colony Immunoblotting for Counting and Isolating Lactobacillus rhamnosus in Intestinal Flora

Abstract:

A specific antibody against pilus protein (FimI) of Lactobacillus rhamnosus (Lr) was prepared, to develop a colony immunoblotting (CIB) counting and isolation method for Lr. BALB/c mice were immunized with recombinant FimI (rFimI) to obtain the antiserum, and the reactivity and specificity of the antiserum were tested by western blotting and whole bacterial dot-blot analysis. The CIB counting method was developed based on the rFimI antiserum, and was used to quantitatively detect Lr in the artificial sample (mouse intestinal flora + Lr) and the fecal samples from mice fed with Lr by gavage. The titer of the obtained antiserum was 1:51,200 and this antiserum showed strong reactivity with both the lysate and whole cells of Lr, but no cross-reactivity with the intestinal flora of mouse. The optimum dilution of rFimI antiserum for CIB was 1:2000, and clear immunoblots were observed on all colony transfer membranes of samples containing Lr. The positive colony number determined using the CIB method agreed with the Lr concentration in the artificial samples, which may reflect the fluctuation tendency of Lr in fecal samples from the mouse fed with Lr by gavage. The established CIB counting method can be used to selectively count and isolate Lr in intestinal samples.