Development Of Quantitative Microparticle Immunoassay For Human Serum Ferritin Based On Full-automatic Chemiluminescence Immune Analyzer

Iron deficiency anemia (IDA) is a type of anemia caused by not having enough storage iron in body because of various reasons. Iron deficiency anemia is one of the most common nutrition deficiency diseases, affecting about1billion people in the world.The progress from iron deficiency to iron deficiency anemia is a slow process, which can divided into three stages:iron deplete(ID), iron deficiency erythropoiesis(IDE), iron deficiency anemia (IDA). Iron deficiency and iron deficiency anemia will not only affect the normal function of the body, and may cause irreversible effects on mental development of fetal and children. Serum ferritin level is associated with body storage iron, which makes serum ferritin as a sensitive and specific biomarker for evaluation storage iron in body and diagnosing iron deficiency anemia in clinical practice. Inspection of serum has important meaning for realizing iron deficiency as early as possible and preventing iron deficiency anemia. The purpose of this research is to establish a human serum ferritin chemiluminescense immunoquantification kit suitable for full-automatic tubular chemiluminescence immune detection system.In this study, E.coli expression system is used to express human ferritin H,L subunit, after purified by heat treating,gel filteration chromatography, ainon exchange (DEAE) chromatography, high purity of rhFTHl,rhFTL was acquired. The purified rhFTHl,rhFTL assemble into24mer nanoparticles similar to native ferritin, which is verified by SDS-PAGE, molecular sieve chromatography, transmission electron microscopy (TEM) analysis.The purified rhFTHl,rhFTL nanoparticles were used to immunize mice, the result indicted the antigenicity is good. Using rhFTL and native liver ferritin as screen antigen to screen hybridoma,22monoclonal antibody cell lines were acquired.16IgG type monoclonal antibody was characterized, among which12monoclonal antibodies have a titer over1million. The12monoclonal antibodies which have good reactivity were selected to screen antibody pair by ferritin standard protein and specimen. After preliminary screening, two antibody pairs were obtained to establish human serum ferritin chemiluminescense immunoquantification kit. Further analysis such as detection sensitivity,detection range and the correlation of testing specimens with contrast reagent was carried out. The result showed that the detection sensitivity of the human serum ferritin quantification kit established based on antibody pair(7B4-7C10) was0.5ng/ml, the detection range was1-2000ng/ml, and the correlation coefficient r2of testing specimens with reagent from Beckman company was over0.95.In summary,recombinant human ferritin antigens and human ferritin specific antibodies were obtained,based on that quantitative microparticle chemiluminescense immunoassay for human serum ferritin was established.The performance of this immunoassay is comparative with imported serum ferritin chemiluminescense immunoquantification kit,which means the quantitative microparticle chemiluminescense immunoassay for human serum ferritin established in this study reached the level of international mainstream reagent.