CN104792989B - 赛庚啶半抗原、抗原及其制备方法和应用 - Google Patents
赛庚啶半抗原、抗原及其制备方法和应用 Download PDFInfo
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- CN104792989B CN104792989B CN201410018901.3A CN201410018901A CN104792989B CN 104792989 B CN104792989 B CN 104792989B CN 201410018901 A CN201410018901 A CN 201410018901A CN 104792989 B CN104792989 B CN 104792989B
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- JJCFRYNCJDLXIK-UHFFFAOYSA-N cyproheptadine Chemical compound C1CN(C)CCC1=C1C2=CC=CC=C2C=CC2=CC=CC=C21 JJCFRYNCJDLXIK-UHFFFAOYSA-N 0.000 title claims abstract description 106
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/70—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种赛庚啶半抗原,相应的人工抗原和单克隆抗体,同时本发明也公开了所述赛庚啶半抗原,相应的人工抗原和单克隆抗体的制备方法及其应用。本发明提供的赛庚啶半抗原是式1所示产物,用式1所示产物与载体蛋白连接可以得到赛庚啶抗原。所述赛庚啶抗原可应用于制备赛庚啶特异性抗体。本发明制备方法简便可行、成本较低,半抗原产率较高。本发明的赛庚啶人工抗原,通过免疫动物可产生了针对赛庚啶的特异性抗体,可用于制备检测赛庚啶残留的酶联免疫检测试剂盒,具有简单、快速、处理样品量大、灵敏度高、特异性强等诸多优点。
Description
技术领域
本发明属于食品安全检测技术领域,具体涉及赛庚啶半抗原、抗原、单克隆抗体制备方法及其应用。
背景技术
赛庚啶(Cyproheptadine)化学名称为1-甲基-4-(5H-二苯并[a,d]环庚三烯-5-亚基)哌啶盐酸盐倍半水合物,别名塞庚定、二苯环庚啶、偏痛定,英文别名2,6-dichloro-N-(2-imidazolin-2-yl)aniline。分子式C21H21N·HCl·3/2H2O;分子量:350.89。赛庚啶为白色或浅黄色结晶粉末,无臭、味微苦。熔点113℃。在甲醇中易溶;在氯仿中溶解,溶液有乳化现象(若以干燥品溶解,溶液即澄明);在乙醇中略溶;在水中微溶,水溶液呈酸性反应;在乙醚中几乎不溶。赛庚啶为人用抗过敏药,对抗体内组胺对血管、支气管平滑肌的作用,从而消除过敏症状,也可用于改善患者的食欲。赛庚啶是一个典型的H1受体阻滞剂,属于抗组胺药,但同时也是5-羟色胺受体拮抗剂,可在下丘脑食欲中枢拮抗5-羟色胺,这可以解释赛庚啶具有刺激食欲的功能。赛庚啶易吸收,主要代谢物是葡萄糖醛酸苷季铵物,以及芳环羟基化、N-去甲基化和杂环氧化产物。