CN104402992B - 一种合成大麻抗原的合成方法及合成大麻抗原的应用 - Google Patents
一种合成大麻抗原的合成方法及合成大麻抗原的应用 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
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- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及免疫检测技术领域,公开了一种合成大麻抗原的合成方法,通过二硫键将JWH系列物质与大分子蛋白质偶联,得到的合成大麻抗原。还公开了运用该种方法合成的合成大麻抗原应用于胶体金检测卡。本发明合成的JWH类似物巯基化后,通过二硫键与大分子蛋白质偶联;其化学键更稳定,有利于抗原的长期保存;更有利于产品的稳定性。本发明的检测抗原,其交联臂要与免疫抗原的交联臂不一样,降低了免疫检测过程中的非特异性结合。此外,具有不同交联臂的检测抗原,保障了检测抗原用于快速免疫检测。本发明没有使用格氏试剂、氢化钠,解决了使用格氏试剂、氢化钠时,操作条件苛刻,以及有一定安全隐患的技术问题。
Description
技术领域
本发明涉及免疫检测技术领域,涉及一种合成大麻抗原的合成方法,尤其涉及运用该种方法合成的合成大麻抗原的应用。
背景技术
合成大麻(synthetic cannabis)是一种新型香料类毒品,是将不同香料和药草,混合不同化学物质制成不同口味品种的低成本化学合成毒品。其商品名有K2、Spice、Genie、Zohai、迷幻鼠尾草等名称。由于这种新型香料中不含有天然大麻活性成分,在二十一世纪初的几年里,世界各国基本都未将其列入毒品检验范围,因而流行欧美,扩散至亚洲。但是近几年的研究表明,合成大麻中的主要精神活性成分JWH-018、JWH-073、JWH-250和CP-47,497及相应的同系物及其代谢物都是大麻素受体的激动剂,其作用与△9-THC一样,有些甚至强于△9-THC的效力。吸食合成大麻后的人们会出现高血压、晕厥、心动过速、幻觉、精神错乱、低钾血症、癫痫和惊恐发作等,此外还可以引起药物依赖性和精神分裂症的发生,以及严重的毒副作用和毒性反应。因此,禁止合成大麻(K2)的流通势在必行。现在世界上不少国家都已经采取了立法行动来禁止或者用别的方式来管制这种香料毒品。例如2009-2011年期间美国、英国、法国、德国、俄罗斯、拉脱维亚、瑞典、爱沙尼亚、意大利、罗马尼亚、乌克兰等国将JWH-018列为管制药品;我国目前还没有开展对这类毒品的研究,但是在实际侦查中,我们已经遇到了相关的案例。这种新型的香料类毒品的吸食和贩卖情况已引起我国以及国际禁毒执法部门越来越多的关注。
对于吸食了合成大麻的患者,在其尿液,血液和唾液中可以检测到JWH系 列和CP-47,497系列物质及其代谢物。检测方法主要是GC-MS、LC-MS、HPLC、ELISA和胶体金法。前三种检测方法仪器精密,价格昂贵,占用空间大,耗时长不利于禁毒执法部门的现场使用。ELISA法通量大,但是不方便随身携带。
此外,现有的合成大麻抗原的合成方法,应用了格氏试剂和氢化钠,这些试剂价格昂贵,条件要求苛刻,且具有较大的安全隐患,不利于工业化生产。现有的检测抗原,其交联臂要与免疫抗原的交联臂一样,在产生抗体的过程中,交联臂很可能属于抗体识别区。
发明内容
本发明针对现有技术的缺点,公开了一种合成大麻抗原的合成方法,还公开了运用该种方法合成的合成大麻抗原的应用。
为了解决上述技术问题,本发明通过下述技术方案得以解决。
一种合成大麻抗原的合成方法,通过二硫键将JWH系列物质与大分子蛋白质偶联,得到的合成大麻抗原的结构如式(Ⅰ)所示:
其中,N=1或2或3;M=1或2。本发明具体方程式参见具体实施例部分。
