Generation Of Multivalent Antibody Against Small Hapten And Studies On Its Bioactivities

The obtaining of antibody against small hapten is very difficult, and under some circumstances, antibody affinity is too low to be used, which extremely limit the application of antibody in detection of hapten. In order to explore the construction of antibody with known sequence, scFv against parathion was produced. We also generated a single chain Fv antibody which retained the characteristic of binding specifically to microcystins-LR (MC-LR). In order to improve the functional affinity, we constructed a tetravalent antibody. These studies provide a basis for the application of genetically engineered antibody in the detection of hapten.Antibody sequence against parathion in GenBank was analyzed, and the heavy chain V-region (VH) and light chain V-region (VL) fragments of it were linked by a linker of 45 nt to produce scFv gene. After being ligated to pET-28a (+), the recombinant plasmid was transformed into E.coli Origami 2 (DE3). Soluble scFv was expressed after being induced by IPTG. SDS-PAGE and Western Blot analyses confirmed the expression of scFv with the molecular weight of about 28 kD. The recombinant protein was purified by metal affinity chromatography using Ni-NTA, and the purity reached 74.8%. ELISA revealed that the expressed recombinant protein could specifically react with parathion. These studies provide a basis for the construction of multivalent antibody with higher specificity and affinity and also provide a theoretical basis for the application of genetically engineered antibody in the detection of pesticide hapten.The cDNAs of heavy chain and light chain genes were amplified by RT-PCR from hybridoma cell secreting anti MC-LR monoclonal antibody. Sequence analysis revealed that the full length of heavy chain is 1473 bp, including partial VH, the entire CH1, CH2, CH3 and 3'noncoding region. VH is 363bp in length, which encodes 121 amino acids and belongs to mouse IgH-V7183 VH5 family. The full length of light chain is 880bp, including partial VL, the entire constant region and 3'noncoding region. VL is 336 bp, encoding 112 amino acids and is a member of mouse IGKV21 subgroup. Both VH and VL contain 4 framework regions, 3 complementarity-determining regions and 2 antibody characteristic Cys, which are the common characters of immunoglobulin variable region, and there are no intragenic stop codons in them. All the results revealed that the obtained genes are mouse antibody heavy and light chain genes.VH and VL were amplified separately and spliced by overlap extension PCR, and the generated scFv fragment was ligated to the expression vector pET-28a(+) to produce an expression vector pET-mc. Sequence analyses revealed that the full length of scFv is 744bp, encoding 248 amino acids. pET-mc was transformed into E. coli Origami 2 (DE3) and induced to express by IPTG. SDS-PAGE and Western Blot analysis showed that scFv was expressed mainly as inclusion body in the cytoplasm of E. coli. Soluble scFv was in its highest content when IPTG concentration was 0.1 mM and temperature was 15℃. The recombinant protein was purified by metal affinity chromatography using Ni-NTA, and the concentration of purified scFv was 0.115 mg/ml. ELISA revealed that the expressed recombinant protein can bind specifically to MC-LR.In order to obtain recombinant Fv antibody with higher affinity, the core-streptavidin was used to crosslink scFv. scFv gene was spliced with gene encoding core-streptavidin, and the spliced product was ligated to pET-28a(+) to generate the expression vector pET-teab. SDS-PAGE and Western Blot analyses showed that target protein was expressed mainly in the form of inclusion body. Soluble expression product was in its highest content when IPTG was 0.1 mM and temperature was 15℃. The recombinant protein was purified by metal affinity chromatography using Ni-NTA, and the concentration of purified scFv was 0.179 mg/ml. Non-reduced SDS-PAGE and Western Blot revealed that the purified protein existed in the form of both tetramer and monomer. Higher molecular weight, aggregated species, however, was not detected. ELISA demonstrated that tetravalent antibody has gained in avidity compared with scFv and monoclonal antibody.