赛庚啶常见的副作用为轻、中度的嗜睡、头昏、乏力、口干等,自2005年起已在国内的饲料产品中发现添加,分析原因可能为通过促进动物食欲以达到增重的目的。食用含有赛庚啶残留的动物源性产品,对人体健康具有一定的危害性,对儿童的作用更为明显,高浓度可直接引发昏迷、呼吸急促、全身无力等中毒症状,严重者则直接导致儿童死亡。赛庚啶并不是一种饲料添加剂,世界卫生组织、我国、美国、欧盟及一些发达国家等都禁止违法使用赛庚啶。2010年12月农业部1519号公告已明确把赛庚啶列入了《禁止在饲料和动物饮水中使用的物质》清单。
目前国内外大多采用高效液相色谱法测定,这些方法特异性强、灵敏度高、但是样品前处理操作步骤繁琐,成本较高,也不适用于大批量样品的筛选检测。免疫化学分析由于在抗原抗体的定性定量方面独特的优势和操作简便快速、成本低、灵敏度较高、分析样本量大的优点弥补了理化分析的不足。赛庚啶人工抗原的制备为这一方法的实施奠定了重要基础。影响免疫化学分析质量的根本因素是抗体的特异性与亲和性,这些性质又决定于免疫半抗原分子的结构,因此免疫半抗原的分子设计与合成就是产生特异性抗体和建立小分子兽药残留快速检测技术的最基础和最关键的步骤。
发明内容
本发明的目的是提供一种赛庚啶半抗原、抗原、特异性抗体制备方法及其应用。
本发明提供的赛庚啶半抗原,其结构为式Ⅰ所示:
本发明还保护式Ⅰ所示化合物的制备方法,包括如下步骤:
将赛庚啶,氯碳酸乙酯和甲苯的混合物回流,冷却后,用盐酸和水洗涤,干燥并浓缩;用正己烷处理,得到浅黄色结晶,重结晶,得到微黄色针状结晶;产物加入水和一水氢氧化锂,pH试纸检测pH=10以上;室温水解反应后用乙酸乙酯萃取,萃取水解反应水相一到两次,除去杂质,再用盐酸调pH值到4,用乙酸乙酯萃取,旋干得到半抗原。
本发明所提供的赛庚啶抗原,是将赛庚啶半抗原与载体蛋白偶联获得的抗原。
所述赛庚啶抗原,其结构为式Ⅱ所示:
本发明还保护所述赛庚啶抗原的制备方法,包括如下步骤:
(1)取式Ⅰ所示产物31.7mg,溶于3ml二甲基甲酰胺DMF中;
(2)加入1ml0.01M PBS助溶,加入碳二亚胺EDC38mg,N-羟基琥珀酰亚胺NHS38mg,室温活化2h;
(3)取载体蛋白100mg溶于10ml的0.1M碳酸氢钠溶液中,将活化物在4℃冰水浴条件下,加入到蛋白中,4℃搅拌反应过夜,置1L0.01MPBS中透析8次,以除去未反应的小分子物质;
(4)分装,于-20℃保存备用。
常用载体蛋白均可采用,如牛血清白蛋白(BSA),卵清蛋白(OVA),人血清白蛋白(HSA),鼠血清白蛋白(MSA),甲状腺蛋白(TG)或血蓝蛋白(KLH)等。
所述赛庚啶抗原可以作为免疫原制备赛庚啶特异性抗体,也可以作为包被原制备酶标板。
所述抗体具体可为单克隆抗体。
式Ⅰ所示产物、所述赛庚啶抗原、所述抗体均可应用于检测赛庚啶。
本发明还公布了应用赛庚啶抗原和赛庚啶单克隆抗体制备得到的酶联免疫试剂盒。
所述酶联免疫检测试剂盒,是由包被有赛庚啶抗原的酶标板、酶标抗体工作液、赛庚啶系列标准品、底物显色液、终止液、浓缩复溶液、浓缩洗涤液。
