Sensitive and specific immunoassays to detect rabbit IL-8 and MCP-1: Cytokines that mediate leukocyte recruitment to the lungs

The alpha- and beta-chemokines such as IL-8 and MCP-1 direct the recruitment of neutrophils and monocytes into the lungs and other tissues. In order to study the roles of IL-8 and MCP-1 in animal models, specific reagents are required that provide accurate measurements of these cytokines in biological fluids. Here we describe the development of sensitive and specific immunoassays for rabbit IL-8 and rabbit MCP-1, and the validation of these assays in rabbit plasma and bronchoalveolar lavage fluid. The sensitivity of each assay is 0.25 ng/ml for IL-8 and 0.1 ng/ml for MCP-1. The rabbit IL-8 assay does not crossreact with rabbit CRO, another alpha-chemokine, and crossreacts only weakly with human IL-8. The rabbit MCP-1 assay does not crossreact with human MCP-1. Anticoagulants interfere with the detection of IL-8 and MCP-1 in plasma, although EDTA has the least inhibitory effect, Heat-sensitive inhibitors in normal rabbit serum interfere with the detection of IL-8 and MCP-1, although autoantibodies to IL-8 and MCP-1 were not detected. Rabbit erythrocytes bind IL-8 and MCP-1, but erythrocyte contamination of bronchoalveolar lavage fluid causes only a small error in the detection of IL-8 and MCP-I, unless the number of erythrocytes approaches the number found in blood. These assays provide sensitive and specific means to detect IL-8 and MCP-1 in rabbit plasma and bronchoalveolar lavage fluid, and demonstrate the importance of using species-specific reagents in animal studies.