CN105646536B - 一种头孢噻呋半抗原及其胶体金检测装置及其制备方法 - Google Patents
一种头孢噻呋半抗原及其胶体金检测装置及其制备方法 Download PDFInfo
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- CN105646536B CN105646536B CN201511016840.8A CN201511016840A CN105646536B CN 105646536 B CN105646536 B CN 105646536B CN 201511016840 A CN201511016840 A CN 201511016840A CN 105646536 B CN105646536 B CN 105646536B
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- ceftiofur
- monoclonal antibody
- haptens
- colloidal gold
- mouse
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
- C07D501/14—Compounds having a nitrogen atom directly attached in position 7
- C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
- C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
- C07D501/24—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids with hydrocarbon radicals, substituted by hetero atoms or hetero rings, attached in position 3
- C07D501/36—Methylene radicals, substituted by sulfur atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
- C07D501/02—Preparation
- C07D501/04—Preparation from compounds already containing the ring or condensed ring systems, e.g. by dehydrogenation of the ring, by introduction, elimination or modification of substituents
-
- 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
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- 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
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- 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
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- G—PHYSICS
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- 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
- G01N33/532—Production of labelled immunochemicals
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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Abstract
本发明提供了一种头孢噻呋半抗原及其胶体金检测装置及其制备方法,所述头孢噻呋半抗原,为具有式(1)所示的化学结构式。采用本发明的技术方案,特异性强,检测灵敏度高,准确性高,重复性好,能更加快速、简便地检测头孢噻呋残留;不需要任何仪器设备,便于携带;试纸条使用简单,无需专业人士操作;试纸制作容易,成本低廉,满足了食品安全检测中对头孢噻呋残留量检测需求。
Description
技术领域
本发明属于生物检测技术领域,尤其涉及一种头孢噻呋半抗原及其胶体金检测装置及其制备方法。
背景技术
头孢噻呋是一种亲脂弱碱性乙胺嘧啶类抑菌剂,其抗菌谱和磺胺类药物相似,但抗菌作用较磺胺类药物强,对大肠杆菌、奇异变形杆菌、肺炎杆菌、腐生葡萄球菌、多种革兰阳性和阴性细菌有效,但对绿脓杆菌感染无效,最低抑菌浓度常低于10mg/L,单用易引起细菌耐药性,故一般不单独使用,主要与磺胺药组成复方制剂。因此它又被称为磺胺增效剂,用于扩大抗菌谱,增强抗菌活性。临床上用于治疗尿路感染、肠道感染、呼吸道感染、菌痢、肠炎、伤寒、脑膜炎、中耳炎、流脑、败血症及软组织感染等。可与长效磺胺药合用于耐药恶性疟的防治。随着磺胺类药物在兽医临床上不合理的使用,甚至滥用,以至于抗菌增效剂类药物在动物源性食品中的残留会不可避免地发生。人类长期食用含有头孢噻呋残留的动物性食品及其制品,将有可能对人类健康产生危害。因此为了加强对动物性食品中这类药物的监控,建立一种准确、快速、方便的头孢噻呋残留量检测方法很有必要。
目前,国内外检测头孢噻呋的方法主要有高效液相色谱法(HPLC),液相-质谱联用(LC-MASS)法和酶联免疫吸附(ELISA)法。在食品安全检测中,往往先用ELISA初筛后再对阳性样本用HPLC或LC-MASS进行确证。上述方法中所用仪器设备安规复杂、成本高、对操作人员技术要求高且不能立即显示结果,因此不适用于商检、防疫、畜牧生产者对怀疑对象进行快速的在线检测和监控。
发明内容
针对以上技术问题,本发明公开了一种头孢噻呋半抗原及其胶体金检测装置及其制备方法,针对现有检测头孢噻呋技术的不足,建立一种检测头孢噻呋残留的快速方法,使其能更加快速、灵敏、简便地检测头孢噻呋残留,满足了快速检测的需要。
对此,本发明采用的技术方案为:
一种头孢噻呋半抗原,其具有式(1)所示的化学结构式:
式(1)。
上述分子式的化学名为(6R,7R)-7-[2-(2-氨基丙酸噻唑-4-基)-(Z)-2-(甲基亚胺基)乙酰胺基]-3-[(2-呋喃基羰基)硫甲基]-3-头孢烯-4-羧酸,其分子式为C22H21N5O9S3,MASS[M+H]峰值为596.4,熔点为249度。
作为本发明的进一步改进,所述的头孢噻呋半抗原采用以下步骤制备得到:
步骤S1:将头孢噻呋钠溶解于甲醇中,加入冰乙酸,于20~30℃下搅拌10~120分钟,其中,所述冰乙酸的摩尔量是头孢噻呋钠摩尔量的1~3倍;再加入水,二氯甲烷萃取,旋干有机相,得到淡粉红色固体;
步骤S2:取步骤S1中得到的淡粉红色固体溶于DMF(二甲基甲酰胺)中,加入碳酸钾及溴丙酸,35~45℃下反应2~8小时,采用二氯甲烷萃取过柱提纯得所述头孢噻呋半抗原。
本发明还公开了一种头孢噻呋免疫抗原,采用如上所述的头孢噻呋半抗原制备得到,将所述头孢噻呋半抗原与DCC(Dicyclohexylcarbodiimide,二环己基碳二亚胺)、NHS(N-羟基琥珀酰亚胺)混合,于4℃下搅拌,离心后取上清液;将人血清蛋白溶于pH 值为8.0的PBS溶液中,加入DMF后搅拌,然后将所述上清液逐渐加入其中,4℃下反应 12h,离心后,取上清液,透析纯化,得到所述头孢噻呋免疫抗原。
作为本发明的进一步改进,所述透析纯化时,在4℃下用生理盐水透析3天,每天更换3次透析液。
本发明还公开了一种头孢噻呋单克隆抗体,采用如上所述的头孢噻呋免疫抗原与免疫Balb/c小鼠经细胞融合,筛选得到分泌头孢噻呋单克隆抗体的杂交瘤细胞,用获得的杂交瘤细胞采用体内诱生腹水法制备得到头孢噻呋单克隆抗体。