本发明依靠免疫学、免疫化学基本原理和残留分析技术手段,设计、合成小分子目标分析物半抗原,并与载体蛋白偶联,制备有效人工抗原,免疫动物制备针对小分子分析物的特异性抗体。利用抗原抗体的特异性免疫学反应,定量的检测样本中微量小分子目标分析物,具有特异、灵敏、准确、快速、方便、廉价等特点。本发明制备方法简便可行、成本较低,半抗原产率较高。本发明克服了现有检测技术中对赛庚啶样品预处理复杂、耗时、且需要大量有机溶剂萃取,以及在检测过程中要用到精密昂贵的检测仪器而不适于推广使用等缺点。本发明的赛庚啶人工抗原,通过免疫动物可产生了针对赛庚啶的特异性抗体,用于快速检测食品中的赛庚啶残留,具有简单、快速、处理样品量大、灵敏度高、特异性强等诸多优点。
附图说明
图1赛庚啶抗原的紫外光谱图(其中BSA为人血清白蛋白);
图2为赛庚啶酶联免疫检测试剂盒标准曲线。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。人血清白蛋白简称HSA。
实施例1、制备赛庚啶半抗原
将3.3g赛庚啶,7.49g的氯碳酸乙酯和17mL甲苯中的混合物回流3.5小时。冷却后,将反应混合物用盐酸和水洗涤,然后干燥并浓缩。用正己烷处理,得到浅黄色结晶,重结晶,将残余物固化从乙醇中,得到微黄色针状结晶。产物加入50ml水,加入一水氢氧化锂8.36g,pH试纸检测pH=10以上。室温水解反应3h以上。用乙酸乙酯100ml萃取,萃取水解反应水相一到两次,除去杂质,再用5mol/L的盐酸调pH值到4,用乙酸乙酯萃取,旋干得到赛庚啶半抗原CHD-OCL。
赛庚啶半抗原的结构式如式Ⅰ所示。
实施例2、赛庚啶人工抗原的制备和鉴定
一、赛庚啶免疫抗原的合成
(1)取CHD-OCL 31.7mg溶于3mlDMF中;
(2)加入1ml0.01MPBS助溶,加入EDC38mg,NHS38mg,室温活化2h;
(3)取BSA 100mg溶于10ml的0.1M碳酸氢钠溶液中,将活化物在4℃冰水浴条件下,加入到蛋白中,4℃搅拌反应过夜;
(4)置1L0.01MPBS中透析8次,得全抗原CHD-OCL-BSA;
(5)分装,于-20℃保存备用。
赛庚啶免疫抗原的结构式如式Ⅱ所示。
二、赛庚啶包被抗原的合成
(1)取CHD-OCL 31.7mg溶于3mlDMF中;
(2)加入1ml0.01MPBS助溶,加入EDC38mg,NHS38mg,室温活化2h;
(3)取OVA 100mg溶于10ml的0.1M碳酸氢钠溶液中,将活化物在4℃冰水浴条件下,加入到蛋白中,4℃搅拌反应过夜;
(4)置1L0.01MPBS中透析8次,得全抗原CHD-OCL-OVA;
(5)分装,于-20℃保存备用。
三、赛庚啶人工抗原的鉴定
按合成赛庚啶免疫抗原和包被抗原反应所用的半抗原、载体蛋白与偶联产物的比例,分别进行紫外(200nm~400nm)扫描鉴定,并通过比较三者在同一波长下的吸光值计算其结合比。产物的紫外光谱图与载体蛋白相比发生了变化,说明半抗原已经与载体蛋白成功偶联制得赛庚啶人工抗原。经计算,赛庚啶半抗原分子与BSA分子的结合比为15.6:1,赛庚啶半抗原分子与OVA分子的结合比为10.6:1。
赛庚啶抗原的紫外光谱图如图1所示。
实施例3、酶标单抗的制备和特异性鉴定
一、赛庚啶单抗的制备
1、用上述制备出的免疫原按100μg/只,以生理盐水溶解免疫原与弗氏完全佐剂等体积混匀,颈背部皮下注射免疫6~8周龄Balb/c雌鼠,初次免疫后第7、14、28天以免疫原与弗氏不完全佐剂等体积混匀,各追加免疫一次,融合前3天以免疫复合物100μg/只,不加弗氏佐剂再追加免疫一次。
