The Preparation, Certification Of Reference Matreials For Ricin And Abrin And Their Rapid Test Methods

Ricin toxin (RT) and abrin toxin (AT) are phytotoxins with similar structure andproperties. Their high toxicity, wide distribution and easy preparation make them theimportant phytotoxic bioterrorism agents listed in the checklist for biological weapons. Therapid test and prevention of both toxins have been a research hotspot in order to meet theurgent needs of defending against biological war and bioterrorism. Therefore, the research onthe preparation, certification of both phytotoxic references and their rapid test methods wascarried out and detailed in this thesis.In the first part, we contruct a simple purification strategy combining the efficientaffinity matrixes with the gel filtration for obtaining the native RT and AT, and analyze theircharacters and definite value.Firstly, raw toxins were extracted from the seeds of castor and jequirity produced inYunnan, Xinjiang and other areas. The crude toxins containing the toxins and agglutinins werepurified by the galactose residues on the affinity matrixes and the difference in molecularweight between the toxins and agglutinins was used to separate them in gel filtration column.By this way, almost all the unrelated proteins were removed and the purity of toxins indifferent producing areas can reach up to more than95%by12%SDS-PAGE.The molecularweight of RT and AT is approximately64kDa and65kDa, respectively.Secondly, the characters of RT and AT were identified. Although the results of proteinN-terminal sequencing and thermal stability test show that the toxins from different producingareas have the same N-terminal sequence and structure, the RT originated from Xinjiang andthe AT originated from Yunnan were selected as the reference candidates because of thehighest score of mass spectrometry. The purified toxins have biological active, the CT50inSMMC-7721cytotoxicity test of ricin and abrin was2.35±0.26ng/ml and0.0032±0.0012ng/ml respectively; the toxicity test in mice showed that the LD50of ricin and abrin wereapproximately15μg/kg and5.3μg/kg respectively.Lastly, the180bottles of purified RT and120bottles of AT were lyophilized andsubpackaged at1mL/bottle. The absolute quantity, homogeneity and stability were analyzedfor the cetification of both toxins. HPLC and ICP-MS/MS were combined to analyze the sulfur in toxins, which allowed fully SI-traceable measurements of protein concentration.According to the results from RT, the content was25.07μg/bottle; the uncertainty was2.77μg/bottle; the relative standard deviation (RSD) of sampling inspection from11bottles ofas-prepared toxins was4.06%. As for AT, the contents was46.79μg/bottle; the uncertaintywas2.33μg/bottle; RSD was4.06%. the activities of bothe toxins were well retained after6months at-80℃.In the second part, the colloidal gold techneque was combined with polyclonalantibodies and monoclonal antibodies to develop ricin and abrin rapid test strips. Moreover,the combination of surface-enhanced Raman scattering (SERS) and the capillary-drivenmicrofluidic chip techneques was used to construct a novel rapid test for abrin.The polyclonal antibodies and the monoclonal antibodies were optimized for pairing.Theanti-ricin polyclonal antibodies was used as capture antibody and the anti-ricin toxin A chain(RTA) monoclonal antibodies was used as detection antibody. The sensitivity of as-preparedricin rapid test strip was16ng/mL in phosphate buffer solution (PBS) solution. No crossreactivity was observed among AT, bovine serum albumin (BSA), staphylococcal enterotoxin(SEB) or other unraleted proteins. Likewise, the anti-abrin polyclonal antibodies was used ascapture antibody and the anti-abrin toxin A chain (ATA) monoclonal antibodies was used asdetection antibody. The sensitivity of as-prepared abrin rapid test strip was3ng/mL inphosphate buffer solution (PBS). No cross reactivity was observed among RT, BSA,SEB orother unraleted proteins. The performance of both strips in toxin-spiked food samples (milk,fruit juice, biscuit, etc) was the same as in PBS-diluted samples. The stability could reach upto12months at room temperature.On the basis of the strips mentioned above, a duplex detection strip for ricin and abrinwas developed with anti-RT toxoid polyclonal antibodies and monoclonal antibodies as thecapture antibodyas well asanti-AT monoclonal antibodies and anti-RT toxoid polyclonalantibodiesas the detection antibody. The results showed that the sensitivity was25ng/mL inPBS-diluted RT, AT and both toxin-mixed samples and50ng/mL in the toxins-spiked foodsamples.Compared with the simplex detection strips, the duplex detection striplowers the costand reduces the equipment load, therefore, it is more convient in wartime.In addition, we tried a proof-of-concept use of capillary-driven SERS-based microfluidicchip for AT detection. Thereinto, Micro-electromechanical Systems (MEMS) techneque wasused to design and fabricate the silicon substrate with an opening structure of micro-pillararray, which had the same capillary-driven force as a nitrocellulose membrane. The anti-AT polycolonal antibodies were coupled with the silicon substrate as capture antibodies andexternal Raman molecues as detection probes to establish a rapid SERS-based dual antibodiessandwich test for abrin, acquiring a limit of detection (LOD) of0.1ng/mL in PBS-dilutedabrin sample, which was30times lower than that of traditional strips.In short, we constructed the process of RT and AT refernce materials,completed thecertification of RT and AT refernce materials, developed rapid sinplex and duplex test stripsfor RT and AT. The sensitivity was3~16ng/mL for RT and25~50ng/mL for AT. Besides, wefirstly demonstrated the design and the proof-of-concept use of a capillary-drivensurface-enhanced Raman scattering (SERS)-based microfluidic chip for AT detection.Thesensitivity of the microfluidic chip was0.1ng/mL, which was30times higher than that ofcolloidal gold-based lateral flow test strips. This technique is expected to develop a noveldetector for toxin-related warfare agent.