Intact anti-LPS IgY is found in the blood after intragastric administration in mice

Abstract

Severe burn injury and cirrhosis often cause the translocation of bacterial endotoxins into blood, leading to systemic damage and even death. Our previous studies have shown that anti-lipopolysaccharide egg yolk antibody (anti-LPS IgY) can neutralize bacterial endotoxins in vitro and in vivo effectively, thereby reducing endotoxin damage. Whether anti-LPS IgY can be absorbed into the blood through the intestinal barrier and neutralize endotoxins in circulation remains unclear. In this study, we used in vivo small animal imaging techniques, protein purification, molecular biology, and mass spectrometry to show that intragastrically administered anti-LPS IgY is detected in the blood of mice as an intact molecule and has the capacity to bind to LPS. Immunohistochemical analysis confirmed that anti-LPS IgY is associated with the intestinal mucosa of mice. However, the route of absorption of this large protein molecule was not determined. This study suggests that anti-LPS IgY can be absorbed into the circulation, with the same molecular mass as purified anti-LPS IgY as a macromolecular protein, suggesting a new strategy for the prevention of damage caused by endotoxins.

Keywords: absorption; egg yolk antibody; infection; lipopolysaccharide; mammalian gut.

Figure 1

Purification of FITC‐labeled IgY by G25‐filled gel chromatography column. P1: FITC‐anti‐LPS IgY; P2: free FITC. Cond, Conductivity curve; UV: UV, scanning curve.

Figure 2

Distribution of FITC‐labeled anti‐LPS IgY after intragastric administration to mice. At 30 min after the oral administration of FITC‐IgY, green fluorescence was observed in the abdominal and thoracic cavities of mice. Clear green fluorescence was accumulated in mice from 30 min to 8 h after intragastric administration. After 8 h, the fluorescence gradually began to weaken and disperse and almost disappeared at 24 h later. At 8 h after intragastric administration, the main organs, such as the intestine, liver, lungs and kidneys, all showed fluorescence distribution. However, the fluorescence intensity in different viscera markedly differed. (A–E) 30 min (A), 1 h (B), 2 h (C), 8 h (D), and 24 h (E) after intragastric administration. (F) The main organs 8 h after intragastric administration (heart, liver, spleen, lungs, kidneys and intestine in the order of top to bottom and left to right).

Figure 3

IgY binding to intestinal mucosal tissue, as shown by immunofluorescence. Fluorescent protein molecules were immunohistochemically detected with sheep anti‐chicken secondary antibodies (A) and shown by immunohistochemistry (B–F). Scale bar: 100 μm (A,B,D,E), and 10 μm (C,F).

Figure 4

Antibody titre of anti‐LPS IgY (absorbance value) in blood harvested from mice. After the administration of anti‐LPS IgY, the plasma of mice had the ability to bind to LPS, as tested by ELISA. The absorbance of the blood from the oral administration group (0.27 ± 0.12) was significantly higher than that of the blood from the normal group (0.05 ± 0.00; P = 0.000) by ANOVA. The ability to bind to LPS was greater in the blood of mice intraperitoneally injected with anti‐LPS IgY absorbance value, 5.77 ± 0.39; the absorbance value of blank control was 0.05 ± 0.01). Control, PBS solution; O‐IgY, mice orally administered IgY; PI‐IgY, mice administered IgY by peritoneal injection; Normal, mice without IgY administration (n = 2).

Figure 5

HPLC‐MS/MS of proteins harvested from mouse blood. Protein with a molecular mass of 186.4 kDa could be detected in the blood of mice after the intragastric administration and intraperitoneal injection of anti‐LPS IgY, a size identical to that of the purified standard anti‐LPS IgY. Stomach, mice orally administered IgY; Abdominal, mice administered IgY by peritoneal injection; Contrast, pure anti‐LPS IgY.

Figure 6

SDS/PAGE (left) and WB (right) of blood harvested from mice. After the intragastric administration of anti‐LPS and IgY, particularly after the intraperitoneal injection of anti‐LPS IgY, proteins with a molecular mass of 186.4 kDa could be detected in the blood of mice by sheep anti‐chicken secondary antibodies. Control, mice without the administration of IgY; O‐IgY, mice orally administered IgY; PI‐IgY, mice administered IgY by peritoneal injection; Standard IgY, pure anti‐LPS IgY. All lanes come from the same gel, but dashed lines indicate where lanes have been spliced together.