抗吗啡疫苗抗体的制备及其特性鉴定

Preparation and characterization of anti-morphine vaccine antibody

目的制备高效价的抗吗啡疫苗的抗体,并对其特性进行鉴定。方法将人工合成的6-琥珀酰吗啡(M-6-S)与BSA在pH9.0碳酸盐缓冲液中交联成M-6-S-BSA。经凝胶柱过滤和硫酸铵法纯化后,用两种不同方案免疫BALB/c小鼠和SD大鼠,用ELISA法和中和抑制试验分别测定抗体的滴度和进行特异性鉴定;用辐射热甩尾(TF)反射试验检测M-6-S-BSA疫苗的免疫效果。结果用合成的M-6-S-BSA疫苗连续免疫小鼠8次,所获抗血清的效价可达1∶200000,大鼠免疫4次后抗血清的效价超过1∶20000。中和抑制试验表明,抗M-6-S抗体对吗啡和与吗啡有共同母环结构的6-单乙酰吗啡、海洛因及可待因具有较高的特异性。免疫大鼠腹腔注射2mg/kg的吗啡后,其甩尾(TF)反射受到明显抑制,最大效应百分率(%MPE)减少了74.7%,TF潜伏期回到基线水平的时间明显快于对照组。结论成功地制备M-6-S-BSA疫苗,用其免疫小鼠和大鼠均可产生高滴度和特异性抗吗啡抗体。该抗体可明显减低大鼠对吗啡的抗伤害感受,为进一步研究吗啡疫苗抗体对抗吗啡的成瘾作用奠定了基础。

AIM： To prepare anti-morphine vaccine antibody with high titer and good specificity and to identity its properties. METHODS： Chemically synthesized 6-succinyl-morphine （M-6-S） was cross-linked to BSA in pH 9.0 carbonate buffer. After being purified by gel filtration and （NH4）2SO4 salting out, the immunogen was used to immunize BALB/c mice and SD rats. The titer and specificity of antisera were identified by ELISA and neutralization inhibition test, respectively. The effect of the immunization was examined by the radiant heat tail-flick （TF） reflex test. RESULTS： The titers of antiserum from immunized BALB/c mice and SD rats were up to 1： 200 000 and over 1 ： 20 000, respectively. Neutralization inhibition test proved that the anti-morphine vaccine antibody had the cross-reactions to following drugs with analogous structure as morphine： 6- monoacetylmorphine, heroin and codeine. The TF reflex test showed： （ 1 ） TF reflexes were notable inhibited after intraperitoneal injection of 2 mg/kg morphine, and （2） percentage of the maximum possible effect （ % MPE） reduced 74.7%, （3） the TF latency recovered to the baseline level much more rapidly compared with the control rats. CONCLUSION： The M-6-S-BSA vaccine has been prepared successfully. The BALB/c mice and SD rats immunized with the vaccine can produce anti-morphine antibody with high titer and satisfactory specificity, which may significantly reduce rats＇ antinociception against morphine.