脱氧雪腐镰刀菌烯醇抗原设计及直接竞争酶联免疫方法
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摘要
脱氧雪腐镰刀菌烯醇(DON)半抗原的分子设计是建立其免疫分析方法的关键步骤,目前,DON半抗原设计仍采用经验法,造成时间和资金的浪费。本文从半抗原设计的最大原则(不改变待分析物的结构性质)出发,明确DON最佳半抗原的设计思想:最大化抗原表位相似度。利用分子模拟技术,通过分析分子的空间构型、电子特性、疏水性质这几个影响抗原-抗体间作用力的因素在DON修饰前后的变化,研究半抗原结构与免疫识别之间的关系,建立DON半抗原设计的理论模拟方法,为小分子半抗原设计提供新的思路,并在此基础上建立了DON的直接竞争酶联免疫分析方法。
根据DON抗原设计流程:选择结合位点→引入连接臂→偶联载体蛋白,首先研究结合位点对DON免疫识别的影响,在DON分子中的4个潜在结合位点(3 ,7 ,15位羟基以及8位酮基)引入相同碳链长度的的连接臂,借助分子模拟软件Hyperchem7.5对DON及半抗原的表位决定参数(空间结构、原子点电荷、疏水常数)进行计算分析,3位半抗原具有与DON最相似的结构性质,表明3位是DON引入连接臂的最佳位点,这与Casale和Ramesh的免疫实验结果一致。
其次,在3位羟基上设计不同的连接臂结构,通过抗原表位相似度模拟筛选得到最佳连接臂结构:丁二酸酐臂。同时合成不同连接臂的DON完全抗原,并将之进行免疫抗体实验,测定抗体的效价以及抑制率,验证分子模拟结果,研究半抗原结构对抗体免疫活性的关系。结果表明B组(顺丁烯二酸酐臂)抗体平均效价:256000,A组(丁二酸酐臂)、C组(邻苯二甲酸酐臂)平均效价:64000; DON浓度为500 ng/ml时对抗体的抑制率分别:A组(66.4 %)>B组(21.1 %)>C组(10.8 %)。抑制率的大小与抗体对DON的特异性相关,A组与DON具有最高的抗原表位相似度,抗体对DON的特异性强,抑制率高;抗体的效价可以反映半抗原分子的抗原性,苯环结构、不饱和键可以增强抗原性,B组连接臂中含有不饱和键,增强了半抗原的抗原性,产生的抗体效价高;C组虽含有苯环结构,确未能产生高效价的抗体,结合其空间结构发现C组半抗原中羧基端与半抗原主体结构非常靠近,半抗原结构可能被载体蛋白屏蔽而影响免疫识别;综合考虑,丁二酸酐臂是DON的最佳连接臂,免疫试验结果与分子模拟具有很好的相关性。
在最佳半抗原基础上,比较BSA和KLH两种载体蛋白的DON完全抗原的免疫原性。通过丁二酸酐衍生法和碳二亚胺法(EDC法)制备不同载体蛋白的DON完全抗原,免疫新西兰长耳兔获得DON抗体,并运用酶联免疫方法(ELISA)对制备的抗体进行研究。
在上述研究结果基础上合成DON-KLH完全抗原,建立DON的直接竞争酶联免疫分析方法,通过一步抗原抗体反应,快速检测食品中DON。检测范围为1 -100 ng/mL。半数抑制率IC50为10 ng/mL,最低检出限为0.56 ng/mL,与T-2毒素等真菌毒素的交叉反应率小于12 %;平均批内变异系数为2.82 %,平均批间变异系数为14.54 %;对玉米粉的加标回收率在80.2-91.1 %,准确性较高;与商品化试剂盒相比具有所需时间短,精密度高,灵敏度高的优点。
本研究表明分子模拟方法对DON半抗原设计有积极作用,指导半抗原设计,减少半抗原设计中时间及资金的投入。不同载体DON免疫抗原的免疫原性不同,DON-KLH免疫原性优于DON-BSA。6本研究所建立的DON的直接竞争ELISA方法,快速、方便,具有较好的实用价值,为进一步研究DON的ELISA检测试剂盒提供了重要的实验依据。
DON hapten design is the key step to establish the immune analysis methods. Currently, DON hapten design still adopt experience, causing the waste of time and money. From half antigen design principle, clear DON best hapten design thought: maximization similarity.of antigen sites.