本发明合成的JWH类似物巯基化后,通过二硫键与大分子蛋白质偶联;其化学键更稳定,有利于抗原的长期保存;更有利于产品的稳定性。本发明将半抗原偶联到既具有反应原性又具有免疫原性的完全抗原---大分子蛋白质上,使 其获得抗半抗原的特异性抗体;解决了JWH系列物质属于半抗原,只具有反应原性,不具有免疫原性的技术问题。
作为优选,JWH系列物质与大分子蛋白质的物质的量比为5-500:1。
作为优选,吲哚和萘甲酰氯在三氯化铝的催化下生成3-(1-萘甲酰基)吲哚;通过溴乙酸乙酯或溴乙酸丙酯或溴乙酸丁酯在3-(1-萘甲酰基)吲哚的N位上引入羧基,再与同型高半胱氨酸硫内酯盐酸盐或者巯基保护的半胱氨酸反应引入巯基,引入巯基后的产物通过二硫键与SPDP活化的蛋白质偶联。本发明选用溴乙酸乙酯或溴乙酸丙酯或溴乙酸丁酯,一方面避免了产生抗交联臂的抗体;另一方面可以将半抗原暴露在蛋白质的外表,而不是被蛋白质遮掩住。
作为优选,大分子蛋白为BSA。
作为优选,将SPDP溶于DMF中,得到SPDP-DMF溶液,并使SPDP在DMF中的浓度为28-35MG/ML;每1ML BSA溶液中滴入0.18-0.3ML SPDP-DMF溶液,室温反应1H,除去未反应的SPDP,得到SPDP-BSA蛋白溶液。
作为优选,通过过脱盐柱除去未反应的SPDP。
作为优选,引入巯基后的产物为(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸;取42ML SPDP-BSA蛋白溶液,滴入(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸,室温反应8-18小时,得到牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((2-羧甲酸)二硫基)丁酸偶联物。
以上提到的合成方法合成的合成大麻抗原应用于胶体金检测卡。
与现有技术相比,本发明的有益效果为:
(1)本发明合成的JWH类似物巯基化后,通过二硫键与大分子蛋白质偶联;其化学键更稳定,有利于抗原的长期保存;更有利于产品的稳定性。
(2)本发明的检测抗原,其交联臂要与免疫抗原的交联臂不一样。在产生抗体的过程中,交联臂不属于抗体识别区,降低了免疫检测过程中的非特异性结合。此外,具有不同交联臂的检测抗原,保障了检测抗原用于快速免疫检测。
(3)本发明没有使用格氏试剂、氢化钠,解决了使用格氏试剂、氢化钠时,操作条件苛刻,以及有一定安全隐患的技术问题。
(4)本发明的合成工艺中所使用的材料和催化剂均为普通产品,因此更具实用性,适于工业生产并能产生经济价值。
(5)而本发明采用的胶体金法具有以下优势:便于携带使用,通量大,耗时短;能为禁毒执法部门及时提供可疑人物的检测结果。
具体实施方式
实施例1
1.3-(1-萘甲酰基)吲哚的制备:在氮气保护下,于圆底烧瓶中将754.8mg的萘甲酰氯溶于16ml无水二氯甲烷,然后加入717mg的无水三氯化铝,搅拌反应20min,溶液呈黄绿色。将351.45mg的吲哚溶于6ml无水二氯甲烷中,再滴入到圆底烧瓶,继续搅拌反应3h。TLC检测反应进程,展开剂石油醚:乙酸乙酯=4:1。反应结束后,溶液呈黄色;将反应体系至于冰水浴中,然后缓慢加入45ml冰水,转移至分液漏斗分离出有机相;用2*45ml二氯甲烷萃取,合并有机相,饱和食盐水洗涤,减压蒸干,柱层析得606mg的3-(1-萘甲酰基)吲哚,得率74.54%。得到的3-(1-萘甲酰基)吲哚的结构如式(Ⅱ)所示:
以下为3-(1-萘甲酰基)吲哚的表征数据:
1H NMR(500MHz,CD3OD)δ:8.331-8.313(1H,m),8.045-8.010(2H,m),7.956-7.940(1H,d),7.651-7.637(1H,m),7.581-7.551(1H,m),7.530-7.468(4H,m),7.298-7.277(2H,m).
