The Preparation Of Antibody Against Neutrophil Gelatinase-associated Lipocalin (NGAL) And Initial Establishment Of Immunoassay For Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Neutrophil gelatinase-associated lipocalin(NGAL) is one member of lipocalin, a25KD protein. In the environment of inflammation and carcinoma, elevated NGAL expression can be detected in many organs such as Gastrointestinal epithelial tissue, the uterus, kidney, lung, large intestine, stomach, salivary gland. In the normal organs such as kidney, lung, large intestine, stomach,only weak NGAL expression can be identified, but these organs were injured, elevated NGAL expression can be detected. The molecular weight of NGAL is small and can be easily detected in the urine. In recent years, a lot of studies have showed that NGAL was a promising biomarker which is highly sensitive and specific. It has been shown to be a novel AKI assessment index with more specificity and sensitivity than traditional index of serum urea, uric acid and creatinine, as well as endogenous creatinine clearance rate(Ccr). Most of the monoclonal antibody for NGAL in domestic experiments is imported and and has poor specificity. The factor mentioned above restricted the studies about NGAL in our clinical work.Objective:To prepare monoclonal antibodies against NGAL. To establish hybridoma cell lines which secrete monoclonal lines against NGAL for the initial establishment of a double antibody sandwich ELSIA for neutrophil gelatinase-associated lipocalin(NGAL)Methods: Mice spleen cells and myeloma cells (SP2/0) were fused after Balb/c mice were immunized with the recombinant protein. By the way of cloning and screening, the hybridoma cell lines were established, with which large-scale monoclonal antibodies produced in mice ascites was prepared. The property of monoclonal antibodies was identified by ELSIA and Western Blot. SDS-PAGE identified the purity of monoclonal antibodies. The reaction conditions were preliminary explored by antibodies matching test after labeling antibodies with HRP.Results:1. which could stably secret specific McAb against NGAL, after these hybridoma cell lines injecting into mice abdominal cavity, the ascites abundant in McAb were obtained.2. SDS-PAGE analysis showed that the antibodies had high purity3. The monoclonal antibodies we obtained were successfully matchedConclusions:1. The monoclonal antibody against human NGAL is prepared with recombinant protein.2. A double antibody sandwich ELSIA For Neutrophil gelatinase-associated lipocalin (NGAL) was established initally...