Study the changes of molecular space configuration, electronic characteristics, hydrophobic property that several influence the antigen-antibody intercropping force factors , before and after DON modified. Research the relationship between hapten structure and immune discrimination. Establish DON hapten theory simulation design method. Provide a new thinking for hapten design. And on this basis, build a DON direct competition enzyme-linked immunosorbent analysis method.
According to the design process: choose binding sites→introduced connection arm→coupling carrier protein. Firstly, study the effect of binding sites to immune discrimination, introduced the same length of carbon chain of connection arm in the four potential binding sites (3, 7, 15-hydroxyl and 8-ketone base),Using molecular simulation software Hyperchem7.5 analysis determines parameters (spatial structure, atomic stereograph.furthermore, hydrophobic constant), 3-hapten is most similar to DON. Show 3-OH is the best DON binding site. Consistent to immune experiment results of Casale and Ramesh. Secondly, designed different connection arm structure in 3-OH, through similarity simulation received the best connection arm: succinic anhydride arm.
Choose 3 DON hapten with different arms structure to do immune antibody test, determination of antibody titer and inhibition rate to verify the molecular simulation results, study relationship of hapten structure and antibody immunity activity.
Group B antibody titer: 256000; Group A, C average titer: 64000. DON concentration for 500 ng/ml of group A antibodies suppression rate respectively: 66.4%, group B: 21.1%, group C: 10.8%. Inhibition rate of antibodies related to the specificity of DON, Group A has the highest similarity with DON, the specificity of strong, DON inhibition rate high, Antibody titer can reflect the carp of hapten. the benzene rings, unsaturated double bond can enhance the carp. Group B containing unsaturated double bond, enhanced the carp, produce high titer antibody, Although group C contains benzene structure, but failed to produce high titer antibody, combined with its spatial structure found: group C carboxyl end is very close to the main structure, may be shielded by carrier protein and affect immune discrimination. Comprehensive consideration, succinic anhydride is the best connected arm to DON .
In basis of the best hapten, compare the immunogenicity effect of vector protein(BSA、KLH).Preparation completely antigen of different carrier protein, to obtain DON antibody. Study antibody using enzyme-linked immunosorbent method (ELISA) and round two color spectrum (CD).
Established DON direct competition enzyme-linked immunosorbent analysis method. Examination scope for 1~100 ng/ml, half the inhibition rate IC50 for 10 ng/mL, minimum detect limit get 0.56 ng/ml, cross reaction rate less than 12 %, average batch coefficient of variation within 2.82 %,average batch coefficient of variation between 14.54 %;the recovery in 80.2-91.1 %.
This study shows that the molecular simulation method has the positive role to DON hapten design, guiding half antigen design, reduce time and capital investment. DON-KLH immunogenicity is superior DON–BSA. Established DON direct competition ELISA method, quick, convenient, and has good practical value.
根据DON抗原设计流程:选择结合位点→引入连接臂→偶联载体蛋白,首先研究结合位点对DON免疫识别的影响,在DON分子中的4个潜在结合位点(3 ,7 ,15位羟基以及8位酮基)引入相同碳链长度的的连接臂,借助分子模拟软件Hyperchem7.5对DON及半抗原的表位决定参数(空间结构、原子点电荷、疏水常数)进行计算分析,3位半抗原具有与DON最相似的结构性质,表明3位是DON引入连接臂的最佳位点,这与Casale和Ramesh的免疫实验结果一致。
其次,在3位羟基上设计不同的连接臂结构,通过抗原表位相似度模拟筛选得到最佳连接臂结构:丁二酸酐臂。同时合成不同连接臂的DON完全抗原,并将之进行免疫抗体实验,测定抗体的效价以及抑制率,验证分子模拟结果,研究半抗原结构对抗体免疫活性的关系。结果表明B组(顺丁烯二酸酐臂)抗体平均效价:256000,A组(丁二酸酐臂)、C组(邻苯二甲酸酐臂)平均效价:64000; DON浓度为500 ng/ml时对抗体的抑制率分别:A组(66.4 %)>B组(21.1 %)>C组(10.8 %)。抑制率的大小与抗体对DON的特异性相关,A组与DON具有最高的抗原表位相似度,抗体对DON的特异性强,抑制率高;抗体的效价可以反映半抗原分子的抗原性,苯环结构、不饱和键可以增强抗原性,B组连接臂中含有不饱和键,增强了半抗原的抗原性,产生的抗体效价高;C组虽含有苯环结构,确未能产生高效价的抗体,结合其空间结构发现C组半抗原中羧基端与半抗原主体结构非常靠近,半抗原结构可能被载体蛋白屏蔽而影响免疫识别;综合考虑,丁二酸酐臂是DON的最佳连接臂,免疫试验结果与分子模拟具有很好的相关性。
在最佳半抗原基础上,比较BSA和KLH两种载体蛋白的DON完全抗原的免疫原性。通过丁二酸酐衍生法和碳二亚胺法(EDC法)制备不同载体蛋白的DON完全抗原,免疫新西兰长耳兔获得DON抗体,并运用酶联免疫方法(ELISA)对制备的抗体进行研究。
