Preparation And Identification Of Chicken-Derived Singlechain Variable Fragment Antibody Against Nglycolylneuraminic Acid

N-glycolylneuraminic(Neu5Gc) is a kind of sialic acids in many mammals, which is occurring in the glycoconjugates of most deuterostome animals. But Neu5 Gc is absent in normal cels of human and chicken. Glycoconjugate-bound Neu5 Gc is a common component of the dietary sources, particularly in most types of red meat. Neu5 Gc will be absorbed into the cell membrane surface, if it enters into hunman body through the dietary. Neu5 Gc is a kind of specific receptor, for example, E.coli K99 can combine Neu5 Gc of intestinal cels and cause disease, serious food poisoning and other symptoms. The incorporation of Neu5 Gc from dietary into human body can produce heterogeneous antibody, the interaction between the Neu5 Gc and the body will result in the chronic inflammation, further promote the occurrence and development of cancer and related diseases. It is reported that the content of Neu5 Gc in tumor tissues was far more than that of the normal human tissue. The incorporation of Neu5 Gc from dietary into human body is associated with certain types of cancer development and can serve as some specific markers of the tumor. Neu5 Gc antibodies can stimulate the cytotoxicity effect of CTL(cytotoxic T lymphocyte). So Neu5 Gc become the new target of tumor diagnosis and the food safety testing. However, due to the limitations of analytical methods to detect it, very little is known about the occurrence and biologic function. So, there is need for the sensitive and specific detection of Neu5 Gc in human tumors tissues and biotherapeutic products about red meat or milk. At present, detection methods on Neu5 Gc are mainly chemical instrument method and immunological method. But these methods have some shortcomings and the insufficiency, the chemical instruments are so expensive, require complex sample preparation and pretreatment the professional operator and so on. The existing immunological method could not recognize the free Neu5 Gc and the combined formulation. So a rapid, simple and sensitive assay is necessary for detecting the free of Neu5 Gc. But the important thing is to acquire an efficient and strong specificity of antibody when establish the immunological detection method.In order to prepare the high titer and specific Neu5 Gc antibody, Neu5 Gc was linked to carrier proteins by carbodiimide(EDC) method. After the immunization of immunogen Neu5Gc-KLH to laying hens, the Ig Y antibody was gained. Neu5Gc-Ig Y was analyzed and identified by ELISA. The results of UV, NAGE showed that the artificial antigen Neu5Gc-KLH and Neu5Gc-BSA were successfully synthesized. The development of the Ig Y antibody titer of 1:10000 was monitored by indirect ELISA. The result showed that the artificial antigen preparation was successfully completed. The complete antigen and immunized highland brown laying hens can be used for the next test. Then, we use the molecular biology and genetic engineering technic to obtain the single chain antibody. Total RNA of the immunized chicken spleen is extracted and reverse transcribed into c DNA. Then, the variable region gene of heavy chain and light chain was amplified based on the c DNA template. The synthetic single chain fragment variable(sc Fv) gene was linked through the gene of(GGGGS)3 by overlap extension PCR(SOEPCR). The sc Fv gene is cloned to phage plasmid vector(p CANTAB5E) and the recombinant phage plasmid is transformed into E.coli TG1 to obtain single antibody gene library of 1.7×107 cfu/m L against Neu5 Gc. Then, the primary library is selected by four cycles of absorbed, eluted, amplified, enriched and elutriated using the phage display technology. Four phage single antibody named AKsc Fvj1, AKsc Fvj2, AKsc Fvj3 and AKsc Fvj4 against Neu5 Gc were obtained by phage ELISA for the qualitative and quantitative analysis and sequencing. Finally, AKsc Fvj3 and AKsc Fvj4 were successfully expressed and established a competitive of inhibition curve of AKsc Fvj4. The optimized working concentration of Neu5Gc-BSA is 4 μg/m L. The best dilution and antibody titer of sc Fv is 1:1600 and 1:3200, respectively. The sensitivity of sc Fv was detected by the competitive blocking ELISA. The liner regression equation is y = 23.12 x + 33.19, with R=0.980. The IC50 is 5.333 μg/m L. The linear range is 0.66~286.42 μg/m L. The detection sensitivity is 0.66 μg/m L.The chicken-derived single chain Variable Fragment antibody against Neu5 Gc would establish a favourable foundation and a novel idea for detecting of the combined Neu5 Gc and free Neu5 Gc of red meat, milk and tumor tissues.