间接酶联免疫吸附法测定血液中抗西尼罗河病毒单克隆抗体MIL94浓度及其应用

Determination of MIL94,a monoclonal antibody against West Nile virus,in cynomolgus monkeys serum and its application by indirect enzyme-linked immunosorbent assay

目的建立食蟹猴血清中抗西尼罗河病毒单抗MIL94的间接酶联免疫吸附(ELISA)定量测定法,并进行食蟹猴体内药动学初步研究。方法抗原包被量为每孔100 ng,酪蛋白封闭液于37℃孵育1 h,血清样品用PBS进行前处理,二抗按1∶10000稀释,3,3′,5,5′-四甲基联苯胺溶液室温显色10 min,硫酸终止反应。12只食蟹猴随机分为2组,每组6只,分别30 min内静脉推注MIL945和100 mg·kg^(-1),不同时间点(1 h~56 d)采血,分离血清,ELISA法测定血清中MIL94浓度。WinNonlin软件计算药动学参数,分析药动学特征。结果该方法的线性范围为0.25~32 mg·L^(-1),板内精密度1.18%~4.62%,板间精密度8.19%~10.20%。食蟹猴静注MIL945和100 mg·kg^(-1),半衰期分别为8±5和(8±5)d,清除率(Cl)分别为0.25±0.08和(0.26±0.009)mL·h^(-1)·kg^(-1),稳态表观分布容积分别为55±21和(74±19)mL·kg^(-1),药时曲线下面积(AUC)分别为951.2±393和(18030±6139)d·mg·L^(-1),AUC与剂量成正比,Cl不随剂量改变而改变。结论建立的间接酶联免疫吸附法的灵敏度、特异性、线性范围、精密度和稳定性等均满足临床前药动学研究需求。初步获得了MIL94在5~100 mg·kg^(-1)的静注剂量范围内,食蟹猴体内的线性药动学特征,为MIL94的研发提供支持。

OBJECTIVE To establish an indirect enzyme-linked immunosorbent assay(ELISA)for the determination of MIL94,a novel monoclonal antibody against West Nile virus(WNV),in the serum of cynomolgus monkeys and to study its pharmacokinetics in monkeys.METHODS The amount of antigen coating was 100 ng/well,the casein blocking solution was incubated at 37℃for 1 h,the serum samples were pretreated with PBS,and the secondary antibody was diluted at the rate of 1∶10000.TMB solution was used for color development for 10 min,and the reaction was terminated by sulfuric acid.Each group of monkeys was administered with MIL94 via iv injection within 30 min at dosage of 5 and 100 mg·kg^(-1),respectively.Blood samples were collected at different time points after administration.The sera were obtained and the concentrations of MIL94 were determined using ELISA method.The pharmacokinetics parameters area under curve(AUC),clearance(Cl),steady state apparent distribution volume(Vss),half time(t1/2)and mean residence time(MRT)of MIL94 were analyzed using WinNonlin software.RESULTS The linear range of the method was 0.25-32 mg·L^(-1).The precision of the intra-and inter-assay was within 1.18%-4.62%and 8.19%-10.20%,respectively.After iv injection at the dosage of 5 and 100 mg·kg^(-1),the terminal t1/2 of MIL94 in cynomolgus monkeys was 8±5 and(8±5)d,Cl was 0.25±0.08 and(0.26±0.09)mL·h^(-1)·kg^(-1),Vss was 55±21 and(74±19)mL·kg^(-1),AUC was 952±393 and(18036±6139)d·mg·L^(-1),respectively.The AUCs were proportional toward the two doses.The plasma clearances of MIL94 did not change with the dose.CONCLUSION An indirect enzyme-linked immunosorbent assay is established for the determination of MIL94 in monkey serum,which can meet the requirements of preclinical pharmacokinetics research in terms of sensitivity,specificity,linear range,precision and stability.The linear pharmacokinetic characteristics of MIL94 in cynomolgus monkeys in the dose range of(5-100)mg·kg^(-1) are identified,which can contribute to the development of MIL94.