Study On The Diagnosis Of Toxoplasmosis With Nanogold Biosensor Based On Recombinant SAG1 Of Toxoplasma Gondii

Objectives To clone and express the truncated fragment of main surface antigen 1?tSAG1?of Toxoplasma gondii and establish nanogold biosensors for the detection of antibodies to Toxoplasma gondii.Methods The nucleic acid sequence from 515 to 1267 in the SAG1coding region was amplified by PCR from Toxoplasma gondii genomic DNA and was cloned into cloning vector pMD18-T.After being identified by clony PCR and DNA sequencing,the tSAG1 gene was subcloned into the expression vector pET-28a?+?.The expression of tSAG1 was induced with IPTG and purified by metal chelating chromatography.The immunological activity of tSAG1 was analyzed by Western blot.Gold nanorods were prepared by seed growth method and coupled with tSAG1to construct a gold nanorods biosensor for the detection of antibodies to Toxoplasma gondii in human serums,Non-parametric test was used to determine the difference in the measured values.Positive values was defined as more than or equal to the mean of the redshift values from normal healthy person plus 2 times standard deviations.Sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency were calculated.Gold nanoclusters were prepared by glutathione method and activated by 1-ethyl-3-dimethylaminop-ropyl carbodiimine hydrochloride and n-hydroxysuccinimide,and then connected with sheep anti-human IgG secondary antibody.The tSAG1was fixed on the pores of the microporous plate to bind with antibodies of Toxoplasma gondii in human serums,after being combined with the anti-human IgG coupled with the nanoclusters,the complexes were reacted with the trisodium citrate solution and the hydrogen peroxide which was diluted by morpholine ethylsulfonic acid-hydrate solution,then the auric acid solution was added to the mixed solution and reacted for 1h.The absorbance at 550 nm was determined by spectrophotometry.Non-parametric test was used to determine the difference in measured values.Positive values is defined as less than or equal to the mean of OD₅₅₀50 values from normal healthy person minus 2 times standard deviations.Sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency were calculated.Results The recombinant expression plasmid of tSAG1 was constructed,and the immunoreactive tSAG1 with molecular weight about 30 kDa was obtainedfromtherecombinantbacteriawheninducedwith isopropyl?-D-thiogalactoside?IPTG?and purified by metal chelating chromatography.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of nanogold biosensor for detection of antibodies to Toxoplasma gondii were 80.9%,90.0%,90.2%,80.6%and 85.2%,respectively.Based on the simulated enzymatic properties of gold nanoclusters,we constructed a visualized surface plasma nanogenic sensor.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of this method were 80.0%,90.0%,88.9%,81.8%and 85.0%,respectively.Conclusions Nanogold biosensor based on recombinant tSAG1 can be used to detect antibodies to Toxoplasma gondii.