ABSTRACT
Ricin is a highly toxic type 2 ribosome-inactivating protein (RIP) which is extracted from the seeds of castor beans. Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far. In this study, by structural modification of a retrograde transport blocker Retro-2, a series of novel compounds were obtained. The primary screen revealed that compound has an improved anti-ricin activity compare to positive control. pre-exposure evaluation in Madin-Darby Canine Kidney (MDCK) cells demonstrated that is a powerful anti-ricin compound with an EC of 41.05 nmol/L against one LC (lethal concentration, 5.56 ng/mL) of ricin. Further studies surprisingly indicated that confers post-exposure activity against ricin intoxication. An study showed that 1 h post-exposure administration of can improve the survival rate as well as delay the death of ricin-intoxicated mice. A drug combination of with monoclonal antibody mAb4C13 rescued mice from one LD (lethal dose) ricin challenge and the survival rate of tested animals is 100%. These results represent, for the first time, indication that small molecule retrograde transport blocker confers both and post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.
ABSTRACT
Objective:To explore the immunity provided to BALB/c mice by immunization with the recombinant ricin B chain protein (rRTB). Methods:Female BALB/c mice were randomly selected into rRTB-vaccinated group and PBS group. Mice were subcu-taneously (s. c. ) injected four times with 14 days immunization time interval. Changes of antibodies (IgG,IgG1and IgG2a) in serum were detected by ELISA. Meanwhile,the expression of IL-4 and IFN-γ were detected by flow cytometry. Results:The mean IgG titers reached 106 after the fourth immunization and a strong secondary response was induced in vaccinated mice when challenged with toxin. There was significant difference between rRTB-vaccinated group and PBS group ( P<0. 05 ) . The same result was shown in IgG1. However,no changed was detected in IgG2a. Meanwhile,there was significant difference for IL-4 between two groups (P<0. 05), while no significant difference for IFN-γwas observed. Conclusion:rRTB can produce higher levels of antigen-specific antibodies ( IgG and IgG1),and cytokine (IL-4) of splenocytes,which means the recombinant protein can induce the Th2-type immune response and trigger a good immune response. rRTB may be a potentially valuable vaccine candidate against human exposure to AT.
ABSTRACT
Objective To develop an up-converting phosphor technology based lateral flow assay ( UPT-LF) to detect ricin toxin ( RT) quickly, accurately and quantitatively.Methods Ricin-monoclonal antibodies were prepared and their affinity was evaluated before four types of monoclonal antibodies with the highest titer were applied to couple with the up-converting phosphor nano-particles ( UCP-NPs) as the bio-conjugate and disperse on the analysis membrane as the test line, respectively.Following systematic optimization to establish the RT-UPT-LF strip, the sensitivity, precision, quantita-tive ability and specificity of RT-UPT-LF were evaluated.Results The detection could be accomplished within 15 min and the detection limit of the RT-UPT-LF assay could reach 0.5 ng/ml within the quantitative detection range of 0.5-1000 ng/ml.Other non-specific toxins at a concentration of 1000 ng/ml did not cause any non-specific reactions.Conclusion The developed RT-UPT-LF strip provides a new means for on-site quantitative detection of ricin toxin.
ABSTRACT
Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain (RTA) and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guidod molecular design method. Its coding sequence was ob- tained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then ex-pressed in E. coli BL21 (DE3), identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for riein was evaluated by competitive ELlSA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coll. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel pep-tide based on riein toxin antibody provides a novel method to develop new generation of ricin antagonists.
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Objective:To clone human anti-ricin antibodies from large phage antibody library.Methods:Panning for a large phage library against ricin toxin was conducted to select specific antibodies against ricin. The binding activities and specificities were tested by ELISA method. Soluble ScFvs were prepared through infecting E coli. HB2151 with the selected phage antibodies and induction with IPTG. Results:Forty positive clones were obtained after 5 rounds of panning, and 12 clones had specific binding ability to ricin toxin. DNA fingerprinting showed 7 different band patterns indicating 7 different positive clones. DNA sequencing showed that variable regions of these ScFvs belonged to different subgroups.Conclusion:Human anti-ricin antibodies were successfully obtained from large phage antibody library.
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Objective: Immunotoxin rRTA:DS27, which was prepared by conjugating DS27 with recombinanl ricin a chain, was compared with ricin:DS27 as immunotoxins. Methods: System analysis were performed regard to their cell-specific cytotoxicity, inhibition on the proliferation of hemoapeutic potential cells, immunogold-labelled intracellular routing and effect of NH_(4)Cl on the cytotoxicity. Results: Results showed that rRTA: DS27 got a more specific cytotoxicity and a weaker inhibition on the proliferation of hemoapeutic potential cells than ricin:DS27, NH_(4)Cl could obsolutelyy enhence the cytotoxicity of rRTA:DS27. Conclusion: rRTA:DS27 had more advantages than ricin: DS27 as immunotoxins.