Measurement of serum and fecal antibodies

Serum IgG and IgA antibodies and fecal IgA antibodies to PorA were assayed by enzyme-linked immunosorbent assays¹⁵. The wells of a Maxisorp immunoplate (Nunc) were coated with carbonate buffer (pH 9.6) containing 1 µg/ml of rPorA at 4 °C for 24 h. The wells were washed four times with PBST and blocked with PBSTB at 37 °C for 1 h. A 1: 100 dilution of lysed blood sample or a 1: 10 dilution of fecal IgA in PBSTb was added to the wells and allowed to react at 37 °C for 1 h. The wells were washed four times with PBST and reacted with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (that recognize IgG or IgA isotypes) (Kirkegaard & Perry Laboratories) diluted 1: 1000 in PBSTb at 37 °C for 1 h. The wells were washed four times with PBST. The substrate, ABTS (Sigma) was added, and after 30 min of incubation at 37 °C, OD_{405  nm} was measured.

In addition, agglutinating antibodies in feces were also assayed by the method of Ueki et al.¹⁷. Fifty ul of suspension of C. jejuni 111 in PBS (containing approximately 5 × 10⁸ CFU/ml) was added to each of a 96 V-shaped-well microtiter plate (Nunc), after which 50 µl of 2-fold serially diluted fecal IgA in PBS was added (with a starting dilution of 1: 10). After incubation at 37 °C for 2 h, the plate was kept at 4 °C for 20 h and the agglutination was read. The highest serum dilution showing agglutination was recorded as the titer.