OBJECTIVE: To establish a double antibody sandwich enzyme-linked immunoadsorbent assay (ELISA) method for measuring the concentration of phospholipid-binding anticoagulation protein (PBAP) from Agkistrodon halys brevicaudus venom in rabbit serum. Compartment model was described and pharmacokinetic parameters were calculated according to the variation of serum concentrations, which provided experimental foundation for clinical research. METHODS: Rabbits were randomly divided into low-dose and high-dose groups (PBAP from Agkistrodon halys brevicaudus venom, 0.4 mg·kg-1, 0.8 mg·kg-1) and negative control group (normal saline) with each group of five rabbits. Those groups were given relevant drugs via ear vein. Blood samples were collected from common carotid artery before administration and 1, 5, 15, 30, 60, 120, 240, 480 min after admistration.Blood samples were added into polyclonal antibodies elisa plate of PBAP from Agkistrodon halys brevicaudus venom with concentration of 100 μg·mL-1 using normal serum before administration as blank control. Serum concentrations of PBAP from Agkistrodon halys brevicaudus venom at different time points were detected by double antibody sandwich ELISA method. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS: The linear range of PBAP was 0.002 4～10 μg·mL-1 (r=0.970 3)with an average recovery of 98.78％. The average intra-assay and inter-assay coefficient of variance (CV) were 7.96％ and 10.94％. The main pharmacokinetic parameters of high-dose group vs. low-close group were as follows: t1/2α: (9.553 ± 4.668) min vs. (9.873 ± 1.433) min; t1/2β: (201.295 ± 66.060) min vs. (205.798 ± 76.834) min; Cmax: (0.685 ± 0.160) mg·L-1 vs. (2.333 ±0.478) mg·L-1; AUC0～∞: (27.114 ± 2.781) mg·min· L-1 and (61.625 ± 11.631) mg·min· L-1. CONCLUSION: The double antibody sandwich ELISA is accurate, sensitive and specific. It is suitable for pharmacokinetic study of PBAP in rabbits.%目的:建立测定家兔血清中短尾蝮蛇毒磷脂结合抗凝蛋白(PBAP)浓度的方法,并考察其药动学特征.方法:取家兔随机分为低、高剂量组(短尾蝮蛇毒PBAP,0.4,0.8 mg·kg(-1))和阴性对照组(生理盐水),每组5只,耳缘静脉单次给予相应药物,给药前及给药后1,5,15,30,60,120,240,480 min分别从颈总动脉取血,加入到短尾蝮蛇毒PBAP抗体包被酶标板(浓度为100 μg·mL(-1))中,以给药前的正常血清作为空白对照,用双抗体夹心-酶联免疫吸附(ELISA)法测定不同时间点血清中短尾蝮蛇毒PBAP的浓度,并用DAS 2.0软件计算其药动学参数.结果:短尾蝮蛇毒PBAP检测浓度的线性范围为0.002 4～10 μ·mL(-1)(r=0.970 3),平均回收率为98.78%,平均板内变异系数为7.96%,平均板间变异系数为10.94%;低、高剂量短尾蝮蛇毒PBAP的主要药动学参数分别为t1/2α(9.553±4.668),(9.873±1.433) min,t1/2α(201.295±66.060)、(205.798±76.834) min,cmax (0.685± 0.160),(2.333±0.478) mg·L(-1),AUC0～8(27.114±1 2.781),(61.625 ±11.631) mg·min· L(-1).结论:ELISA法准确、灵敏、特异性强,可用于短尾蝮蛇毒PBAP在家兔血清中的药动学研究.
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