Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY).

Isolation of Nile crocodile (Crocodylus niloticus) serum immunoglobulin M and Y (IgM and IgY). J Immunol Methods. 2019 Dec 11;:112724 Authors: Abdool FS, Coetzer THT, Goldring JPD Abstract Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affin...
Source: Journal of Immunological Methods - Category: Allergy & Immunology Authors: Tags: J Immunol Methods Source Type: research