No abstract

Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 g of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

Serum immunoglobulin transudation into the murine gut after intragastric immunization with the model antigen ovalbumin and cholera toxin adjuvant was investigated with regard to the mucosal sampling technique applied. The levels of serum-derived immunoglobulin A (IgA) turned out to be lowest in feces, intermediate in gut lavage fluid specimens, and highest in filter wick-collected samples. However, these levels did not exceed 2% of total and specific IgA in any mucosal sample type, except after the administration of very high antigen doses (>1 mg of antigen per g of body weight), when transudation rates of up to 31% could be measured in filter wick-collected samples from individual animals. Luminal IgG was plasma transudate and/or bile borne and appeared to be reabsorbed at the mucosa to some extent.In order to exert its full protective power, mucosal immunoglobulin A (IgA) must be dimeric (13) and carry the secretory component (12), which is provided by the epithelia from which it is released. When specific IgA levels at mucosal surfaces are measured, it is therefore important to discriminate between transudated monomeric and locally produced, actively secreted dimeric IgA, as only the latter is able to carry the secretory component with which the immune complex is immobilized in mucus (12). Since the way in which mucosal secretions are collected may have a considerable influence on the extent of contamination with monomeric serum IgA, we investigated the contributions of serum-borne IgA and IgG in feces, intestinal lavage fluid, and filter wick-collected local intestinal secretions, the three most commonly used samples obtained after experimental mucosal immunization (5). MATERIALS AND METHODSImmunization and sample production. For immunization, groups of six female BALB/c mice (age, 8 weeks; Charles River Wiga, Sulzfeld, Germany) were gavaged four times on days 0, 21, 35, and 49 with 0.2, 2, or 20 mg ovalbumin (Calbiochem, Bad Soden, Germany) plus 10 g of cholera toxin (List Biological Laboratories, Campbell, Calif.) in 300 l of 3% (wt/vol) sodium bicarbonate or with buffer alone (controls). Ten to 11 days after the last immunization, feces, blood, intestinal lavage fluid, and local intestinal secretions were collected and processed as described previously (2, 6).Determination of transudation marker and immunoglobulin concentrations. Murine serum albumin (MSA) was chosen as the transudation marker and was assayed by capture enzyme-linked immunosorbent assay. Each well of highbinding enzyme immunoassay plates (Corning Costar, Bodenheim, Germany) were coated with 75 l of 40 ng of goat anti-mouse albumin (Bethyl, Montgomery, Tenn.) per ml in 10 mM sodium phosphate (pH 7.0)-10 mM NaCl overnight at 4°C, washed three times with Dulbecco's phosphate-buffered saline (D-PBS) containing 0.05% (vol/vol) Tween 20, and blocked with D-PBS containing 5% (wt/vol) nonfat dry milk (PBS-Blotto) for 5 h at room temperature. After four washes, 75 l of serially diluted samples and immunoglobulin-free MSA standard (ICN, Eschwege, G...