优选的,所述头孢噻呋单克隆抗体采用以下方法制备得到:使用所述头孢噻呋免疫抗原并鉴定后免疫4只6周龄昆明小鼠,加强免疫三次后,采血测效价,待血清效价不再上升,用两倍剂量的抗原不加佐剂免疫小鼠,三天后脱颈致死小鼠,在无菌条件下取脾脏制备脾细胞,与生长旺盛的小鼠骨髓瘤细胞按8:1的比例混合于50mL离心管,加入30mL无血清IPMI1640培养基,1100r/min离心5分钟弃上清,将细胞团轻轻振松,置于37摄氏度水浴中。把 1mL50%(体积百分比)PEG-4000缓缓加入细胞中,在1分钟内滴完,同时轻轻搅动底部沉淀,静置1分钟后,前30秒沿管壁缓慢匀速加入无血清培养基 1mL,后30秒加入2mL,然后快速加入27mL终止融合过程,1100r/min离心5分钟,弃上清,用HAT选择性培养基重悬后加到已铺有饲养细胞的96 孔细胞培养板中,37摄氏度、体积百分比5%的CO2条件下培养。7天后换成HT培养液,待孔内的杂交细胞数量达到300个以上时,用间接ELISA(enzyme linkedimmunosorbent assay,酶联免疫吸附测定)法筛选,选择强阳性、抑制效果好、细胞生长旺盛的孔进行有限稀释克隆化,经3次以上的克隆培养和检测,均呈阳性的孔内细胞即为分泌单克隆抗体的杂交瘤细胞,将杂交瘤细胞扩大培养以备单克隆抗体的制备;然后,采用体内诱生腹水法生产头孢噻呋单克隆抗体。
作为本发明的进一步改进,所述体内诱生腹水法的步骤为:对小鼠腹腔注射液体石蜡油 0.5mL/只,7天后腹腔注射所述杂交瘤细胞3~5×106/只,10天后,待小鼠腹部明显膨大时收集腹水,用正辛酸-硫酸铵沉淀法来纯化腹水得到所述头孢噻呋单克隆抗体。
本发明还公开了一种头孢噻呋胶体金检测装置,其包括反应杯和检测试纸,所述检测试纸包括底板,以及底板上依次铺设的样品垫、硝酸纤维素膜和吸水纸;所述反应杯里含有胶体金标记的如上所述头孢噻呋单克隆抗体,所述硝酸纤维素膜上设有检测线和控制线,所述检测线为采用如上所述的头孢噻呋免疫抗原在硝酸纤维素膜上进行喷涂制得。
作为本发明的进一步改进,所述控制线为采用兔抗鼠IgG进行喷涂制得,所述检测线和控制线相互平行。优选的,所述检测线和控制线距离至少0.5cm。
优选的,所述兔抗鼠IgG是通过以下方法获得:
用载体蛋白结合抗原免疫新西兰白兔,免疫剂量为50μg~100μg/次,背部皮下分多点注射,其中所述载体蛋白为常规的载体蛋白,优选人血清白蛋白、卵清蛋白或牛血清白蛋白;首免,用合成的人工抗原与等量弗氏完全佐剂乳化;加强免疫,用合成的人工抗原与等量弗氏不完全佐剂乳化,连续免疫4~5次,每次间隔4~8周,最后一次免疫后10~15天,以ELISA法测其定效价达到105以上时,采血并分离收集高免血清,以饱和硫酸铵盐析法提取兔抗鼠IgG抗体,-20℃冻存备用。
本发明还公开了如上所述的头孢噻呋胶体金检测装置的制备方法,其包括以下步骤:
步骤A:将头孢噻呋钠溶解于甲醇中,加入冰乙酸,于20~30℃下搅拌10-120分钟,其中,所述冰乙酸的摩尔量是头孢噻呋钠摩尔量的1~3倍;再加入水,用二氯甲烷萃取,旋干有机相,得到淡粉红色固体;将淡粉红色固体溶于DMF中,加入碳酸钾及溴丙酸,35~45℃下反应2~8小时,采用二氯甲烷萃取过柱提纯得所述头孢噻呋半抗原;利用碳二亚胺法将所述头孢噻呋半抗原与载体蛋白牛血清偶联,制备得到头孢噻呋免疫抗原;
优选的,所述头孢噻呋免疫抗原的制备步骤为将所述头孢噻呋半抗原与DCC、NHS混合,于4℃下搅拌,离心后取上清液;将人血清蛋白溶于pH 值为8.0的PBS溶液中,加入DMF后搅拌,然后将所述上清液逐渐加入其中,4℃下反应 12h,离心后,取上清液,透析纯化,得到所述头孢噻呋免疫抗原。
步骤B:将所述头孢噻呋免疫抗原与免疫Balb/c小鼠经细胞融合,筛选得到分泌头孢噻呋单克隆抗体的杂交瘤细胞,用获得的细胞诱导小鼠产生腹水,纯化后获得头孢噻呋单克隆抗体;
步骤C:用柠檬酸三钠与氯金酸反应制备胶体金;将胶体金与所述头孢噻呋单克隆抗体混合形成金标抗体,离心复溶后得到胶体金标记的头孢噻呋单克隆抗体;