2、按常规方法进行,取免疫小鼠的脾细胞与处于对数生长期的小鼠骨髓瘤细胞(SP2/0)混合,然后在45s内缓慢加入预热的融合剂(PEG4000)进行融合,用HAT培养基悬浮均匀,再加入适量的饲养细胞,培养于96孔培养板,于37℃,5%CO2培养箱中培养,5天后用HT培养基半换液,9天时候进行全换液。
3、细胞融合后,待细胞长到培养孔面积的1/4时,采用分步筛选法筛选杂交瘤细胞。初选采用间接ELISA方法,以包被抗原(预先用方阵法常规滴定其最佳包被浓度和阳性血清稀释度)包被酶标板,加入被测孔培养上清,孵育,清洗后加入羊抗鼠IgG-HRP和IgM-HRP,OPD进行显色反应。筛选出的阳性孔再用间接竞争ELISA方法筛选,先将细胞上清与100μg/mL的赛庚啶等体积混合,37℃水浴作用30min,再加入到包被好的酶标板中。同时用PBS取代赛庚啶作对照,其余步骤同上。若经赛庚啶阻断后的OD450nm值下降到对照孔的50%以下,则判为阳性,经2~3次检测都为阳性的孔,立即用有限稀释法进行亚克隆化。
4、将2~3次亚克隆建株后的杂交瘤细胞扩大培养,收集上清液用间接ELISA测定效价,冻存;并取8~10周龄Balb/c小鼠腹腔注射液体石蜡0.5mL/只,7~10日后腹腔注射杂交瘤细胞1~2×106/只,7~10日后抽取小鼠腹水,离心取上清,测定效价,并冻存备用。
二、酶标抗体的制备
(1)称取辣根过氧化物酶(HRP)2mg溶解于0.5mL水中,加入0.5mL 0.06mol/L NaIO4溶液,4℃避光作用30min;
(2)加入160mmol/L的乙二醇0.5mL,室温作用30min;
(3)加入步骤一制备的赛庚啶单抗2mg,混匀后装入处理过的透析袋中,置1000mL的0.05mmol/L碳酸钠缓冲液中透析,4℃过夜;
(4)透析液吸至10mL的离心管中,加0.25mL 5g/L的NaBH4溶液,混匀后置4℃2h;
(5)加入等体积的饱和硫酸铵溶液,4℃作用30min后4℃下3000r/min离心25min,弃上清;
(6)将沉淀溶于1.5mL0.02mol/L pH 7.4的PBS中,吸入透析袋内,在0.02mol/L pH 7.4PBS透析,4℃过夜(中途更换PBS 3次);
(7)将透析袋中液体吸至微量离心管中,4℃下10000r/min离心30min,将上清液吸出,加等量甘油,混匀,-20℃保存备用。
三、酶标赛庚啶抗体的鉴定
赛庚啶标准品购自Sigma公司。
用方阵滴定法确定赛庚啶包被抗原和步骤一制备的单抗的工作浓度,赛庚啶包被抗原的工作浓度为0.6μg/mL,单克隆抗体的工作浓度为1:5000。
用不同浓度的赛庚啶标准品溶液做实验溶液,其浓度如下:0、0.05、0.15、0.45、1.35、4.05μg/L。采用8组平行试验(n=8)。间接竞争性ELISA方法:
(1)用上述工作浓度的赛庚啶抗原包被酶标板,将赛庚啶标准品实验溶液与酶标抗体溶液同时加入酶标板微孔中,同时设置空白孔(将添加的抗体溶液换成高纯水,其它一致)和阴性对照孔(将标准品实验溶液用PBS溶液代替,其它一致),25℃避光环境中反应30min;
(2)倒出孔内液体,用洗涤液洗涤3~5次,将酶标板倒置在吸水纸上拍干;
(3)加入底物显色溶液到酶标板微孔中,25℃避光环境中反应15min;
(4)加入终止液,轻轻振荡混匀,用酶标仪在波长450nm处测定OD值。
