Preparation Of The Monoclonal Antibodies Against Acrolein And Establishment Of ELISA Detecting Method

Objectives: To produce the ascitictype monoclonal antibodies(MAbs) againstAcrolein based on the positive hybridoma cell strain. To optimize and establish anEnzyme-Linked Immunosorbent Assay(ELISA) for detecting Acrolein.Methods: Acrolein complete antigen was synthesized by using the Directsynthesis method. Ballb/C female mice of6~8weeks were immunized with thecomplete antigen and five immunity had a good response. Cell fusion was carried byusing PEG method. Through four times subcloning, Monoclonal antibody hybridomacell1G7G6was obtained，which was able to product IgG2b antibody subtypes，andwere used to produce ascitictype MAbs. Based on these monoclonal antibodiesproduced，Acrolein ELISA conditions including coating buffer，coating condition，blocking time and reaction time were optimized. Then the indirect competitive ELISAfor detecting Acrolein was established.Results: The complete antigen of Acrolein were synthesized successfully. Themonoclonal antibodies titer was up to8×104，with low cross reactivity with otherAcrolein analogues. The optimum conditions of ELISA were given as follows：Jetmicrowell-plate was coated with the antigen in CBS(pH9.6，0.05M).The plate wasincubated at4℃for16hours.5%skimmed milk powder in PBST blocking the plateat37℃for1hours could reduce the non-specific adsorption. Primary antibody andsecond antibody were diluted in PBST and incubated at37℃for1hours，TMB wasused to develop the colour for10min. Under optimized conditions，the sensitivity ofELISA was43.2ng/ml(IC50)，the detection limits (LOD）was1.8ng/ml(IC10)， thelinear concentrations ranged was from4.4to417.2ng/mL.Conclusions: The high affinity and high specific McAbs against Acrolein wereprepared. This is the first time, a rapid，sensitive and specific indirect competitiveELISA for Acrolein detection was optimized and established.