Preparation And Clinical Significance Of Monoclonal Antibody Immunotoxin Against Endotoxin Lipopolysaccharide Rceptors CD14

Research background:Septic shock is the main reason of death in pediatric intensive care unit,it mainly includes microcirculation disturbance caused by toxins of Microorganism and multi-organs function failure due to severe disease.Septic shock is very troublesome in clinic treatment,and it's pathogenesy is not yet identified.People explained the pathogenesy of shock by micro-circulation theory in past.They think that shock is a syndrome of acute micro-circulation disturbance.Shock will cause organ's blood hypoperfusion and cell's functional disorder.Some scholars suggest the theory of inflammation out of control for the past few years.They think that septic shock is organism response to infectivity factor and routine anti-infection can not contain this processes.There are a great quantity of cytokines in the development of septic shock.They interact to format a lot of positive feedback loop,and induce "water fall effect".CD14,the receptor of LPS,plays capital role in pathologic response of septic shock.It recognizes and combines with LPS or LPS/LBP compound,thus mediates cell response inducded by LPS.Endotoxin is one of these main mediator that can induce shock and organ failure.Some abroad studies indicated that blocking endotoxic generation or inhibiting it's activity can alleviat a series of reaction of organism,and decrease fatality rate of this disease.We carried out the study on immunotoxin against CD14 due to less study on this field in domestic now.Objective:This study plans to crosslink 2F9 and Genistein in vitro by Sulfo-SANPAH,and completes preparation of 2F9-Gen.To study 2F9-Gen's targetic lethal effect to mononuclear cell in vitro and provide a mediator treatment tool for septic shock possibly.Material and methods:1.Identificat the subtype of 2F9To detIect the subtype of 2F9 with flow cytometry by fluorescent labeling.2.Preparation and purify and identification of 2F9 ascitesTo prepare ascites with routine methods,and to purify 2F9.To analyses the molecular weight of 2F9's heavy chain and light chain after SDS-PAGE,and to estimate antibodie's purity.3.Preparation of 2F9-FITCTo mark pure 2F9 with FITC by improvement Marsshall method,and to remove redundant FITC by dialysis.To determine it's OD495 and OD280 with ultraviolet spectrophotometer.4.Blockade test of 2F9 antibodyTo observe the blocking result of 2F9 to 2F9-FITC and standard CD14-FITC,1ml supernatant of 2F9 hybridoma cell was used during the experiment the through FCM.5.Preparation of 2F9-GenTo crosslink 2F9 and Genistein in vitro by Sulfo-SANPAH. 6.2F9-Gen's targetic lethal effect to mononuclear cell in vitro.To obtain appropriate amount peripheral blood of health child,and dissociate lymphocyte,and add in PBS,2F9,2F9-Gen,Gen,and eventually use Annexin VFITC apoptosis kit to detect after cultivated 24 hours.Result:1.Identification of the subtype of 2F9The heavy chain subtype of 2F9 is IgG1,the light chain belongs toκ.2F9 antibody subtype is mouse IgG1κ.2.Preparation and purification of 2F9 ascitesHybridprotein was separated from 2F9 antibody by chromatographic analytical column.Through SDS-PAGE gel electrophoresis,a heavy chain(molecular weight 57.92KDa) and a light chain(30.29KDa) were detected.The purified 2F9 was obtained.3.Preparation of 2F9-FITC and blocking test of 2F9 antibody2F9-FITC was prepared successfully by improved Marsshall method with the same Specificity and sensitivity compared with standard CD14-FITC by FCM.4.Detection of the concentration of 2F9-SANPAHThe concentration of 2F9-SANPAH is 0.35mg/ml(0.0019886mmol/L).5.Preparation of 2F9-GemThe concentration of 2F9-Gen is 0.15mg/ml(0.0008571mmol/L).6.2F9-Gen's targetic lethal effect to mononuclear cell in vitro2F9-Gen and Gen both have lethal effect to mononuclear cell in vitro,but 2F9-Gen has better effect than Gen.Conclusions:1.Success to prepare and purify 2F9 antibody.2.Success to prepare and purify 2F9-FITC. 3.Success to prepare the immunotoxin(2F9-Gen).4.2F9-Gen has good effect of killing monocytes in vitro,and provide a new way of antimediator therapy to septic shock possibly,but the mechanism needs further research.