Development of a Novel Procedure for Preparation of Equine Immunoglobulin F(ab′)_2 against Tetanus Toxin

Objective To develop a novel procedure for preparation of equine immunoglobulin F(ab′)2 against tetanus toxin.Methods Tetanus toxoid(TT) and recombinant tetanus toxin C fragment(rTT-C) were prepared and purified,then mixed at a ratio of 2 ∶ 1.Horse were immunized with the mixture TT / rTT-C and,when the serum antibody titer reached 3 500 IU / ml,the sera were collected and inactivated,from which the foreign protein was removed,then digested with pepsin.The prepared F(ab′)2 was purified by DEAE column chromatography,and analyzed for neutralizing efficacy,safety and stability.Results The concentrations of purified TT and rTT-C were 70 000 Lf / L and 4 ～ 5 mg / L,while the purities were more than 95% and more than 96%,respectively.The recovery rate,purity and neutralizing antibody titer of purified TAT-F(ab′)2 were 1.4%,more than 91% and more than 15 000 IU / ml respectively,while the safety and stability met the requirements in Chinese Pharmacopeia(VolumeⅢ,2005 edition).The validity period was determined as 18 months temperately.Conclusion A novel procedure for preparation of equine immunoglobulin F(ab′)2 against tetanus toxin was developed,and the quality of product was equal to or even higher than that of imported product,which provided a technical support for upgrading of equine antiserum of the same kind.