在上述研究结果基础上合成DON-KLH完全抗原,建立DON的直接竞争酶联免疫分析方法,通过一步抗原抗体反应,快速检测食品中DON。检测范围为1 -100 ng/mL。半数抑制率IC50为10 ng/mL,最低检出限为0.56 ng/mL,与T-2毒素等真菌毒素的交叉反应率小于12 %;平均批内变异系数为2.82 %,平均批间变异系数为14.54 %;对玉米粉的加标回收率在80.2-91.1 %,准确性较高;与商品化试剂盒相比具有所需时间短,精密度高,灵敏度高的优点。
本研究表明分子模拟方法对DON半抗原设计有积极作用,指导半抗原设计,减少半抗原设计中时间及资金的投入。不同载体DON免疫抗原的免疫原性不同,DON-KLH免疫原性优于DON-BSA。6本研究所建立的DON的直接竞争ELISA方法,快速、方便,具有较好的实用价值,为进一步研究DON的ELISA检测试剂盒提供了重要的实验依据。
DON hapten design is the key step to establish the immune analysis methods. Currently, DON hapten design still adopt experience, causing the waste of time and money. From half antigen design principle, clear DON best hapten design thought: maximization similarity.of antigen sites.
Study the changes of molecular space configuration, electronic characteristics, hydrophobic property that several influence the antigen-antibody intercropping force factors , before and after DON modified. Research the relationship between hapten structure and immune discrimination. Establish DON hapten theory simulation design method. Provide a new thinking for hapten design. And on this basis, build a DON direct competition enzyme-linked immunosorbent analysis method.
According to the design process: choose binding sites→introduced connection arm→coupling carrier protein. Firstly, study the effect of binding sites to immune discrimination, introduced the same length of carbon chain of connection arm in the four potential binding sites (3, 7, 15-hydroxyl and 8-ketone base),Using molecular simulation software Hyperchem7.5 analysis determines parameters (spatial structure, atomic stereograph.furthermore, hydrophobic constant), 3-hapten is most similar to DON. Show 3-OH is the best DON binding site. Consistent to immune experiment results of Casale and Ramesh. Secondly, designed different connection arm structure in 3-OH, through similarity simulation received the best connection arm: succinic anhydride arm.
Choose 3 DON hapten with different arms structure to do immune antibody test, determination of antibody titer and inhibition rate to verify the molecular simulation results, study relationship of hapten structure and antibody immunity activity.
Group B antibody titer: 256000; Group A, C average titer: 64000. DON concentration for 500 ng/ml of group A antibodies suppression rate respectively: 66.4%, group B: 21.1%, group C: 10.8%. Inhibition rate of antibodies related to the specificity of DON, Group A has the highest similarity with DON, the specificity of strong, DON inhibition rate high, Antibody titer can reflect the carp of hapten. the benzene rings, unsaturated double bond can enhance the carp. Group B containing unsaturated double bond, enhanced the carp, produce high titer antibody, Although group C contains benzene structure, but failed to produce high titer antibody, combined with its spatial structure found: group C carboxyl end is very close to the main structure, may be shielded by carrier protein and affect immune discrimination. Comprehensive consideration, succinic anhydride is the best connected arm to DON .
In basis of the best hapten, compare the immunogenicity effect of vector protein(BSA、KLH).Preparation completely antigen of different carrier protein, to obtain DON antibody. Study antibody using enzyme-linked immunosorbent method (ELISA) and round two color spectrum (CD).
Established DON direct competition enzyme-linked immunosorbent analysis method. Examination scope for 1~100 ng/ml, half the inhibition rate IC50 for 10 ng/mL, minimum detect limit get 0.56 ng/ml, cross reaction rate less than 12 %, average batch coefficient of variation within 2.82 %,average batch coefficient of variation between 14.54 %;the recovery in 80.2-91.1 %.
This study shows that the molecular simulation method has the positive role to DON hapten design, guiding half antigen design, reduce time and capital investment. DON-KLH immunogenicity is superior DON–BSA. Established DON direct competition ELISA method, quick, convenient, and has good practical value.
引文
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