Contribution of Serum Immunoglobulin Transudate to the Antibody Immune Status of Murine Intestinal Secretions: Influence of Different Sampling Procedures

Abstract

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...

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