Screening And Evaluation Antitoxic Activity Of Compounds Against Ricin

Ricin is a ribosome inactivating protein（RIP）and consists of two disulfide-linked polypeptides chains:RTA and RTB.Ricin is considered to be a great threat to the public safty for its unique properties such as high toxicity,stable properties,easy accessibility.Ricin can quickly inhibits protein synthesis and the cell or tissue damage begins within several hours after exposed to ricin,however,the sufferer do not show any intoxication signs,which maks the treatment difficult.Symptomatic therapies are effective in some case of intoxication but not effective for all cases especially for lethal dose toxic exposure.Objective:Screening and compare the antitoxic activity of antibodies,adjuvants of Rivax vaccine and small molecular compounds.Contents:1)Screening effective monoclonal antibodies and small molecule inhibitors against ricin and then evaluating the possible effect of drug combination;2)Comparing the effect of Rivax vaccine compatible with adjuvants.Methods:1)Monoclonal antibodies（McAbs）were prepared by hybridoma technique.Subtypes and characteristics of McAbs were detected by ELISA assay.The neutralization activity of McAbs was measured by TNT^?T3 coupled reticulocyte lysate cell-free kits.The protective ability of McAbs was detected in vitro and in vivo.In vitro,the viability of MDCK cells was measured by RTCA.In vivo,The viability of ICR mice was calculated.2)The effect of Rivax vaccine with adjuvants was evaluated in vivo.Mice were immunized with Rivax or Rivax mixed with Alum adjuvant,polysaccharide PCP-I or G1a respectively and then treated with 1 lethal dose,3 lethal dose or 9lethal dose of ricin,respectively.The viability of immunized mice after exposure to ricin was calculated.3)The effect of small molecule inhibitors were evaluated in vitro and in vivo.In vitro,the viability of MDCK cells was detected by CCK-8 kits after treated with compounds or ricin.In vivo,the viability of intoxcation ICR mice pretreated with inhibitor was calculated.Results:1)Five McAbs recognized ricin holotoxin were obtained.8-1-1-8 and 13-2-4 recognize RTB subunit;S11 recognize RTA;AB6 recognize neither RTA nor RTB.The result from the cell-free system showed that only S11 could block the inhibition of protein synthesis by ricin.The result of real time cell analysis（RTCA）showed that S11 and AB6 could alleviate the cytotoxicity induced by ricin with an EC₅₀ value of 26.79 ng·mL^-1 and 11.19 ng·mL^-1 respectively.In vivo results suggested that S11 and AB6 could protect mice from twice lethal dose of ricin exposure.2)Compare with Rivax group,the viability of compatibility of adjuvant groups showed significant increase.After co-immunized with Rivax and AL（OH）₃or PCP-I adjuvant,mice could against 9 lethal dose of ricin with a viability of90.90%and 50%,respectively.When mice were co-inoculated with Rivax and G1a adjuvant,immunized animal could against three lethal dose ricin with a viability of 77.78%.3)Both through concurrent administration and preventive administration,the inhibitor re705c and re706c could inhibit the cytotoxicity induced by ricin with an EC₅₀ 0.67±0.14μmol×L^-11 and 0.39±0.08μmol×L^-11 in concurrent administration and 17.53±1.37 nmol×L^-11 and 0.44±0.08μmol×L^-1 in preventive administration.When pretreated with 50 mg×Kg^-11 re705c,the survival time of poisoned mice was significantly prolonged.Mice treated with AB6 or S11 after toxication with 2.5 times of lethal dose ricin,the viability of mice was 30%and73%.When combination of re705c,the viability of mice increased up to 90%and100%,respectively.Conclusions:1)S11 and AB6 have good protective effect against lathal dose ricin both in vitro and in vivo and could be a candidate against ricin.2)Adjuvant alum and PCP-I can protect mice from 9 lathal dose of ricin exposure and could be the candidate adjuvants for Rivax vaccine.3)Re705c could prolong survival time of mice after exposure to lethal dose of ricin and increase the protective effect of AB6 when combination with it.