Quantification of Immunoglobulin G as an Alternative Method to the Miles Evans blue Assay to Measure Vascular Hyperpermeability induced by Infection and Sterile Inflammation

Abstract

Vascular hyperpermeability is conventionally evaluated by the Miles Evans blue assay. The assay is based on the intravenous injection of Evans blue dye which binds non-covalently to albumin and remains within the blood vasculature. As a consequence of the vascular hyperpermeability associated with inflammation, the dye bound to albumin, extravasates from blood vessels along with macromolecules to accumulate in interstitial tissues at sites of inflammation. In the mouse model, Evans blue dye is injected intravenously through the lateral tail vein, a procedure that has a significant failure rate, even when performed by highly trained personnel and requires extensive practice. We developed a simpler and easier assay that quantifies endogenous plasma immunoglobulin G antibodies instead of Evans blue dye bound to albumin, as a measure of vascular hyperpermeability to macromolecules; the assay does not require intravenous injection and uses tissue homogenates as samples that can be used in conjunction with other assays, thereby reducing the number of experimental animals needed. With increased vascular permeability during inflammation or other diseased states, immunoglobulin G, like albumin, also leaks from the vasculature, extravasates into tissues, and accumulates at inflammatory foci. In this work, we compared the Miles Evans blue assay with the immunoglobulin G assay to evaluate the increase in vascular permeability associated with inflammation due to cutaneous infection with Bacillus anthracis and to topical application of the pro-inflammatory agent, phorbol 12-myristate 13-acetate. We found the immunoglobulin G assay easier to perform and requires fewer animals when additional tissue samples are required.