Glycyrrhizic Acid, Naringin Monoclonal Antibody Preparation And Establishing The Enzyme-linked Immunoassay Method

ObjectiveTo observe the monoclonal antibodies of GA and NAR, and establish the enzyme linked immunosorbent assays based on the monoclonal antibodies.MethodsThe hapten of GA and NAR is prepared by sodium periodate oxidization which transform the neighbor hydroxide group to ketone group. Then the hapten was coupled to a carrier such as BSA and OVA which give the antigenicity to the peaoniflorin to prepare the artificial antigen. The structure of artificial antigen is identified by ultraviolet spectrophotometry (UV).The artificial antigen was fused with adjuvant. Prepare antiserum against GA and NAR by immune the mice through hypodermic and intraperitoneal injection. The antiserum was collected by cutting tails. The titers were determined by indirect ELISA, and the specificity was determined by the complete indirect ELISA.BALB/c mice were immunized with the prepared conjugate, and spleen cells from the mice were isolated and fused with SP2/0myeloma cells at ratio of10:1by PEG4000, and selected with hypoxanthine-aminopterin-thymidine (HAT) medium. Antibodies to GA and NAR were screened by indirect ELISA and limited dilution. Mabs were producted by ascites method. The enzyme-linked immuno sorbent assay was established. And related indicators such as specificity, sensitivity cross reaction rate and recovery rate of the method were tested.ResultIn this study, we successfully synthesized the artificial antigents of GA, NAR and carrier protein conjugates. Then, we prepared the monoclonal antibody (Mab) against GA and NAR through hybridoma technology. Based on the purified anti-GA Mab, anti-NAR Mab we established enzyme linked immunosorbent assay (ELISA) method to quantify GA and NAR. The main results are as following.Artificial antigents for immunization (GA-BSA, NAR-BSA) and coating (GA-OVA, NAR-OVA) were synthesized through sodium periodate oxidization. The reaction product was identified by UV scan. The results showed that GA/NAR and carrier proteins condensate successfully. Balb/c mice were immunized with the GA-BSA, after the fifth immunization, the Balb/c mice were selected by indirect ELISA. The results showed that the anibody titers in all of the mice serum were overl:12800. The spleen cells from the mouse were fused with myeloma cells. After three times of limiting dilution, we obtained hybridoma cell line GA-Mab-DF5which can secrete Mab against GA. It was injected into mice’s abdominal cavity to induce the production of MAbs.Balb/c mice were immunized with the NAR-BSA, after the fifth immunization, the Balb/C mice were selected by indirect ELISA. The results showed that the anibody titers in all of the mice serum were overl:6400. The spleen cells from the mouse were fused with myeloma cells. After three times of limiting dilution, we obtained hybridoma cell line NAR-Mab-AB6which can secrete Mab against NAR. It was injected into mice’s abdominal cavity to induce the production of MAbs.An indirect competitive elisa (ic-elisa) with the MAbs to detect GA was established.Under optimum conditions, the standard curve equation was y=-0.31n(x)+1.3594, R2=0.9986. The Linear Range was at4-64μg/ml,IC10was4.59μg/ml, the sensitivity of the Mabs to GA were about17.14μg/ml.The Mabs had low cross-reactivity with other compounds.A indirect competitive elisa (ic-elisa) with the MAbs to detect NAR was established.Under optimum conditions, the standard curve equation was y=-0.4111n(x)+2.5018,R2=0.998. The Linear Range was at14-225ng/ml, IC10was19.70ng/ml, the sensitivity of the Mabs to GA were about85.45ng/ml.The Mabs had low cross-reactivity with other compounds.ConclusionThe final results showed that we can detect GA and NAR by IC-ELISA established by application monoclonal antibody against GA and NAR.