以OD值为纵坐标,以赛庚啶实验溶液浓度的log10值为横坐标,绘制半对数标准曲线图。标准曲线具有完整的反S形状,并具有上平台和下平台,标准曲线的平行测定次数8次,实验重复性良好,相对标准偏差(变异系数)均在10%以内。
根据标准曲线得出半数抑制量(IC50),确定检测灵敏度。
抑制率用以下公式计算:
式中:ODmax:为不加标准品时的吸光值,ODx为标准品x时的吸光值,ODmin为空白对照孔的吸光值。
由上述公式计算得赛庚啶抗体在缓冲液中的半数抑制量(IC50)为0.2μg/L。
实施例4、检测赛庚啶的酶联免疫试剂盒及其制备
一、酶联免疫试剂盒由下述物质组成:
(1)包被赛庚啶半抗原的酶标板;
(2)酶标赛庚啶抗体工作液:实施例3中所述酶标抗体溶液;
(3)赛庚啶标准品:赛庚啶标准品溶液浓度分别为0、0.05、0.15、0.45、1.35、4.05μg/L;
(4)底物显色液:由A液和B液组成,A液为2%过氧化脲的水溶液,B液为1%四甲基联苯胺(TMB)的水溶液;
(5)终止液:0.2M硫酸水溶液;
(6)浓缩洗涤液:每1升所述洗涤液是按照如下方法配制得到的:将10mL吐温-20、5g叠氮化钠和990mL磷酸盐缓冲液混合,得到所述洗涤液;所述磷酸盐缓冲液的浓度为0.01M,pH值为7.4;
(7)浓缩复溶液:0.04mo1/L的磷酸盐缓冲液,将浓缩复溶液用水稀释至20倍体积,即为样品稀释液。
二、包被有赛庚啶半抗原的酶标板及其制备
包被赛庚啶半抗原的聚苯乙烯酶标板:用0.05M的碳酸盐溶液将抗原稀释至0.6μg/mL,包被96孔聚苯乙烯酶标板,每孔100μL,37℃温育2h,倾去包被液,用洗涤液洗涤3次,每次10s,拍干,然后在每孔中加入150μL封闭液,37℃温育2h,倾去孔内液体,干燥后用铝膜真空密封保存。
包被缓冲液:pH9.6,0.05mo1/L的碳酸钠缓冲液;
封闭液:每1升封闭液按照如下方法配制:将5mL马血清、1g叠氮化钠、30g酪蛋白混合,用磷酸盐缓冲液溶解并定容至1000mL,得到封闭液;其中,磷酸盐缓冲液的浓度为0.02M,pH值为7.2。
三、试剂盒检测方法
(一)尿液样品前处理
(1)量取尿液样本400μL至2mL聚苯乙烯离心管中,3000g离心10min;
(2)吸取上清与样品稀释液按1:1体积混合;
(3)取50μL混合液用于检测。
(二)用试剂盒检测
1、标准曲线的制作
向包被有赛庚啶半抗原的酶标板微孔中加入赛庚啶标准品溶液50μL,然后加入酶标抗体工作液100μL/孔,轻轻振荡混合均匀,用盖板膜盖板后置25℃避光环境中反应30min。小心揭开盖板膜,将孔内液体甩干,加入洗涤工作液250μL/孔,充分洗涤4~5次,每次间隔10s,泼掉板孔内洗涤液,用吸水纸拍干。加入底物A液50μL/孔、底物B液50μL/孔,轻轻振荡混匀,25℃恒温箱避光显色15min,每孔加入终止液50μL,轻轻振荡混匀,用酶标仪,测定每孔吸光度值。
用每个浓度的标准品溶液的吸光度平均值(B)除以第一个标准品溶液(0标准)的吸光度值(B0)再乘以100%,得到百分吸光度值。以赛庚啶标准品浓度(μg/L)的半对数值为X轴,百分吸光度值为Y轴,绘制标准曲线图。得到的标准曲线如图2所示。
2、样品中赛庚啶浓度的测定
用每个检测样本溶液的吸光度平均值(B)除以第一个标准品溶液(0标准)的吸光度值(B0)再乘以100%,得到百分吸光度值。相对应每一个检测样本溶液的百分吸光度值,再根据标准品溶液的浓度值换算出样本溶液中赛庚啶的残留量,最后再乘以各样品前处理过程的稀释倍数,即可计算出样品中赛庚啶的浓度。