步骤D:将步骤C得到的胶体金标记的头孢噻呋单克隆抗体分装至反应杯中,低温干燥;将所述头孢噻呋免疫抗原在硝酸纤维素膜上进行喷涂制得检测线,将兔抗鼠IgG在硝酸纤维素膜上进行喷涂制得控制线;
步骤E:沿同一方向依次铺设的样品垫、硝酸纤维素膜和吸水纸并粘附在底板上,组装得到所述检测试纸。
优选的,所述头孢噻呋免疫抗原的制备方法为:取头孢噻呋半抗原 0.1mmol溶于2mL DMF(二甲基甲酰胺)中,搅拌加入27.5mg DCC和14.4mgNHS。4℃下磁力搅拌反应过夜,离心后上清液A液,称取人血清蛋白 (HSA)140mg 溶于10mL 浓度0.1mol/L的pH为8.0的PBS(磷酸盐缓冲液)中。加入DMF 1mL,搅拌溶解制备B液,磁力搅拌下,A液逐渐滴B液中,4℃下反应12h。离心后,取上清液,4℃下用生理盐水透析3d,每天更换3次透析液,将得到的所述头孢噻呋免疫抗原以1mg/mL的浓度分装于0.5mL 离心管中,冻存于 -20℃冰箱中备用。
其中,优选的,所述胶体金的制备方法优选为:取1%(质量百分比)氯金酸溶液1ml,加99ml超纯水成终浓度0.01%(质量百分比)的氯金酸溶液,加热沸腾后,取1%(质量百分比)柠檬酸三钠1.6ml一次性迅速加入煮沸的氯金酸溶液中,继续加热至溶液由淡黄色转为蓝黑色最终变为亮红色,颜色稳定后继续加热5min,室温冷却,补充失水至原体积。
优选的,所述胶体金标记的头孢噻呋单克隆抗体制备步骤优选为:调节胶体金溶液pH值至8.0,用恒速搅拌器均匀搅拌,同时逐滴加入头孢噻呋的单克隆抗体,1小时后加入抗体量相当的PEG,充分反应30分钟后加入抗体量相当的BSA,加完后,继续搅拌30分钟。在9000rpm下离心30分钟获得均一性金标抗体沉淀,再加PNPB重悬得到胶体金标记的头孢噻呋单克隆抗体。
作为本发明的进一步改进,还包括将粘好的所述检测试纸切成等宽度的试纸条,将所述反应杯及试纸条密封干燥保存。
本发明检测原理是利用样品垫形成的毛细管虹吸效应,使被检测物质首先与胶体金标记的头孢噻呋单抗发生竞争形式的结合,其结果是,当胶体金标记的头孢噻呋单克隆抗体过量时,多余的头孢噻呋单克隆抗体泳动到检测线,与头孢噻呋免疫抗原结合并显色;而与检测物结合的胶体金标记的头孢噻呋多抗,其V区结合位点被检测物质占据,只能跨越检测线泳动到质控线以C区位点与兔抗鼠 IgG 抗体非特异性结合,同检测线进行比色而得到检测结果。
本发明的有益效果:
第一,采用本发明的技术方案,特异性强,检测灵敏度高,灵敏度可达0.5ppm,CV值小于15%,准确性高,重复性好,能更加快速、灵敏、简便地检测头孢噻呋残留。
第二,采用本发明技术方案的头孢噻呋胶体金检验装置,不需要任何仪器设备,便于携带,检测成本低;试纸条使用简单,无需专业人士操作;试纸制作容易,成本低廉,储存方便,稳定性好,在室温下至少可以保存六个月。
第三,本发明的技术方案的胶体金检测装置,应用层析式免疫胶体金原理,通过检测试纸中的检测线与质控线线比色,来半定量检测样品中的头孢噻呋残留量,在短时间内快速准确地检测出样品是否含有头孢噻呋,以确定头孢噻呋是否超标,能够满足食品安全对头孢噻呋残留量检测需求,适用于屠宰企业及政府检测机构。
附图说明
图1是本发明一种实施例头孢噻呋半抗原的MASS谱图。
具体实施方式
下面对本发明的较优的实施例作进一步的详细说明。:
实施例1
头孢噻呋半抗原的制备,采用以下步骤制备:
步骤S1:将0.1mM 头孢噻呋钠置于甲醇中溶解,加入1~3倍头孢噻呋钠摩尔量的冰乙酸,于室温下搅拌30分钟;加入水,用二氯甲烷萃取;旋干有机相,得到淡粉红色固体。
步骤S2:取步骤S1中得到的淡粉红色固体溶于DMF中,加入碳酸钾及溴丙酸,于40℃反应2-8小时,采用二氯甲烷萃取过柱提纯得所述头孢噻呋半抗原。
对得到的所述头孢噻呋半抗原经过测试,MASS[M+H]峰值为596.4,如图1所示;其熔点为249度。
实施例2
利用头孢噻呋半抗原制备头孢噻呋免疫抗原,采用以下步骤:
