Immunomagnetic Separation Technology For Purification Of Toxoplasma Gondii Tachyzoites

Objective: For the diverse purposes of Toxoplasma and toxoplasmosis approach andantigen preparation, it is very important to obtain highly purified Toxoplasma gondiitachyzoite quickly from Toxoplasma gondii infected host specimens. It is the ultimategoal to remove host cell components as much as possible, without damage of theparasites from mechanical or chemical factor or without any adverse effect on theviability and virulence of parasites. Contamination of the host cells or lower recovery inpurification in the peritoneal exudates, to some extent, is unavoidable with currentlyused methods，to the present approach was aimed at generation of a new method forseparation of Toxoplasma gondii tachyzoites by immunomagnetic technique, and toobtain the highly purified tachizoites without contamination of host cells and loss ofparasite viability for the diverse purposes of Toxoplasma and toxoplasmosis approach.Methods: The mouse were infected with T.gondii Wh3strain (genotype China1)fromthe heart, brain, tongue of the stray cats. When the mouse fall ill, the peritoneal fluidswere obtained, centrifuged three times and washed by sterilized NS.T.gondii solubleantigen was prepared and used to immunize rabbits three times to obtain polyclonal rabbit anti-Toxoplasma antibodies IgG.The density of Antigenwas detected by BCAmethod. The antibodies were determined by IHA indirect hemagglutination withToxoplasma gondii,purified by Protein A Sepharose CL-4Baffinity chromatography andidentified by SDS-PAGE.The mouse peritoneal exudate containing parasites tachyzoiteswas subjected to the antibody-coated immunomagnetic beads named goat anti-rabbitIgG microbeads. The test was carry out following instructions and a little improvement.The purity, cell clearance ratio and recovery were tested. Toxoplasma gondii tachyzoiteswere stained and observedby microscope. The viability was detected by the MTS kit ofcell proliferation and toxicity detection. The490nm absorbance was detected bymicroplate reader. When the mouse were infected with quantitative tachyzoites, thevirulence to mice was tested.Results: Antigen density determined by BCA method was0.7mg/ml. The molecularweight of light and heavy chains from purified IgG antibody was25kDa and50kDa.Immunomagnetic separation technique gave rise to as high as98.2%of the tachyzoitespurity, the removal rate of host cells reached to96%and the recovery rate of tachyzoiteswas73.5%. The ratio of Toxoplasma gondii tachyzoites and mouse cells was53.6:1. Aftertachyzoites and mouse cells Wright staining, we observe peritoneal fluid smear showstachyzoites and a few mouse cells before magnetic separation. The separation ofsuspensions smear shows a large number of tachyzoites, mice perspective cells wassignificantly reduced after magnetic separation. The viability of tachyzoites was95.6%when detected by the MTS kit of cell proliferation and toxicity detection, and noattenuation of viability and virulence to mice was noted. Toxoplasma gondii beforedeath showed clinical signs of acute infection. A lot of Toxoplasma gondii tachyzoitesand false cysts was seen.It indicated immunomagnetic sorting had no effect on thevirulence. Conclusions: A high purity of T.gondii tachyzoites was achieved by immunomagnetictechnique and no negative impacts of the novel method on the parastes viability andvirulence were found, suggesting that the immunomagnetic separation,which is fast andsimple to carried out, might be useful in the immune and molecular approach ofToxoplasma and toxoplasmosis.