| According to the common structure of O,O-dimethyl organophosphorus pesticides, the generic hapten (H1,3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid) was designed with roxyphenylpropionic acid and 0,0-dimethyl phosphorochloridothioate. H1 was covalently attached to BSA as immunogen and to OVA as homologous coating antigen with the active ester method. BALB/c female mice (8 weeks old) were immunized with the H1-BSA conjugate. The dose consisted of 100μg of conjugate intraperitoneally injected. One week after the third booster injection, mice were tail-bled and antisera were tested for anti-hapten antibody titer by a noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) using a homologous coating antigen (H1-OVA). The mice showing higher serum reactivity were selected as the donors of spleen cells for hybridoma production. Two hybridoma cell lines that can secrete broad-specific McAbs were established by cell fusion technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity of McAbs were determined by noncompetitive indirect ELISA, the class and subclass of McAbs were identified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determined by competitive indirect ELISA. The McAb affinity of D12-B5 is 1.709×109L/mol. The class and subclass of D12-B5 were IgGl andκ. In the competitive indirect ELISA, the I50 of Parathion-methyl、Chlorpyrifos-methyl、Fenthion、Tolclofos-methyl、Fenitrothion and Malathion was respectively 42.1μg/mL,60.2μg/mL, 75μg/mL,13.58μg/mL,117μg/mL and 172.9μg/mLTo study the effect of heterologous coating antigens on immunoassay sensitivity, eight haptens designed were four pesticides’ derivatives. The result showed that the haptens whose structure were similar to analytes could not improve the sensitivity, exactly the sensitivity of generic-antibody was not associated with the homology of heterologous coating antigen and analyte. Whereas the combination of mAb with coating antigen (H7-OVA) gave the lowest I50 values. According to the analysis of heterologous coating antigens, we choosed the H7-OVA as the coating antigen. Under the optimization of the ionic strength and pH, the standard curves were estblished by competitive indirect ELISA. The I50 values of specific to parathion-methyl was 0.165μg/mL, showing the lowest detection limit of 0.0144μg/mL, the I50 value achieved for tolclofos-methyl was 0.95μg/mL, with the lowest detection limit of 0.033μg/mL.The total RNA of spleen cells and hybridoma cell was isolated with trizol reagent, and the variable region genes were amplified with variable region primers by RT-PCR (reverse transcriptase-polymerase chain reaction). The variable region genes of heavy chain and light chain were strung together by SOE-PCR (splicing by overlap extension PCR) and the ScFv gene was constructed. After cloned into T vector, the recombinant ScFv gene was identified by PCR and sequencing. The sequencing result shows that the ScFv gene consists of about 750 bp, with more than 300 bp of heavy chain variable region gene and light chain variable region gene, which were linked by a 45 bp peptide.On Discovery Studio, with homologous protein-structure-prediction, a three-dimensional model of ScFv was mimiced according to variable region gene. The three-dimension of ScFv antibody provided a method for improving antibody affinity, investigating antibody structure and analyzing function of light, as well as heavy chain in antibody activity. |
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