四、试剂盒检测效果评价
(一)准确度和精密度试验
向不含赛庚啶的尿液样品中添加赛庚啶标准品,使赛庚啶标准品在样品中的终浓度分别为0.1、0.2、0.4μg/L;将添加后的样品分别按照实验三中所述方法进行前处理,得到检测样本溶液。
从三个不同批次的试剂盒中各抽取3个试剂盒进行检测,检测方法如实验三中所述,每个实验重复5次,分别计算变异系数。结果分别见表1。
表1 准确度和精密度试验结果
批内变异系数:同一次测定中各平行样本的变异系数。
批间变异系数:同一样本在不同批次测定结果的变异系数,取其平均值。
结果表明:尿液样品的平均添加回收率在85.9~108.6%,批内变异系数在6.1~8.4%,批间变异系数在7.0~9.8%。
(二)试剂盒保存期
试剂盒保存条件为2~8℃,经过15个月的测定,试剂盒的最大吸光度值(0标准),50%抑制浓度、赛庚啶添加实际测定值均在正常范围之内。考虑到运输和使用过程中,会有非正常保存条件出现,将试剂盒在37℃保存的条件下放置9天,进行加速老化实验,结果表明该试剂盒的各项指标完全符合要求。考虑到试剂盒冷冻情况发生,将试剂盒放入-20℃冰箱冷冻9天,测定结果也表明试剂盒各项指标完全正常。从以上结果可得出试剂盒可以在2~8℃至少可以保存12个月以上。
(三)交叉反应率试验
选择与赛庚啶结构或功能相似的其他药物进行交叉反应试验,通过各种药物的标准曲线分别得到其50%抑制浓度。用下式计算试剂盒对其它类似物的交叉反应率。与其他药物的交叉反应率越小,说明赛庚啶酶联免疫检测试剂盒对赛庚啶的检测特异性越好。结果见表2。
表2 赛庚啶试剂盒交叉反应率
药物名称 | 交叉反应率(%) |
赛庚啶 | 100.0 |
氯雷他定 | <1 |
酮替芬 | <1 |
试验结果表明,本发明试剂盒对弗雷他定、酮替芬的交叉反应率均小于1%,所以试剂盒对赛庚啶的特异性好,即本发明试剂盒可以检测赛庚啶。
Claims (4)
1.一种赛庚啶半抗原,为式Ⅰ所示产物:
2.式Ⅰ所示产物的制备方法,包括如下步骤:将3.3g赛庚啶,7.49g的氯碳酸乙酯和17mL甲苯中的混合物回流3.5小时;冷却后,将反应混合物用盐酸和水洗涤,然后干燥并浓缩;用正己烷处理,得到浅黄色结晶,重结晶,将残余物固化从乙醇中,得到微黄色针状结晶;产物加入50ml水,加入一水氢氧化锂8.36g,pH试纸检测pH=10以上;室温水解反应3h以上;用乙酸乙酯100ml萃取,萃取水解反应水相一到两次,除去杂质,再用5mol/L的盐酸调pH值到4,用乙酸乙酯萃取,旋干得到式Ⅰ所示的赛庚啶半抗原。
3.一种赛庚啶抗原,是将式Ⅰ所示产物和载体蛋白偶联得到的偶联物。
4.权利要求3所述赛庚啶抗原的制备方法,包括如下步骤:(1)取式Ⅰ所示的赛庚啶半抗原31.7mg溶于3mlDMF中;(2)加入1ml0.01MPBS助溶,加入EDC 38mg,NHS 38mg,室温活化2h;(3)取载体蛋白100mg溶于10ml的0.1M碳酸氢钠溶液中,将活化物在4℃冰水浴条件下,加入到蛋白中,4℃搅拌反应过夜;(4)置1L0.01M PBS中透析8次,得所述赛庚啶抗原;(5)分装,于-20℃保存备用。
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