Preparation Of Monoclonal Anitbodies Against Ricin

Ricin is a ribosome-inactivating protein composed of two glycoproteins chains A(RTA)and chains B (RTB)linked by a disulfide bond. Supported by RTB, RTA can easilypenetrate into cells, and then inactivate60S ribosomal subunits by an N-glycosidic cleavage toblock protein synthesis and therefore induce cell death. The amino acid sequences andsecondary structures of ricin are basically clear. RTB has two galactose-binding or galactoseresidues-binding sites. These two sites could recognize the galactose residues on cellularmembrane and then mediate the endocytosis of holotoxin into cell. RTA and RTB have1and2oligosaccharide branched chains with terminal mannose residue respectively which can bindto reticular endothelial cells especially macrophages. Macrophages surface are rich inmannose receptors which make it take up ricin preferentially. This is essential for the toxiceffect of ricin. Due to its high toxicity, ricin is regarded as a high terrorist risk for the publicand has been considered as a potential agent of biological warfare or terrorist attack. Ricin hasbeen classified as a―B‖weapon (with a moderate threat) by Centers for Disease Control ofUSA.Because ricin inhibits protein synthesis very quickly, the cell or tissue damage beginswithin several hours although signs of intoxication might not be noted, making therapydifficult. There is no specific antidote for ricin poisoning. For ricin intoxication therapy, bothearly diagnosis and specific antidote are very important. In order to establish theimmunoanalysis method and screen the effective antitoxic antibody for the ricin poisoningdetection and therapy, respectively, we aimed to prepare the monoclonal antibodies againstRTB and RTA.Method: The expressed vectors, pET28a-RTB and pET28a-RTA, were constructed. RTAand RTB were respectively expressed in E.coli. BL21and then purified by nickel affinitychromatography column. They were used as antigens to immunize mice and Enzyme-linkedimmunosorbent assay (ELISA) was used to screen positive hybridoma cells which couldsecrete monoclonal antibody against ricin. Both ELISA and Western blot were used to analyze the epitopes recognized by antibodies.Results: RTA and RTB were successfully expressed and purified. Using these twoantigens, eight hybridoma cells secreting mAbs against RTB (3-2-16，14-1，3-2-16，2-28-5-6，8-1-1-8，13-2-4，7-8-24，9-2) and4cells (1-1,13-4,4-2-15-4,2-07) secreting mAbs againstRTA were obtained. The ascidic fluid were prepared and mAbs were purified by Protein GSepharose4fast flow column. RTA, RTB and ricin were usedrespectively as antigen todetermine the antigen-antibody reactivity by ELISA. The result indicated that all eightantibodies against RTB could well recognize ricin and RTB with high specificity. However,they could not to be used to establish the Sandwich ELISA to analyze ricin. Within fourantibodies against RTA, two of them (1-1,4-2-15-4) could weakly bind with RTB andantibodies13-4，2-07had specific binding activity with RTA. Antibodies13-4and2-07wereused to establish an enzyme-linked immunosorbent assay (ELISA) using glycoprotein as acoating antigen.Conclusion: In this research, the prokaryotic expression vectors, pET28a-RTB andpET28a-RTA, were constructed and the recombinant proteins were expressed in E.Coli. TheRTA and RTB were purified by Ni2+affinity chromatography and then used as antigens toprepared antibodies, respectively. Eight hybridoma cells secreting antibodies against RTB andfour cells secreting antibodies against RTA were screened. All purified antibodies could not beused to establish the method of SandwichELISA (antibody-ricin-HRP antibody). But twoantibodies,13-4,2-07could be used to establish an ELISA (glycoprotein-ricin-antibody-HRPGAM) method for ricin detection.Ricin B chain helps A chain into the cell membrane, itself is not toxic, we can useantibodies against the B chain, to study the structure, properties and mechanism of action ofricin. We will screen pairing antibodies from A chain and B chain antibodies, and we hope tofind the paired antibodies,develop a kit, for qualitative and quantitative detection of ricin.Finally, we will also perform functional studies to see if there is neutralizing antibodies against the A chain can be of great significance in the treatment of acute phase of ricin poisoning.