| DON hapten design is the key step to establish the immune analysis methods. Currently, DON hapten design still adopt experience, causing the waste of time and money. From half antigen design principle, clear DON best hapten design thought: maximization similarity.of antigen sites.Study the changes of molecular space configuration, electronic characteristics, hydrophobic property that several influence the antigen-antibody intercropping force factors , before and after DON modified. Research the relationship between hapten structure and immune discrimination. Establish DON hapten theory simulation design method. Provide a new thinking for hapten design. And on this basis, build a DON direct competition enzyme-linked immunosorbent analysis method.According to the design process: choose binding sites→introduced connection arm→coupling carrier protein. Firstly, study the effect of binding sites to immune discrimination, introduced the same length of carbon chain of connection arm in the four potential binding sites (3, 7, 15-hydroxyl and 8-ketone base),Using molecular simulation software Hyperchem7.5 analysis determines parameters (spatial structure, atomic stereograph.furthermore, hydrophobic constant), 3-hapten is most similar to DON. Show 3-OH is the best DON binding site. Consistent to immune experiment results of Casale and Ramesh. Secondly, designed different connection arm structure in 3-OH, through similarity simulation received the best connection arm: succinic anhydride arm.Choose 3 DON hapten with different arms structure to do immune antibody test, determination of antibody titer and inhibition rate to verify the molecular simulation results, study relationship of hapten structure and antibody immunity activity.Group B antibody titer: 256000; Group A, C average titer: 64000. DON concentration for 500 ng/ml of group A antibodies suppression rate respectively: 66.4%, group B: 21.1%, group C: 10.8%. Inhibition rate of antibodies related to the specificity of DON, Group A has the highest similarity with DON, the specificity of strong, DON inhibition rate high, Antibody titer can reflect the carp of hapten. the benzene rings, unsaturated double bond can enhance the carp. Group B containing unsaturated double bond, enhanced the carp, produce high titer antibody, Although group C contains benzene structure, but failed to produce high titer antibody, combined with its spatial structure found: group C carboxyl end is very close to the main structure, may be shielded by carrier protein and affect immune discrimination. Comprehensive consideration, succinic anhydride is the best connected arm to DON .In basis of the best hapten, compare the immunogenicity effect of vector protein(BSA,KLH).Preparation completely antigen of different carrier protein, to obtain DON antibody. Study antibody using enzyme-linked immunosorbent method (ELISA) and round two color spectrum (CD).Established DON direct competition enzyme-linked immunosorbent analysis method. Examination scope for 1~100 ng/ml, half the inhibition rate IC50 for 10 ng/mL, minimum detect limit get 0.56 ng/ml, cross reaction rate less than 12 %, average batch coefficient of variation within 2.82 %,average batch coefficient of variation between 14.54 %;the recovery in 80.2-91.1 %.This study shows that the molecular simulation method has the positive role to DON hapten design, guiding half antigen design, reduce time and capital investment. DON-KLH immunogenicity is superior DON–BSA. Established DON direct competition ELISA method, quick, convenient, and has good practical value. |
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