Preparation Of Recombinant Single-chain Antibody Against GCA And Studies On Immunoassay

Glycocholic acid(GCA),the secondary cholic acid,is a derivative of steroid acid.GCA is the conjugated cholic acid produced by bile acid and glycine in the liver.It is one of the major components of bile acids.GCA has the effect of promoting the emulsification of lipids and accelerating the digestion and absorption of lipid materials.The concentrations of GCA in the blood and urine are associated with the severity of hepatocyte damage and the bile acid metabolism.Therefore,the development of effective methods for monitoring the GCA can provide important evidence for the identification,diagnosis,treatment and prognosis analysis of various liver diseases.At present,the detecting methods for GCA include high performance liquid chromatography(HPLC),liquid chromatography-mass spectrometry(LC-MS),immunoassay and so on.Immunoassay is based on the specific binding between antibodies and antigens.It has the advantages of high sensitivity,specificity,simplicity,speed and cheapness,and is especially suitable for screening the large samples.Antibodies are one of the most important factors that affect the specificity and sensitivity of the immunoassay.The traditional antibodies include polyclonal antibodies(pAb)and monoclonal antibodies(mAb).However,there are some drawbacks in these two kinds of antibodies,such as the difference quality between batches to batches in the preparation of polyclonal antibodies;and time-consuming,high cost and complex steps in the preparation of monoclonal antibodies.By phage display technology,it can be isolated the recombinant antibodies with high affinity from the antibody library,and can be obtained the encoding sequence of the antibody by DNA sequencing.Single-chian variable fragment(scFv)is one of the most common format of recombinant antibodies,which is the smallest unit that can maintain the effective binding ability of antibody.scFv consists of a variable region of heavy(VH)and light(VL)chains,which are joined together by a flexible peptide linker.scFv has many advantages,such as prokaryotic expression and genetic engineering modification,which has already been widely used in the medical diagnosis,disease treatment,environmental monitoring and food safety analysis.In this study,the high affinity scFv antibodies were isolated by phage display technique with GCA as a target.The main results are as follows:1.Construction of scFv phage display library and panning1.1 After immunized the chicken with immunogen GCA-BSA,the phage display library was constructed with the capacity of 3.34×107cfu.By the GCA competing elution method,the phage display library was conducted four rounds of panning under the harsh conditions.The results were confirmed by phage-ELISA and the positive clones were analysed by DNA sequencing.Finally,four unique scFv sequences were obtained.2.The prokaryotic expression of anti-GCA scFv antibody and its application2.1 The phagemid scFv-G11 with the highest sensitivity was used for prokaryotic expression and purification.Based on the purified scFv antibody,the indirect competitive ELISA for the GCA in urine was establised,with the IC50 of 0.06 g/mL,the linear range(IC20-IC80)of 0.02-0.18 g/mL,the detection limit(LOD)of 0.01 g/mL,the cross reaction rate for TCA of 40%,the cross reaction with other similar structure of cholic acid lower than 0.58%.The recovery in urine was 86~123%,and the results were compared with LC-MS/MS.The results showed that the correlation was good(R2 = 0.99).3.Preparation and application of biotinylated scFv antibody3.1 The scFv antibody was labeled with biotin using a chemical and an enzyme method,respectively.It was found that The enzymatic method proved superior giving sensitive scFv-biotin preparations.Based on the enzymatical biotinylated scFv antibody,an indirect competitive BA-ELISA was establised,with the IC50 of 0.42 g/mL,the linear range(IC20-IC80)of 0.14?1.24 g/mL,the detection limit(LOD)of 0.07 g/mL,the cross reaction rate for TCA of 43.75%,the cross reaction with other similar structure of cholic acid lower than 1.88%.The recovery in urine was 110.8?125.6%,and the results were compared with LC-MS/MS.The results showed that the correlation was good(R2 = 0.99).In conclusion,four unique scFv antibodies with high sensitivity were isolated by phage display technique.In addition,the scFv antibody with highest sensitivity was labeled with biotin by an enzyme method.Based on the scFv antibody and biotinylated scFv antibody,the ELISA and BA-ELISA were established,'respectively.In this work,the developed immunoassay can be used to effectively detect GCA,and it is of great significance for the monitoring of liver function.