Extraction and purification of anti-proteinase 3 [PR3] antibodies from egg yolk

Recently it has been reported, that immunoglobulin Y [IgY] can be used instead of polyclonal antibodies extracted from mammals [IgG] for the purpose of diagnosis and therapy. These antibodies are found to have better properties in terms of specificity and ease of large-scale production. In addition, IgY binds neither to mammalian complement or Fc-receptors nor does it interfere with rheumatoid factors [RF], which has proven to be advantageous in many immunological tests. Proteinase 3 [PR3], a constituent of azurophil granules of neutrophils, is the target antigen for most anti-neutrophil cytoplasmic antibodies [c-ANCA] in Wegener granulomatosis [WG]. Capture ELISA was found to be the method of choice in case of c-ANCA determination for the diagnosis and management of WG. However, in this method, the reaction of RF with the Fc portion of IgG in capture ELISA leads to false positive in the assay of c-ANCA and is found to be the most important short-comings of available diagnostic immunochemical tests using mammalian antibodies. To avoid such unwanted interactions, laying hens were inoculated with PR3, and IgY was purified from egg yolk by acidic extraction with chloroform. The aqueous phase was treated with sodium sulphate and the precipitate collected, was dissolved in buffer and was purified using a T-gel chromatography method. The prepared IgY-anti-PR3 was used to set up a capture ELISA. Our results showed that the prepared IgY-anti-PR3 had good titer [lu g/ml in a coating system] and specificity. Hence, IgY based immunoassay would be a useful alternative to mammalian IgG antibody used in PR3 immunoassays