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Purification of high-abundant proteins from plasma has been well established,which often involves multiple processing steps including cold ethanol precipitation and sequential column chromatography.For low-concentration proteins,such as certain specific immunoglobulin,the conventional process often results in waste of the valuable plasma,low recovery rate and purification-fold.Thus,development of effective method for purification of low-concentration from plasma is necessary.In this study,an immunoaffinity chromatography method was developed to purify the specific immunoglobulin with high recovery rate.Three kinds of immunoaffinity chromatography media were prepared,and then were used for purification of the tetanus,hepatitis B and rabies immune globulin,respectively.The tetanus toxoid,hepatitis B vaccine and rabies vaccine were used as affinity ligands and coupled on the Sepharose 6FF,Sepharose 4FF and poly (styrene-divinylbenzene) microsphere.The effect of flow rate,sample volume,eluate and flow rate on the immunoaffinity chromatographic process was investigated.It was found that high flow rate would result in the low protein yield due to the decreased antigen-antibody interaction time.Increase of sample volume could affect the protein deformation and protect the activity.Due to the strong affinity between antigen and antibody,conventional eluate such as high concentration salt and alkaline solution could not totally elute the specific immunoglobulin.After optimization,1M acetic acid solution was used as the eluate.In optimized chromatographic conditions,the three kinds of immune globulin could be effectively purified from plasma.The recovery and purification-fold were as follow: 47% and 92 for tetanus immune globulin,45% and 120 for hepatitis B immune globulin,29% and 50 for rabies immune globulin.To increase the protein activity recovery,several amphiphilic polymers were used as protective reagents during chromatographic process.It was found presence of polyol in the mobile phase could increase the protein activity recovery.By addition of polyethylene glycol (PEG),the activity recovery could reach to 67% for tetanus immune globulin,53% for hepatitis B immune globulin and 34% for rabies immune globulin.The protective effect of PEG on protein activity might result from the high hydrodynamic radius of PEG shield the specific immunoglobulin from binding on the non-specific adsorption sites.Then,protein denaturation caused by the non-specific adsorption could be reduced by addition of PEG in equilibrated buffer.Immunoaffinity chromatography was tested as facile and effective method for purification of specific immunoglobulin from plasma.Selection of the matching antigen as affinity ligand,proper eluate solution and optimization of chromatographic process are very important for different plasma proteins.Addition of proper protective reagents might increase the protein activity recovery.