免疫指纹图谱法研究双黄连注射剂中绿原酸的致敏性
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摘要
目的免疫指纹图谱法研究双黄连注射剂中绿原酸的致敏性,并建立高效、快速筛查中药注射剂过敏原的检测方法。方法通过人工合成绿原酸完全抗原并用紫外光谱对其鉴定,所得抗原免疫动物制备抗体血清并对其分离纯化,用酶联免疫法检测绿原酸是否呈阳性,再用免疫指纹图谱法研究双黄连注射剂中绿原酸的免疫原性。结果紫外光谱的结果显示绿原酸抗原合成成功;酶联免疫法的结果显示绿原酸组颜色明显较阴性组深而呈阳性反应;运用免疫指纹图谱法检测绿原酸在双黄连注射剂中的致敏性,发现用IgE、IgG吸附前后绿原酸指纹图谱特征峰发生显著性变化,其吸附率分别为44.55%、38.42%。结论用酶联免疫法初步锁定绿原酸为致敏原,而免疫指纹图谱法则进一步确定了绿原酸为双黄连注射剂中的致敏性成分,同时建立了高效、快速检测中药注射剂中致敏性成分的研究方法。
Objective To determine the sensitivity of chlorogenic acid in Shuanghuanglian injection by immune fingerprinting, and to establish an effective and rapid method to detect allergen in traditional Chinese medicine injection. Methods Complete antigen of chlorogenic acid was synthesized and identified by ultraviolet spectra. The antigens were immunized with animals to prepare antibody serum and purified. Enzyme-linked immunoassay was used to detect whether chlorogenic acid was positive or not. The immunogenicity of chlorogenic acid in Shuanghuanglian injection was studied by immun fingerprinting. Results Ultraviolet spectrum showed successful chlorogenic acid antigen synthesis. Enzyme-linked immunoassay showed that the chlorogenic acid group was darker than that of the negative group and showed a positive reaction. The sensitivity of chlorogenic acid in Shuanghuanglian injection was detected by immune fingerprinting, and significant changes were found in the characteristic peaks of chlorogenic acid fingerprinting before and after IgE and IgG absorption, with absorption rates of 44.55% and 38.42%, respectively. Conclusion Enzyme-linked immunoassay may preliminarily identify chlorogenic acid as the allergenic agent, and immune fingerprinting method may confirm the allergenic components in Shuanghuanglian injection. An efficient and rapid method for the detection of allergenic components in traditional Chinese medicine injection is established.
Objective To determine the sensitivity of chlorogenic acid in Shuanghuanglian injection by immune fingerprinting, and to establish an effective and rapid method to detect allergen in traditional Chinese medicine injection. Methods Complete antigen of chlorogenic acid was synthesized and identified by ultraviolet spectra. The antigens were immunized with animals to prepare antibody serum and purified. Enzyme-linked immunoassay was used to detect whether chlorogenic acid was positive or not. The immunogenicity of chlorogenic acid in Shuanghuanglian injection was studied by immun fingerprinting. Results Ultraviolet spectrum showed successful chlorogenic acid antigen synthesis. Enzyme-linked immunoassay showed that the chlorogenic acid group was darker than that of the negative group and showed a positive reaction. The sensitivity of chlorogenic acid in Shuanghuanglian injection was detected by immune fingerprinting, and significant changes were found in the characteristic peaks of chlorogenic acid fingerprinting before and after IgE and IgG absorption, with absorption rates of 44.55% and 38.42%, respectively. Conclusion Enzyme-linked immunoassay may preliminarily identify chlorogenic acid as the allergenic agent, and immune fingerprinting method may confirm the allergenic components in Shuanghuanglian injection. An efficient and rapid method for the detection of allergenic components in traditional Chinese medicine injection is established.
引文
[1]张雨恬,王韧,李文姣,等.中药注射剂(类)致敏成分检测技术进展[J].中南药学,2017,15(9):1185-1190.
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[10]林辉,刘屏,王建勋,等.绿原酸人工抗原的合成及反应条件的优化[J].中国中药杂志,2009,34(15):1906-1909.
[11]包小燕,贺福元,曾姣丽,等.芦丁完全抗原的合成鉴定及免疫原性分析[J].中国中药杂志,2013,38(3):397-401.
[12]Ferreira AM,Faustino VFM,Mondal D,et al. Improving the extraction and purification of immunoglobulin G by theuse of ionic liquids as adjuvants in aqueous biphasic systems[J]. J Biotechnol,2016,236:166-175.
[13]贺福元,邓凯文,曾姣丽,等.中药注射剂致敏成分群筛查方法研究的现状与免疫综合法[J].中国中药杂志,2012,37(19):2836-2841.
[14]邹莉,石园,刘高峰.中药注射剂的药物相互作用研究进展[J].中南药学,2016,14(11):1183-1187.
[2]刘爽,乔艳,刘高峰.中药注射剂不良反应与不合理用药问题分析[J].中南药学,2016,14(11):1177-1182.
[3]于烨,胡宗苗,邓颖颖,等.双黄连注射剂中黄芩苷致敏家兔后血清中特异性抗体的测定[J].世界中西医结合杂志,2017,12(3):345-348.
[4]董立财,王艳宏,吕邵娃,等.中药注射剂致敏物质筛选方法[J].中国实验方剂学杂志,2011,17(4):237-239.
[5]杨华,赵艳娇,耿凤英,等. 838例中药注射剂不良反应分析[J].实用药物与临床,2007,10(4):228-229.
[6]曾姣丽,贺福元,唐昱,等.双黄连注射剂中黄芩苷致敏原性的研究[J].中国实验方剂学杂志,2012,18(1):102-106.
[7]赵华.双黄连注射剂的不良反应及其影响因素研究[J].中国卫生产业,2013,10(36):103-104.
[8]张信岳,陈爱君,李钦,等.绿原酸主动全身过敏与被动皮肤过敏的实验研究[J].中国中医药科技,2011,18(2):127-128.
[9]胡宗苗,张恩户,刘继平,等.双黄连注射剂中绿原酸致敏家兔后血清中特异性抗体的测定[J].中药药理与临床,2016,32(4):70-73.
[10]林辉,刘屏,王建勋,等.绿原酸人工抗原的合成及反应条件的优化[J].中国中药杂志,2009,34(15):1906-1909.
[11]包小燕,贺福元,曾姣丽,等.芦丁完全抗原的合成鉴定及免疫原性分析[J].中国中药杂志,2013,38(3):397-401.
[12]Ferreira AM,Faustino VFM,Mondal D,et al. Improving the extraction and purification of immunoglobulin G by theuse of ionic liquids as adjuvants in aqueous biphasic systems[J]. J Biotechnol,2016,236:166-175.
[13]贺福元,邓凯文,曾姣丽,等.中药注射剂致敏成分群筛查方法研究的现状与免疫综合法[J].中国中药杂志,2012,37(19):2836-2841.
[14]邹莉,石园,刘高峰.中药注射剂的药物相互作用研究进展[J].中南药学,2016,14(11):1183-1187.