Preparation Of Human FGF23 Monoclonal Antibody And Establishment Of A Double-antibody Sandwich ELISA Method

Chronic kidney disease（CKD）has now become a global public health problem,characterized by high morbidity,high medical costs and high mortality,which seriously threaten human health and quality of life.Because of the insidious manifestation of the disease in the early stage,it is difficult to be detected and it gradually progresses to renal failure.Therefore,the early diagnosis and prevention of chronic kidney disease is an urgent problem that needs to be solved globally.At present,blood creatinine is often used as an index to evaluate renal function in clinical practice,but it lacks high predictive value and cannot accurately and timely monitor the occurrence of CKD.The reason is probably due to the stronger compensatory effect of the kidneys.Therefore,when the kidney is slightly damaged,the blood creatinine cannot be changed in time.Studies have shown that when the blood creatinine changes significantly,the kidney parenchyma has been damaged by 40%to 50%.Therefore,to improve the early diagnosis rate of CKD,the emergence of new kidney injury markers is needed.Human fibroblast growth factor 23（hFGF23）is a bone-derived hormone that mainly acts on its target organ,the kidney.As a new biomolecular marker for early diagnosis of renal damage,hFGF23 has a large number of clinical basic research support.Compared with the existing clinical markers of blood creatinine,hFGF23 can detect the occurrence of CKD earlier without requiring simultaneous combination Comprehensive judgments such as B-ultrasound imaging examinations require only separate measurement.Therefore,hFGF23 can be used as a serological detection method for early diagnosis of CKD.At the same time,the effectiveness of CKD treatment can be monitored by detecting the levels of hFGF23 in plasma before and after treatment.This study hopes to be able to prepare an anti-hFGF23 monoclonal antibody with strong specificity and high sensitivity,which will lay the foundation for the establishment of a new detection method for chronic kidney disease.This study optimized the induced expression conditions of the successfully constructed recombinant human p ET28a-hFGF23 engineering bacteria,and determined the optimal expression conditions of hFGF23 protein.The hFGF23 protein was separated and purified by Ni-NTA affinity chromatography.Use the blood phosphorus reabsorption test to detect the biological activity of the protein.Mice were immunized with hFGF23 protein,and anti-hFGF23monoclonal antibodies were prepared by cell electrofusion.The properties of the antibodies were identified by indirect enzyme-linked immunosorbent assay,western blotting and surface plasmon resonance technology.Monoclonal antibodies are labeled with horseradish peroxidase to detect enzyme-labeled antibody titer by indirect ELISA,and pair them with unlabeled monoclonal antibodies to screen out the best antibody pairing combination,and initially establish a sandwich ELISA detection method.The main experimental results are as follows:（1）It was determined that the best induction time of recombinant human p ET28a-hFGF23engineering bacteria was when the bacterial solution OD₆₀₀=0.8,and the final concentration of IPTG was 1.0 m M at 37°C for 4 hours;The target protein was found to exist in the form of inclusion bodies by SDS-PAGE electrophoresis analysis.After the protein was denatured and renatured,it was separated and purified by Ni-NTA affinity chromatography column to obtain hFGF23 protein with a purity of more than 90%;（2）After cell electrofusion,8 hybridoma cell lines that can stably secrete anti-hFGF23antibodies were obtained,and they were named 1F11,4H12,6H6,7C8,7G3,8D10,12H3 and13D3;The antibody titer can reach up to 1×10⁶;The antibody subclass identification results are:the three antibody subclasses of 1F11,7C8 and 8D10 are Ig G1 type;the 7G3 and 13D3 antibody subclasses are of Ig G2a type;the antibody subclass of 4H12 is Ig G2b.The dissociation constants of antibodies 1F11,4H12,7C8,7G3,8D10 and 13D3 determined by SPR experiment were 26.9 n M,120 n M,220 n M,68 n M,58 n M and 185 n M respectively;（3）Using modified sodium periodate method to label monoclonal antibodies 1F11,4H12and 7G3,two pairs of combinations were screened through antibody pair screening:1F11 and7G3,4H12 and 7G3,Among them,the best matching antibody pair combination is 1F11 and7G3;The best working concentration of capture antibody is 2μg/m L,and the best working concentration of detection antibody 7G3 is 1:1600;the best incubation time for antigen and detection antibody is 1 h.Using this method to detect standard hFGF23 protein,the lowest detectable concentration was 17.4 ng/m L.Specific experiments show that the detection method only reacts with hFGF23,and does not cross-react withFGF19 and FGF21.The precision was evaluated by intra-assay variation and inter-assay variation,and the coefficient of variation was less than 15%.