13CNMR(CD3OD)δ:195.218,140.142,138.782,138.358,135.244,131.985,131.155,129.429,127.833,127.409,127.382,126.955,126.534,125.743,124.796,123.675,122.958,119.282,113.158。
ESI m/z=270(M-1)。
2.2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯的制备:在氮气保护下,将620mg碳酸钾,400mg3-(1-萘甲酰基)吲哚和446mg溴乙酸乙酯先后加入到10ml无水DMF中,60℃搅拌反应6h。TLC监测反应进程,展开剂石油醚:乙酸乙酯=3:1;柱层析纯化得480mg的2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯,得率87.65%。得到的2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯的结构如式(Ⅲ)所示:
以下为2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯的表征数据:
1HNMR(500MHz,CD3OD)δ:8.369-8.365(1H,m),8.027-8.010(1H,d),7.976-7.960(1H,d),7.912-7.896(1H,d),7.603-7.587(1H,d),7.540(1H,s),7.506-7.461(2H,m),7.432-7.412(1H,m),7.374-7.355(1H,m),7.317-7.298(2H,m),4.929(1H,s),4.880(1H,s),4.132-4.089(2H,m),1.184-1.156(3H,t)。
13CNMR(CD3OD)δ:194.758,169.508,142.006,139.831,139.208,135.192,131.891,131.269,129.403,127.887,127.870,127.420,127.106,126.491,125.718,125.083,124.109,123.319,119.016,111.302,62.870,48.486,14.355。
ESI m/z=358(M+1)。
3.2-(3-(1-萘甲酰基)吲哚-1-)乙酸的制备:将2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯溶于10ml乙醇,然后加入1.9ml 4N的NaOH,加热回流6小时。TLC监测反应,展开剂石油醚:乙酸乙酯=2:1。反应结束后反应体系由浅黄色逐渐变为墨绿色。减压蒸去溶剂,残留物用1N HCl调节pH=1,然后用乙酸乙酯萃取3遍。合并有机相,饱和食盐水洗涤,减压蒸干得410mg的2-(3-(1-萘甲酰基)吲哚-1-)乙酸,得率92.39%。得到的2-(3-(1-萘甲酰基)吲哚-1-)乙酸的结构如式(Ⅳ)所示:
以下为2-(3-(1-萘甲酰基)吲哚-1-)乙酸的表征数据:
HNMR(MHz500,CD3OD)δ:8.363-8.346(1H,m),8.104-8.088(1H,d),8.016-8.000(1H,d),7.946-7.930(1H,d),7.743-7.727(1H,m),7.601(1H,s),7.579-7.563(1H,m),7.549-7.471(2H,m),7.442-7.425(1H,m),7.320-7.295(2H,m),4.687(2H,s)。
13CNMR(CD3OD)δ:194.813,142.630,140.048,139.487,135.242,131.986,131.126,129.365,128.117,127.787,127.344,127.269,126.617,125.747,124.643,123.769,123.128,118.216,111.697,51.690。
ESI m/z=328(M-1)。
4.(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺的制备:取344mg的2-(3-(1-萘甲酰基)吲哚-1-)乙酸于100mL的三口烧瓶中,溶解于5ml干燥的二氯甲烷中,然后加入1.2eq的EDCI,3eq的三乙胺和1eq的同型高半胱氨酸硫内酯盐酸盐,室温搅拌反应过夜;TLC监测反应进程。反应基本结束后, 蒸干溶剂,柱层析纯化得356mg的(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺,得率80.18%。得到的(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺的结构如式(Ⅴ)所示:
以下为(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺的表征数据:
1HNMR(500MHz,CDCl3)δ:8.462-8.446(1H,m),8.161-8.144(1H,d),7.946-7.930(1H,d),7.881-7.864(1H,d),7.656-7.642(1H,m),7.501-7.441(2H,m),7.355-7.286(4H,m),7.050-7.036(1H,d),4.619-4.615(2H,m),4.446-4.392(1H,m),3.202-3.192(1H,m),3.087-3.073(1H,m),2.544-2.495(1H,m),1.845-1.756(1H,m)。
13CNMR(CDCl3)δ:204.558,192.593,167.220,139.232,138.525,137.486,133.888,130.757,130.516,128.460,127.164,126.961,126.576,126.417,125.827,124.775,124.496,123.555,123.000,118.681,110.150,59.272,49.763,36.731,27.421。
ESI m/z=429(M+1)。
上述的反应过程如下所示:
实施例2
与实施例2的区别特征在于:2-(3-(1-萘甲酰基)吲哚-1-)乙酸乙酯的制备时,用溴乙酸丙酯代替溴乙酸乙酯。
实施例3
合成大麻抗原的合成方法,包括以下步骤,
A.取90mg实施例1制备的(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺溶于3mL甲醇,再加入0.9mL 1N的NaOH水溶液,在室温搅拌8min后,加入0.9mL 1M的盐酸,得到(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸;
(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸的结构如式(Ⅵ)所示:
B.将SPDP溶于DMF中,得到SPDP-DMF溶液,并使SPDP在DMF中的浓度为28mg/mL;
每1mL BSA溶液中滴入0.18mL SPDP-DMF溶液,室温反应1h,除去未反应的SPDP,得到SPDP-BSA蛋白溶液;
C.取42mL步骤B制得的SPDP‐BSA蛋白溶液,滴入(S)‐2‐(2‐(3‐(1‐萘甲基)吲哚‐1‐)乙酰胺)‐4‐巯基丁酸,室温反应8小时,得到牛血清蛋白‐(S)‐2‐(2‐(3‐(1‐萘甲基)吲哚‐1‐)乙酰胺)‐4‐((2‐羧甲酸)二硫基)丁酸偶联物。
牛血清蛋白‐(S)‐2‐(2‐(3‐(1‐萘甲基)吲哚‐1‐)乙酰胺)‐4‐((2‐羧甲酸)二硫基)丁酸偶联物的结构如式(Ⅷ)所示:
上述的反应过程如下所示:
实施例4
合成大麻抗原的合成方法,与实施例2的区别在于:步骤A中,(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺的用量为110mg,NaOH水溶液的用量为1.1mL,室温搅拌12min,盐酸的用量为1.1mL。步骤B中,SPDP-DMF溶液中,SPDP在DMF中的浓度为5mg/mL,SPDP-DMF溶液用量为0.3mL。步骤C中,室温反应18小时。
实施例5
合成大麻抗原的合成方法,与实施例2的区别在于:步骤A中,(S)-2-(3-(1-萘甲酰基)吲哚-1-)-N-(2-氧基四氢噻吩-3-)乙酰胺的用量为100mg,NaOH水溶液 的用量为1mL,室温搅拌10min,盐酸的用量为1mL。步骤B中,中,SPDP在DMF中的浓度为30mg/mL,SPDP-DMF溶液用量为0.2mL。步骤C中,室温反应12小时。
实施例6
实施例6为与实施例1不同的另外一种方案,具体如下:
1.2-(3-(1-萘甲酰基)吲哚-1-)丁酸乙酯的制备:在氮气保护下,将310mg碳酸钾,200mg的3-(1-萘甲酰基)吲哚和256mg溴丁酸乙酯先后加入到10ml无水DMF中,60℃搅拌反应6h。TLC监测反应进程,展开剂石油醚:乙酸乙酯=3:1;柱层析纯化得233mg的2-(3-(1-萘甲酰基)吲哚-1-)丁酸乙酯,得率82.04%。2-(3-(1-萘甲酰基)吲哚-1-)丁酸乙酯的结构如式(Ⅸ)所示:
