Bovine natural antibody IgM inhibits the binding of human norovirus protruding domain to its HBGA receptors

Figures

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  Fig. 1. Identification of bovine IgM as the functional protein that inhibits HuNoV P protein–histo‐blood group antigen (HBGA) interactions. (A and B) Two separate components (component 1 and component 2) of bovine colostrum rich in the target protein were treated with water dialysis, followed by recovery of the supernatant and precipitation fractions. The recovered fractions were then analyzed for blocking effects against GII.4 HuNoV P protein–HBGA interactions by ELISA (A). The two components were analyzed by two‐dimensional‐polyacrylamide gel electrophoresis (B). Arrows indicate the IgM spots that do not occur in the supernatant of component 2 (dotted arrow). The data are presented as the mean ± SD values, Student's t‐test; ***, P < 0.001. All experiments were repeated at least three times. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Fig. 2. Identification of IgM in bovine colostrum samples as the functional protein that inhibits HuNoV P protein–histo‐blood group antigen (HBGA) interactions. Four bovine colostrum samples were treated with water dialysis, followed by recovery of the supernatant and precipitation fractions. The recovered fractions were then analyzed by ELISA for blocking effects against GII.4 HuNoV P protein–HBGA interactions. The data are presented as the mean ± SD values, Student's t‐test; ***, P < 0.001. bCM 1, bCM sample 1; bCM 2, bCM sample 2; bCM 3, bCM sample 3; bCM 4, bCM sample 4. All experiments were repeated at least three times. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Fig. 3. Bovine colostrum (bCM) sample lacking IgM showed significantly reduced blockage abilities against HuNoV P protein–histo‐blood group antigen (HBGA) interactions. (A and B) The IgM of bCM was specifically removed by co‐immunoprecipitation. The IgM was detected by western blot and Coomassie brilliant blue staining (A). Blocking effects of IgM and non‐IgM fractions of bCM against GII.4 HuNoV P protein–HBGA interactions were detected by ELISA (B). The data are presented as the mean ± SD values, Student's t‐test; *, P < 0.05; ***, P < 0.001. All experiments were repeated at least three times. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Fig. 4. Pure IgM inhibiting GII.4 HuNoV P protein–histo‐blood group antigen (HBGA) interactions. (A) Serial diluted bovine IgM was coated to the wells of microtiter plates. The coated IgM was detected by rabbit anti‐IgM antibody. P‐GST fusion protein (5 μg·mL−1) and GST (21 μg·mL−1) were incubated with coated IgM after being blocked with 5% nonfat milk in PBST, and blocking buffer was used as the negative control. Bound P‐GST and GST proteins were detected by rabbit anti‐GST antibody. #, Optical density at 450 nm (OD450) outranged the capacity of the instrument. (B) Blocking effects of commercial IgM against GII.4 HuNoV P protein–HBGA interactions were detected by ELISA. The data are presented as the mean ± SD values, Student's t‐test; ***, P < 0.001. All experiments were repeated at least three times. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Fig. 5. Fetal bovine serum (FBS) could inhibit GII.4 HuNoV P protein–HBGA interactions. (A) FBS samples were dialyzed with water; supernatants and precipitates were collected, and their inhibition rates were measured by ELISA. (B) IgM in FBS precipitates was specifically removed by co‐immunoprecipitation, and the blocking effects of IgM and non‐IgM fractions of FBS P against GII.4 HuNoV P protein–HBGA interactions were detected by ELISA. The data are presented as the mean ± SD values, Student's t‐test; ***, P < 0.001. All experiments were repeated at least three times. [Colour figure can be viewed at wileyonlinelibrary.com]