Establish Of Immunoassay For Ricin & Expression Of Ricin B Chain Protein And Its Antigenicity

This paper reported that 4D8,4D12 and 1H4 are McAb anti-ricin, which purificated by protein-G chromatographic column, and ELISA data showed that their antigenic determinant were located on ricin A china. we labered 4D8 with horse radish peroxidase(HRP) and FITC respectively, then established ELISA,IMS-ELISA,surface plasma resonance(SPR) to detect ricin. test data indicated:1.The optimum condition of sandwich ELISA assays for ricin is: diluted RT polyclonal antibody to 45 ug/ml as capture Ab, diluted HRP-4D8 to 4000 times as detection Ab, chose 5%skimmed milk+2%PEG20000 as blockage, add TMB substrate solution after wash away detection Ab, incubation at 37℃for 20min. read OD₄₅₀. This method don't have cross-reaction with other resemble toxion, minimum detectable concentration is 5ng/ml, The detection range is 0.1-1ug/ml, standard recovery rate to purified water is 107.6%.2.The optimum condition of IMS-ELISA assays of ricin is: coupling beads with polyclonal antibody anti-ricin, add PBST in EP tube to diluted beads after block, add assay sample incubate for 1h at 37℃with slow tilt rotation. Resuspension with micropipettor every 15min, diluted HRP-4D8 to 4000 times, add 200μl as detection Ab, add TMB substrate solution 100μl after wash away detection Ab, incubat at 37℃for 20min. read OD₄₅₀. This method don't have cross-reaction with other resemble toxion, minimum detectable concentration is 50ng/ml, The detection range is 0.2-10ug/ml, standard recovery rate to purified water,pear jam and milk respectively is 95%,93%,68%.3.Kinetic analysis base on SPR result showed the affinity to ricin:4D8 KA=3.16×10¹² ,KD=3.16×10^-12;4D12 KA=1.53×10¹³, KD=6.55×10^-14;2F KA=4.1×10⁸, KD=2.44×10^-9;1C KA=1.86×10¹⁰, KD=5.38×10^-11. Establish the metherd base on SPR assay for ricin and abrin, The detection range for ricin is 0-2000ng/ml, for abrin is 250-2000ng/ml; minimum detectable concentration for ricin is 0.05ng/ml, for abrin is 0.5ng/ml; And the effect of detect single toxion is equal to mixture of ricin and abrin.4. Establish the metherd base on FCM and beads assay for ricin. The detection range for ricin is 10-1000ng/ml, minimum detectable concentration for ricin is 0.1ng/ml.5. The DNA of RTB was cloned from the ricin DNA by PCR and inserted into the carrier pMD18-T, The fusion expression vector RTB-pET28 was contructed and transformed to E.coli BL21 after sequenced wand. The expressed fusion protein after induction with IPTG at 37℃. SDS-PAGE and Western blotting analysis showed that the 31KD fusion protein was expressed correctly in E.coli. and the bioactivity was detected by indirect ELISA. The results provided reliable basis for the specificity of ELISA detect ricin.