2.2-(3-(1-萘甲酰基)吲哚-1-)丁酸的制备:将2-(3-(1-萘甲酰基)吲哚-1-)丁酸乙酯溶于10ml乙醇,然后加入1.9ml 4N的NaOH,加热回流6小时。TLC监测反应,展开剂石油醚:乙酸乙酯=2:1。反应结束后反应体系由浅黄色逐渐变为墨绿色。减压蒸去溶剂,残留物用1N HCl调节pH=1,然后用乙酸乙酯萃取3遍。合并有机相,饱和食盐水洗涤,减压蒸干得206mg的2-(3-(1-萘甲酰基)吲哚-1-)丁酸,得率95.37%。2-(3-(1-萘甲酰基)吲哚-1-)丁酸的结构如式(Ⅹ)所示:
3.巯基保护的半胱氨酸(即巯基保护的Cys)的制备方法:500mg Cys溶于15ml DMF中,然后加入1.15g三苯基氯甲烷,搅拌2天后拉杆,过柱,淋洗剂为二氯甲烷:甲醇=10:1。得到1.205g巯基保护的Cys,得率80.32%。
取206mg的2-(3-(1-萘甲酰基)吲哚-1-)丁酸,溶于2.06ml二氯甲烷中,加入129mgEDCI和77mg NHS,搅拌活化过夜。反应完成以后,先将314mg巯基保护的Cys和95μL三乙胺溶于3ml二氯甲烷中,然后将活化完成的2-(3-(1-萘甲酰基)吲哚-1-)丁酸慢慢滴入其中。滴加完成以后,室温搅拌反应6小时。TLC监测反应,展开剂为二氯甲烷:甲醇=10:1。反应完成后,加入10ml二氯甲烷,用15ml*2水和15ml饱和食盐水洗涤,有机相用无水硫酸钠干燥,减压蒸干,柱层析得到300mg的2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-(三苯甲基硫)丙酸,收率74.07%。2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-(三苯甲基硫)丙酸的结构如式(Ⅺ)所示:
以下为2‐(4‐(3‐(1‐萘甲酰基)‐1‐吲哚‐1‐)丁酰胺)‐3‐(三苯甲基硫)丙酸的表征数 据:
1HNMR(500MHz,CDCl3)δ:1.935(4H,s)2.577-2.609(1H,m)2.664-2.703(1H,m),3.941(2H,s),4.321-4.329(1H,d),6.029-6.042(1H,d),7.091-7.154(3H,m),7.169-7.184(6H,m),7.243-7.270(2H,m),7.316-7.331(7H,m),7.371-7.397(2H,m),7.431-7.461(2H,m),7.585-7.599(1H,m),7.836-7.907(2H,m),8.139-8.155(1H,d),8.288-8.300(1H,d)。
13CNMR(CDCl3)δ:25.419,33.389,46.115,51.115,67.187,77.016,77.270,77.526,110.445,117.806,122.869,123.159,123.999,124.875,126.013,126.136,126.523,127.011,127.078,128.204,128.436,129.618,130.299,130.834,133.877,137.211,138.316,138.960,144.436,172.028,173.584,192.531。
m/z=701.2(M-1)。
4.2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-巯基丙酸的制备:将2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-(三苯甲基硫)丙酸溶解于乙醇中,加入6-10当量的6N HCl,室温搅拌过夜。TLC监测反应,反应完成以后,减压蒸干溶剂,柱层析得到2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-巯基丙酸。2-(4-(3-(1-萘甲酰基)-1-吲哚-1-)丁酰胺)-3-巯基丙酸的结构如式(XII)所示:
上述的反应过程如下所示:
实施例7
抗原在胶体金检测试纸条的应用:
用柠檬酸钠还原四氯化金制备直径在20-40nm的胶体金,然后将胶体金标记到抗合成大麻的抗体到上,用λmax的吸光值(OD)来表示金标抗体的浓度。
将金标抗体上样到金标机上,将一定OD值的金标抗体均匀地喷聚酯膜(金标垫)上,喷好后放入37度烘箱烘8h;切成合适的大小,放入装有干燥机的铝箔袋内,室温存放备用。