取头孢噻呋半抗原 0.1mmol 溶于2mL的DMF中,搅拌加入27.5mg DCC和14.4mgNHS。4℃下磁力搅拌反应过夜,离心后上清液为A液,称取人血清蛋白 (KLH)140mg溶于10mL浓度为0.1mol/L的pH为8.0的PBS中。加入DMF 1mL,搅拌溶解制备B液,磁力搅拌下,A液逐渐滴入B液中,4℃下反应 12h。离心后,取上清液,4℃下用生理盐水透析3天,每天更换3次透析液。得到的全抗原以1mg/mL的浓度分装于0.5mL离心管中,冻存于-20℃冰箱中。
实施例3
利用头孢噻呋免疫抗原制备头孢噻呋单克隆抗体,采用以下步骤:
使用头孢噻呋免疫抗原并鉴定后免疫4只6周龄昆明小鼠,加强免疫三次后,采血测效价,待血清效价不再上升,用两倍剂量的抗原不加佐剂免疫小鼠,三天后脱颈致死小鼠,在无菌条件下取脾脏制备脾细胞,与生长旺盛的小鼠骨髓瘤细胞按8:1的比例混合于50mL的离心管中,加入30mL的无血清IPMI1640培养基,1100r/min离心5分钟弃上清,将细胞团轻轻振松,置于37℃水浴中;把 1mL的体积浓度为50%的PEG-4000缓缓加入细胞中,在1分钟内滴完,同时轻轻搅动底部沉淀,静置1分钟后,前30秒沿管壁缓慢匀速加入无血清培养基 1mL,后30秒加入2mL,然后快速加入27mL终止融合过程,1100r/min离心5分钟,弃上清,用HAT选择性培养基重悬后加到已铺有饲养细胞的96 孔细胞培养板中,37摄氏度、体积百分比5%的CO2条件下培养。7天后换成HT培养液,待孔内的杂交细胞数量达到300个以上时,用间接ELISA法筛选,选择强阳性、抑制效果好、细胞生长旺盛的孔进行有限稀释克隆化,经3次以上的克隆培养和检测,均呈阳性的孔内细胞即为分泌单克隆抗体的杂交瘤细胞,将杂交瘤细胞扩大培养以备单克隆抗体的制备。
然后,采用体内诱生腹水法生产头孢噻呋单克隆抗体。具体步骤为:选4只经产昆明小鼠,腹腔注射液体石蜡油 0.5mL/只,7天后腹腔注射杂交瘤细胞3~5×106/只,10天后,待小鼠腹部明显膨大时收集腹水。用正辛酸-硫酸铵沉淀法来纯化腹水,得到头孢噻呋单克隆抗体,经紫外测定头孢噻呋单克隆抗体的含量。
实施例4
头孢噻呋胶体金检测装置的制备,包括以下步骤:
(1)胶体金的制备
取1%(质量百分比)氯金酸溶液1ml,加99ml超纯水成终浓度0.01%(质量百分比)的氯金酸溶液,加热沸腾后,取1%(质量百分比)柠檬酸三钠1.6ml一次性迅速加入煮沸的氯金酸溶液中,继续加热至溶液由淡黄色转为蓝黑色最终变为亮红色,颜色稳定后继续加热5min,室温冷却,补充失水至原体积。
(2)胶体金标记头孢噻呋单克隆抗体的制备
调节胶体金溶液pH值至8.0,用恒速搅拌器均匀搅拌,同时逐滴加入头孢噻呋单克隆抗体,1小时后加入抗体量相当的PEG,充分反应30分钟后加入抗体量相当的BSA,加完后,继续搅拌30分钟。在9000rpm 下离心30 分钟,获得均一性的金标抗体沉淀,再加PNPB重悬备用,得到胶体金标记头孢噻呋单克隆抗体。
(3)胶体金检测装置的制备
在硝酸纤维素膜上喷涂头孢噻呋免疫抗原溶液形成检测线T线,采用喷涂兔抗鼠IgG形成控制线T线。所述检测线和控制线相互平行,两者距离0.5cm。
然后,在底板上,沿同一方向依次将样品垫、喷涂有头孢噻呋免疫抗原的检测线T线和喷涂有兔抗鼠IgG的控制线T线的硝酸纤维素膜和吸水纸依次搭接粘连,其中,检测线靠近样品垫,所述控制线靠近吸水纸,将粘好的所述检测试纸切成等宽度的试纸条;反应杯内加入胶体金标记头孢噻呋单克隆抗体,冻干;将所述反应杯及试纸条密封干燥保存。
实施例5
样品中头孢噻呋的检测,方法为:
取新鲜鸡组织样品绞碎,头孢噻呋残留经乙腈和丙酮提取,正己烷除脂肪,取样置于反应杯中,室温孵育10分钟,随后插入试纸条,室温孵育3分钟。取出试纸条,轻轻刮除试海绵垫,进行结果判读。若T线与C线同时显示紫红色条带,则结果为阴性;若T线颜色比C线浅或C线显色而T线不显色,则结果为阳性;若C线、T线均不显色,则检测装置已失效。
实施例6
头孢噻呋胶体金检测装置的灵敏度检测。
通过加标实验,发现本发明中头孢噻呋胶体金检测装置的灵敏度可达0.5ppm,CV值小于15%。
实施例7
头孢噻呋胶体金检测装置的特异性实验。
