Production and characterisation of monoclonal antibodies recognising ovine IgE.

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.

A technique for the isolation and purification of viable mucosal mast cells/globule leukocytes from the small intestine of parasitised sheep.

Romney sheep, 1-2 years old, immunized by at least three anthelmintic abbreviated infections of 80-100,000 Trichostrongylus colubriformis larvae usually produced high numbers of intestinal mucosal mast cells/globule leukocytes (MMC/GLs). In isolating these cells, the importance of maintaining the intestine at 37 degrees C, removal of mucus with dithiothreitol, enzymatic dispersion and careful in vitro handling procedures for maximising cell viability are emphasised. The MMC/GLs were separated from most contaminant cells by using a Percoll discontinuous gradient. MMC/GLs collected at the 60/100% Percoll interface were passed through a complement coated nylon wood column to remove the contaminating eosinophils. Viable MMC/GLs were able to grow in vitro in the presence of Concanavalin A and survive in culture for up to 30 days. The MMC/GLs were readily identified by ultraviolet light microscopy after staining with auramine O.

Cervine T-lymphocyte growth factors and their measurement in tuberculosis.

Research into the composition and function of the immune response in domesticated ruminants has tended to focus on the ovine and bovine systems. With the recent domestication of deer, health problems have developed which require a fundamental knowledge of the immune function in exotic ruminants. In this report it is shown that although recombinant human and mouse interleukin-2 (IL-2) were capable of stimulating cervine T-cell proliferation, optimal proliferation was only achieved using recombinant bovine IL-2. While some phylogenetic restriction of IL-2 cross-reactivity was found, in some cases this could be overcome by using high concentrations of recombinant IL-2. Using cervine T-cell blasts it was possible to assay in vitro T-cell growth factor (TCGF) production by lymphocytes isolated from deer naturally exposed to tuberculosis Mycobacterium bovis). Differences were found in the amount of TCGF present in the supernatants of antigen-activated cells isolated from severely diseased animals, those with limited disease and non-diseased animals.