取实施例3制备的合成大麻抗原(牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((2-羧甲酸)二硫基)丁酸偶联物(即Ⅷ))、牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((羧甲酸)硫代)丁酸偶联物(即XIV)、BSA-2-(3-(1-萘甲酰基)吲哚-1-)乙酸偶联物(即XV)为研究对象。
牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((羧甲酸)硫代)丁酸偶联物的结构如式(XIV)所示:
BSA-2-(3-(1-萘甲酰基)吲哚-1-)乙酸偶联物的结构如式(XV)所示:
将Ⅷ、XIV、XV稀释到合适浓度,用点膜机点到硝酸纤维素膜上相应T线位置,37度烘干12h。
样品垫---玻璃纤维经过缓冲液和表面活性的处理,37度烘干10h备用。
按金标垫、样品垫、硝酸纤维素膜,吸水纸,大卡组装成胶体金检测试纸卡见figure 2。
配制相应浓度的小分子溶液,加入到样品垫上,5min后读取结果。
产品对018-H、018-B、073-H、073-P的Cutoff值的要求分别是300ng/ml、50ng/ml、300ng/ml和50ng/ml。
由表一和表二可知:牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((2-羧甲酸)二硫基)丁酸偶联物(即Ⅷ)符合QC标准,且热稳定性好,检测抗原合格;牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((羧甲酸)硫代)丁 酸偶联物(即XIV)只有检测018-H时符合QC标准,且热稳定性差,检测抗原不合格;BSA-2-(3-(1-萘甲酰基)吲哚-1-)乙酸偶联物(即XV)全部都不符合QC标准,且热稳定性差,检测抗原不合格。
表一:三种抗原对四种小分子的检测灵敏度结果
备注1:四种小分子溶于阴性的唾液中
QC标准:Neg≥G8;-50%﹥G4;+50%﹤G3.5。
表二:三种抗原的加速稳定性结果
备注1:将组装好的检测卡55℃烘72小时;
备注2:四种小分子溶于阴性的唾液中;
QC标准:Neg≥G8;-50%﹥G4;+50%﹤G3.5。
总之,以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所作的均等变化与修饰,皆应属本发明专利的涵盖范围。
Claims (6)
1.一种合成大麻抗原的合成方法,其特征在于:通过二硫键将JWH系列物质与大分子蛋白质偶联,得到的合成大麻抗原的结构如式(Ⅰ)所示:
其中,n=1或2或3;m=1或2,JWH系列物质与大分子蛋白质的物质的量比为5-500:1,吲哚和萘甲酰氯在三氯化铝的催化下生成3-(1-萘甲酰基)吲哚;通过溴乙酸乙酯或溴乙酸丙酯或溴乙酸丁酯在3-(1-萘甲酰基)吲哚的N位上引入羧基,再与同型高半胱氨酸硫内酯盐酸盐或者巯基保护的半胱氨酸反应引入巯基,引入巯基后的产物通过二硫键与SPDP活化的蛋白质偶联。
2.根据权利要求1所述的合成大麻抗原的合成方法,其特征在于:大分子蛋白为BSA。
3.根据权利要求2所述的合成大麻抗原的合成方法,其特征在于:将SPDP溶于DMF中,得到SPDP-DMF溶液,并使SPDP在DMF中的浓度为28-35mg/mL;每1mLBSA溶液中滴入0.18-0.3mLSPDP-DMF溶液,室温反应1h,除去未反应的SPDP,得到SPDP-BSA蛋白溶液。
4.根据权利要求3所述的合成大麻抗原的合成方法,其特征在于:通过过脱盐柱除去未反应的SPDP。
5.根据权利要求3所述的合成大麻抗原的合成方法,其特征在于:引入巯基后的产物为(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸;取42mL SPDP-BSA蛋白溶液,滴入(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-巯基丁酸,室温反应8-18小时,得到牛血清蛋白-(S)-2-(2-(3-(1-萘甲基)吲哚-1-)乙酰胺)-4-((2-羧甲酸)二硫基)丁酸偶联物。
6.根据权利要求1-5任意一项所述的合成方法合成的合成大麻抗原应用于胶体金检测卡。
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