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目的:制备河豚毒素(TTX)的小鼠多克隆抗体,研究抗体中和TTX的效果,以探讨开发TTX抗素素的可能。方法:以中国鲎血蓝蛋白(TTH)为载体,通过甲醛与TTX化学连接,制成人工抗原(TTXTTH)并免疫BALB/c小鼠;以福氏佐剂诱导腹水抗体。酶联免疫分析方法检测血清和腹水抗体质量;将TTX与抗体在体外共温育而被中和,然后腹腔注射于KM小鼠,作体内攻毒实验,检验抗体的抗毒活性,并计算抗体的免疫当量。结果:经人工抗原免疫并福氏佐剂诱导,获得了20株具有不同亲和力的腹水抗体,与血清抗体具有同质性,其表观亲和力为10-4～10-7M。抗体可在体外和体内、完全或部分地中和毒素的毒性,有效保护中毒动物免于死亡。抗体体外中和毒素,最高可保护1.5×LD的TTX攻击,动物全部活存(1LD=14μg/kp,i.p.);抗体的最高免疫当量达1300μgTTX/L腹水;抗体的抗毒活性与抗体滴度、亲和力和质量因子显著相关(分别为P<0.05,P<0.01,P<0.001)。抗体在体内预防4d时,可保护1.3×LD的TTX攻击动物活存。结论:TTX小鼠腹水多克隆抗体可在体外和体内有效中和毒素,其抗毒活性依赖于抗体质量,提示了免疫防治对抗河豚毒素中毒的希望。研究提出的抗体“质量因子”是评价抗毒素质量的有效参数。
OBJECTIVE: To prepare mouse polyclonal antibodies against tetrodotoxin (TTX) and to study the effect of neutralizing TTX on the antibody to explore the possibility of developing TTX antibiotics. Methods: The human antigens (TTXTTH) were immunized with BALB / c mice by using TTH as the carrier, and formaldehyde and TTX were chemically linked to each other. The ascites was induced by Freund ’s adjuvant. Enzyme-linked immunosorbent assay (ELISA) was used to detect the antibody quality in serum and ascites fluid. TTX and antibody were co-incubated in vitro and neutralized, then injected into KM mice intraperitoneally for in vivo challenge experiment to test the antitoxic activity of the antibody and to calculate the antibody Of immune equivalent. Results: After induced by artificial antigen and induced by Freund ’s adjuvant, 20 ascites antibodies with different affinities were obtained, which were homogeneous with the serum antibodies and their apparent affinities were 10-4-10-7M. Antibodies can completely and partially neutralize toxin toxicity in vitro and in vivo, effectively protecting the poisoned animal from death. Antibodies neutralize toxins in vitro and protect up to 1.5 × LD of TTX challenge. All animals survived (1LD = 14μg / kp, ip). The highest antibody equivalent of ascites was 1300μgTTX / L ascites. The antitoxic activity and antibody titer , Affinity and quality factors were significantly correlated (P <0.05, P <0.01, P <0.001, respectively). The antibody protects the live viability of TTX challenge animals at 1.3 x LD at 4 days of in vivo prophylaxis. CONCLUSION: The polyclonal antibody against ascites in TTX mice can neutralize toxins effectively in vitro and in vivo, and its antitoxic activity depends on the antibody quality, suggesting the hope of preventing and treating tetrodotoxin poisoning. The proposed antibody “quality factor” is an effective parameter to evaluate the antitoxin quality.