Screening And Identification Of Genetically Engineered Antibodies Against Metabolites Of Estrone And Progesterone

Female sex hormones include estrogen and progesterone,which are very important for women's development and reproductive function.Estrogen consists of estrone,estradiol and estriol.Progesterone is the main progesterone.Estrogen and progesterone levels are indispensable clinical indicators in detecting reproduction.Estrone and progesterone are contained in routine detecting of sex hormones.They are of great significance in diagnosis of infertility,observation of therapy,prognosis and the mechanism research.Routine detecting methods of sex hormones take serum as the sample,which brings psychological pressure in patients with safety risks.Therefore,there is a great demand for the development of rapid non-invasive methods to monitor the level of sex hormones.Estrone-3-glucuronide(E1G)and pregnanediol-3-glucuronide(PdG)are urine metabolites of estrone and progesterone,respectively.Recent studies have found that their concentration in urine are highly correlated with serum concentration,which makes it possible to monitor the level of estrone and progesterone by urine.However,both E1G and PdG have low molecular weight and weak immunogenicity.In addition,they are metabolites that exist in mammals themselves.It is difficult to obtain antibodies or antibodies with high specificity and affinity by traditional immune methods.Antibodies have become the limitation for the development of such detection products.With the development of genetic engineering technology and recombinant antibody technique,the third-generation antibodies,especially the antigen binding fragments(Fab),have shown great potential in preparation antibodies of small-molecule antigen.Combined with in vitro display,high affinity and specificity antibodies can be rapidly screened with high throughput,simple preparation process and fast production.It can be used in prevention,diagnosis,treatment,detection and other fields.In this study,phage display was used to construct Fab antibody libraries for E1G and PdG,respectively.Through 3 and 4 rounds of screening and enzyme linked immunosorbent assay(ELISA),anti-E1G Fabs and anti-PdG Fabs were obtained and validated.The sequences were also analyzed.The main results are as follows:1.Mice were immunized with antigens,E1G-BSA and PdG-BSA,and the change tendency of antibody titers was detected dynamically.The results showed that both antigens could give rise to producing antibodies with high titers.Antibodies titers reached peak at the 9th week.The maximum titer is 1:32000 to 1:64000 and1:16000 to 1:32000,respectively.Antibodies producing in mice lasted about 6 weeks.2.Spleens were taken from mice at the 9th week.Total RNA of splenocytes were extracted and the cDNA was synthesized by reverse transcription.After 3 rounds of gene splicing by overlap extension PCR,gene of Fab was obtained.The recombinant vector,pComb3X?-Fab,was constructed by digestion and ligation.Two Fab libraries with size of 1.13×10~7 and 1.03×10~6 were transformated by electrporation.The recombinant rates were about 78.57%and 92.31%,respectively,and the background ligation rates were both 0%.3.After 3 and 4 rounds of panning,enriched positive clones were randomly picked for soluble fragment ELISA and comparative ELISA.Some antibodies with high affinity to E1G and PdG were obtained,from which 9 clones with better affinity were selected for Sanger sequencing and IMGT/V-QUEST.It showed that antibodies against E1G or PdG had similar structural characteristics.Subsequently,two clones with good affinity,E1G-F6 and E1G-F8,were selected to test the cross-reactivity to three small molecule compounds similar to E1G.The results confirmed that the Fab was indeed able to recognize small molecular compounds with infinitesimal molecular differences.