MAb A7C4 was found out to localize within the cell surface and internalize but only when bound to abrin. terrorist use.1 Abrin, of the flower is one of the most lethal flower toxins known. Owing to its high toxicity, relative ease of purification and convenience, it is regarded as a biohazardous agent.2 Abrin belongs to the family of type II ribosome inactivating proteins (RIPs). It is a heterodimeric protein consisting of the A chain which harbors the N-glycosidase activity and B chain which is a galactose-specific lectin.3,4 The B chain binds to the cell surface terminal galactose residues and facilitates the access of the toxin inside the cell. The A chain, depurinates the universally conserved -sarcin loop of the 28S?rRNA and inhibits protein synthesis5 that activates the unfolded protein response leading to apoptosis.6,7 Despite the high toxicity, treatment for abrin poisoning is symptomatic.8 Currently you will find no antidotes available for managing abrin poisoning. Certain chemical compounds such as N-acetyl-L-cysteine and Trolox which prevented abrin-induced cell death have been proposed for management of abrin intoxication, although the effects of these compounds have not been examined.2 On the other hand, passive immunization with neutralizing antibodies has proven to be a specific and effective mode of defense against poisoning by several biological toxins.9-12 The only known neutralizing monoclonal antibody (mAb) namely D6F10 for abrin was reported from our laboratory.13 This mAb was found to inhibit the enzymatic activity of the abrin A chain and also conferred safety to mice agglutinin (APAA) was constructed.14 The recombinant construct retained the folding domains 7-Methoxyisoflavone of ABA, enabling us to carry out mutagenesis studies 7-Methoxyisoflavone to identify RGS7 the key amino residues. Since the chimera included the epitope related to the mAb D6F10 it was explored like a potential vaccine candidate (unpublished data). Although demonstrated to be effective against administration of lethal doses of abrin in mice, the epitopes related to the response against the chimera are not known. In an effort to delineate the neutralizing epitopes on abrin, we founded hybridoma using the splenocytes from mice immunized with the chimera and characterized the mAbs. With this study we statement one mAb, namely A7C4 which inhibited toxin activity on cells and more importantly, in mice. Toward understanding the mechanism of immunoneutralization of abrin by this antibody, we mapped the core epitope identified by mAb A7C4. Interestingly, the mAb A7C4 neither prevented 7-Methoxyisoflavone the binding of abrin to cell surface nor did it inhibit the enzymatic activity of ABA as identified in cell free system, thereby suggesting that mAb A7C4 rescues abrin toxicity by a different mechanism. Results Reactivity of mAb A7C4 to different RIPs Twelve mAbs were generated to the ABA-APAA chimera. Of these the mAb A7C4 was selected based on its ability to save cells from abrin-mediated cytotoxicity. MAb A7C4 bound to abrin but not to APA in ELISA suggesting the epitope of mAb A7C4 lies within 1 to 123?amino acids of abrin (Fig.?1). The mAb did not bind to the closely related type II RIP ricin, demonstrating that mAb A7C4 recognizes an epitope unique to abrin. Open in a separate window Number 1. Reactivity of mAb A7C4 to abrin, agglutinin and ricin. Wells of ELISA plates were coated with 500 ng/100 l per well of any one of the proteins, chimera, abrin, APA or ricin and incubated with 100 l of the hybridoma tradition supernatants for 2?h, followed by incubation with the secondary antibody-HRP conjugate. After adding the TMB substrate and preventing the reaction with 1M H2SO4, readings were taken at 450?nm. Polyclonal mouse antiserum to the chimera, and no-antigen wells served as the positive and negative control respectively. Each pub represents the imply SEM. Statistical analysis was carried out using One of the ways ANOVA followed by Turkey’s multiple assessment test (*p < 0.05). Neutralization of abrin-induced cytotoxicity by mAb A7C4 The apoptotic human population in cells treated with either abrin only or along with mAb A7C4 was measured. HeLa (adherent tradition) and Jurkat (suspension tradition) cell lines were taken for this study. The choice of the two was based on the studies carried out earlier.