在阴性的鱼肉组织中,分别加入头孢噻呋0.5ppm,四环素、青霉素、头孢匹林、红霉素、林可霉素、泰乐菌素、链霉素、卡那霉素、庆大霉素、磺胺二甲嘧啶各20ppm,按实施例5的操作步骤进行检测,发现本装置对头孢噻呋有优异的选择性,与以上添加药物无交叉反应。
实施例8
头孢噻呋胶体金检测装置的保质期实验。
用三批常规生产的产品分别做保质期实验,放置于室内室温环境保持,每隔1个月取12个装置,用质控样本检测,分别做阴性,分别为0.25ppm,0.5ppm和1ppm浓度的样品,重复三次,观察数据变化,考察保质期时间。阴性显色从14个月开始下降,在1年时间内产品品质无明显变化,因此确定保质期为1年。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (9)
1.一种头孢噻呋半抗原,其特征在于:其具有式(1)所示的化学结构式:
2.一种头孢噻呋免疫抗原,其特征在于:采用权利要求1所述的头孢噻呋半抗原制备得到,将所述头孢噻呋半抗原与DCC、NHS混合,于4℃下搅拌,离心后取上清液;将人血清蛋白溶于pH值为8.0的PBS溶液中,加入DMF后搅拌,然后将所述上清液逐渐加入其中,4℃下反应6~24h,离心后,取上清液,透析纯化,得到所述头孢噻呋免疫抗原。
3.根据权利要求2所述的头孢噻呋免疫抗原,其特征在于:所述透析纯化时,在4℃下用生理盐水透析3天,每天更换3次透析液。
4.一种头孢噻呋单克隆抗体,其特征在于:采用权利要求2或3所述的头孢噻呋免疫抗原与免疫Balb/c小鼠经细胞融合,筛选得到分泌头孢噻呋单克隆抗体的杂交瘤细胞,用获得的杂交瘤细胞采用体内诱生腹水法制备得到头孢噻呋单克隆抗体。
5.根据权利要求4所述的头孢噻呋单克隆抗体,其特征在于:所述体内诱生腹水法的步骤为:对小鼠腹腔注射液体石蜡油0.5mL/只,7天后腹腔注射所述杂交瘤细胞3~5×106/只,10天后,待小鼠腹部明显膨大时收集腹水,用正辛酸-硫酸铵沉淀法来纯化腹水得到所述头孢噻呋单克隆抗体。
6.一种头孢噻呋胶体金检测装置,其特征在于:包括反应杯和检测试纸,所述检测试纸包括底板,以及底板上依次铺设的样品垫、硝酸纤维素膜和吸水纸;所述反应杯里含有胶体金标记的如权利要求4所述的头孢噻呋单克隆抗体,所述硝酸纤维素膜上设有检测线和控制线,所述检测线为采用权利要求2或3所述的头孢噻呋免疫抗原在硝酸纤维素膜上进行喷涂制得。
7.根据权利要求6所述的头孢噻呋胶体金检测装置,其特征在于:所述控制线为采用兔抗鼠IgG进行喷涂制得,所述检测线和控制线相互平行。
8.根据权利要求6或7所述的头孢噻呋胶体金检测装置的制备方法,其特征在于,其包括以下步骤:
步骤A:将头孢噻呋钠溶解于甲醇中,加入冰乙酸,于20~30℃下搅拌10~120分钟,其中,所述冰乙酸的摩尔量是头孢噻呋钠摩尔量的1~3倍;再加入水,二氯甲烷萃取,旋干有机相,得到淡粉红色固体;将淡粉红色固体溶于DMF中,加入碳酸钾及溴丙酸,35~45℃下反应2~8小时,采用二氯甲烷萃取过柱提纯得所述头孢噻呋半抗原;利用碳二亚胺法将所述头孢噻呋半抗原与载体人血清蛋白偶联,制备得到头孢噻呋免疫抗原;
步骤B:将所述头孢噻呋免疫抗原与免疫Balb/c小鼠经细胞融合,筛选得到分泌头孢噻呋单克隆抗体的杂交瘤细胞,用获得的细胞诱导小鼠产生腹水,纯化后获得头孢噻呋单克隆抗体;
步骤C:用柠檬酸三钠与氯金酸反应制备胶体金;将胶体金与所述头孢噻呋单克隆抗体混合形成金标抗体,离心复溶后得到胶体金标记的头孢噻呋单克隆抗体;
步骤D:将步骤C得到的胶体金标记的头孢噻呋单克隆抗体分装至反应杯中,低温干燥;将所述头孢噻呋免疫抗原在硝酸纤维素膜上进行喷涂制得检测线,将兔抗鼠IgG在硝酸纤维素膜上进行喷涂制得控制线;
步骤E:沿同一方向依次在底板上铺设样品垫、硝酸纤维素膜和吸水纸并粘附在底板上,组装得到所述检测试纸。
9.根据权利要求8所述的头孢噻呋胶体金检测装置的制备方法,其特征在于:还包括将粘好的所述检测试纸切成等宽度的试纸条,将所述反应杯及试纸条密封干燥保存。
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