Purification of serum IgM in Acanthopagrus latus and preparation of rabbit sera anti-IgM

The serum IgM of Acanthopagrus latus was purified by using Sepharose-6B gel filtration chromatography,Phenyl Sepharose FF hydrophobic interaction chromatography and rProtein A Sepharose affinity chromatography. The purified IgM obtained through the three methods was then compared. By the methods of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography the IgM was obtained at the purity of only 56%. While through combination of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography,we obtained a bit higher purity at about 69%. The purity reached approximately 89% when using rProtein A Sepharose affinity chromatography. However,the rProtein A Sepharose affinity chromatography suffered relatively high cost. Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography could be good choices for the purification of the IgM in terms of cost and performance. It was revealed that molecular weights of the heavy- and light- chains of IgM were 73.6 and 26.5kD,respectively. The IgM purified by rProtein A Sepharose affinity,chromatography was used to prepare rabbit anti-IgM sera. The antibody activity of anti-sera was tested by Western Blot and double agar diffusion.Indirect ELISA revealed that the anti-serum titer reached 1∶25 600. It is considered that the purified IgM and developed anti-sera provide a foundation for further studies in relation to fish immune response.