A. T. J. Bianchi

The use of a double antibody sandwich ELISA and monoclonal antibodies for the assessment of porcine IgM, IgG and IgA concentrations

Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) have been developed for the assessment of IgM, IgG and IgA concentrations in porcine serum. Isotype-specific monoclonal antibodies (mAb) were used for coating and detection. The DAS-ELISAs were examined for their ability to detect each isotype and for the assay variations. A computer programme was used to verify the parallelism of the slope of each serum sample with the slope of standard/reference serum, a prerequisite for reliable estimation of Ig concentrations. The DAS-ELISAs are easy to perform and highly specific, have adequate detection levels (ranging from 7 to 50 ng ml-1) and are very reproducible, as illustrated by the inter- and intra-assay variation coefficients (ranging between 4.9 and 7%). To illustrate the applicability of the ELISAs we assessed Ig concentrations in pig sera sampled from birth to young adulthood.

The influence of a water-in-oil emulsion on humoral immunity

A strategy for research of immunostimulants is best served choosing a way by which the immune response can be studied without being complicated by a combination of effects originating both in adjuvant and antigen. This means that adjuvant and antigen preferably are applied with an interval and by different routes. In our model an adjuvant (a W/O emulsion) was applied intraperitoneally, whereas the antigen was injected intravenously. The stimulatory effect on the splenic plaque forming B-cell response depended on the dose of antigen, on the interval between adjuvant and antigen application, on the mouse strain used, and on the quality of the antigen with respect to the intrinsic adjuvanticity of the antigens.

Research of the application of a water-in-oil emulsion for the prevention of post-weaning diarrhoea and oedema disease in piglets

A stable water-in-oil emulsion was injected intraperitoneally (i.p.) in piglets about 5 days before weaning to prevent post-weaning diarrhoea (PWD) and oedema disease (OD). So far more than 200,000 piglets have been treated with this adjuvant on a number of farms. On these farms the mortality rate due to PWD and OD decreased, whereas the need for antibiotic treatment declined. Experiments involving alternate application of adjuvant and physiological saline, or adjuvant treatment and no treatment at all, showed a statistically significant positive effect of adjuvant application. The effect of i.p. adjuvant application on specific and non-specific defence mechanisms were examined in well defined rat- and mouse-models, to throw light upon the mechanisms behind the observed adjuvant effect in piglets.

Modification of immune induction by adjuvant.

The role of adjuvanticity in the modification of the immune induction was studied by separate application of antigen and adjuvant in mice. An antigen specific plaque forming cell (PFC) test and a delayed type hypersensitivity (DTH) assay were used for these studies. Antigen (SRBC) and adjuvant were applied separately in time and place. A stable well defined water-in-oil emulsion without antigen was used as adjuvant. Our studies have shown that intraperitoneal (i.p.) injection of this adjuvant stimulates the intravenous (i.v.) induced PFC response, while the DTH response is suppressed. This effect can be demonstrated even when adjuvant has been injected i.p. 5 days prior to i.v. antigen injection. The stimulation of the PFC response depends on the dosage of i.v. injected antigen.

Influence of the time of weaning on the spontaneous (background) immunoglobulin production in the murine small intestine

Shortly after birth, the mucosal B cell compartment is only partially developed. Maternal immunity, transferred via placenta or milk from mother to offspring, protects young animals from infectious diseases. The ontogeny of the mucosal B-cell compartment has been studied only partially. The small intestine (SI) of many newborn mammals contains Peyer’s patches, which develop in the absence of antigenic stimulation. The newborn intestinal tract contains few plasma cells or intraepithelial lymphocytes and the occurrence of those cells does depend on antigenic stimulation. The effect of maternal antibodies on the development of the B-cell compartment in the SI has not been reported, although circulating maternal antibodies have been reported to suppress not only systemic immune responses, but antigen-specific responses in the intestine before weaning as well. In this paper we describe the influence of the time of weaning on the development of the murine B-cell compartment in the SI.

Enhancement of enteric mucosal immune responses by cholera toxin or cholera toxin B-subunit

Cholera toxin (CT) and cholera toxin B subunit (CTB) are used as carrier-proteins for the induction of mucosal immune responses (1,2). Both CT and CTB adhere to the intestinal epithelium (GM1 molecules) and cause immunestimulation by enhanced antigen-uptake by the epithelium. Next to the enhanced antigen-uptake CT has adjuvant effects on the immune response. We determined the stimulatory effect of coupling CT and CTB to the antigen ovalbumin on the intestinal immune response against ovalbumin after intraduodenal booster immunization. Intestinal responses were measured by quantitating the OA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine.

Induction of a combined mucosal and systemic anti-ovalbumin response.

It is generally accepted that a relatively independent mucosal immune system exists next to a systemic immune system (1). Antigen presentation by the mucosal route can evoke mucosal immune responses(2), while the systemic immune response is suppressed. Contrary to the latter oral tolerance phenomenon (2) parenteral immunization for induction of efficient systemic immunity, sometimes has a suppressive effect on the mucosal immune response (3,4).

Development of the intestinal B and T cell compartment in the pig

The number of intestinal immunoglobulin secreting cells (Ig-SC) of mice rises steeply during the first 3 weeks after weaning (1). In adult mice, the majority of all Ig-SC are located along the small intestine (SI) (2). About 99 percent of these intestinal Ig-SC secrete IgA, but also in the period before weaning IgA predominates among the Ig-SC (1).

The Involvement of T-Cells in the Stimulatory Effect of a Water-in-Oil Emulsion on the B-Cell Response

Originally the immunostimulatory effect of water-in-oil (W/O)emulsions, used as adjuvant, has been explained by depot formation resulting in a chronic stimulation of the immune system by small amounts of released antigen (1,2). Later studies by ourselves (3,4) clearly demonstrated that other mechanisms are involved also.

Isolation and enumeration of isotype specific plaque forming cells from the murine intestine to study the development of the intestinal B cell background response.

Quantification of (specific) IgA in faeces using the ELISA technique (1,2) and localization of antibody containing cells (ACC) by immunohistology (3,4) are important methods to investigate the in vivo expression of the enteric humoral immune response. These methods have improved our knowledge of the expression of the mucosal immune system, but they have several restrictions. Immunohistological studies give a direct insight in the in vivo expression of the mucosal immune system itself, but quantitative data are very difficult to obtain and the number of ACC does not reflect the number of B cells secreting antibodies (5) contributing to the intestinal IgA response. Moreover, IgA measured in faeces is not merely a reflection of locally (intestinally) produced and secreted IgA, since systemically produced IgA can also contribute to the IgA levels in faeces (6). Knowledge about the quantitative expression of the enteric humoral immune response can be obtained by isolation of lymphocytes from the intestine which can be tested subsequently for their capacities to secrete (antigen-specific) IgA.