免疫胶体金技术

,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,免疫胶体金技术,Immune colloidal gold technique,胶体金标记技术的相关定义,免疫胶体金技术,是以胶体金作为示踪标志物应用于抗原抗 体的一种新型的免疫标记技术。,胶体金标记,实质上是蛋白质等高分子被吸附到胶体金颗粒表面的包被过程。,胶体金技术的发展,Development of Immune colloidal gold technique,1939-,雏形,Kausche,等把烟草花叶病毒吸附到金颗粒上在电子显微镜下观察金离子呈高电子密度。,1971-,作为标记物应用于免疫组织化学研究,Faulk,等首先将兔抗沙门菌抗血清与胶体金颗粒结合，用直接免疫细胞化学技术检测沙门菌的表面抗原。,1974-,实现间接免疫金染色法,Romano,将胶体金标记到马抗人的,IgG,上，实现了间接免疫金染色法,胶体金技术的种类及优点,快速免疫金渗滤法,(immuogold filtration assay,IGFA),即穿流式,(flow through),的固相膜免疫测定。,主要由两部分组成：膜渗滤装置和标记结合物。前者为一塑料小盒，其中填满吸水性物质，面上紧贴放置一片吸附有抗体,(,以双抗体夹心法测抗原为例,),的硝酸纤维膜，标记结合物为免疫金。,A,：金标记抗体,B,：标本中的抗原,C,：包被抗体,D,：,NC,膜,E,：吸水材料,F,：塑料盒,免疫层析法,(immunochromatography,，,ICA),是继,IGFA,之后发展起来的另一种固相膜免疫测定，与,IGFA,利用微局限性膜的过滤性能不同，免疫层析法中滴加在膜一端的样品溶液受膜的毛细管作用,(,基于层析作用的横流（,lateral flow,）,),向另一端移动。移动过程中被分析物 与固定在膜上某一区域的受体,(,抗原或者抗体,),结合而被固相化，无关物质则越过该区域而被分离，然后通过标记物显色来判定试验结果，以胶体金为标记物的实验称为胶体金免疫层析试验。,免疫胶体金技术的特点,优点,：,检测方法简单而快速，数分钟即可得出结果；,不需仪器设备，操作人员不需特殊训练；,试剂稳定，适用于单份测定；,无污染。,GICA,的特点是单一试剂，一步操作。小型实验室即有条件开发生产。,干燥包装的试剂可在室温保存一年以上。,成为目前,“,病人身边检验,”,（,point-of-care testing,，,POCT,）中广为应用的方法。,最近由于原料的精选和制作工艺的改进，已能制备出可用于定量测定的,GICA,试剂。,Roche,公司生产的用于急性心肌梗死诊断的肌钙蛋白和肌红蛋白的,GICA,试剂和匹配的简便测读器,Cardiac Reader,，其精密度和准确性均符合定量测定要求。,不足：,金免疫测定中应用的是单份试剂，难于进行质量控制。即使是同一批生产的试剂，也很难保证每个试剂的同一性。因此金免疫测定一般只能用于定性试验。,其定量检测和灵敏度的提高还有赖于新原料和新材料的开发与应用。,Colloidal Gold Synthesis,Solution color varies extensively with particle size,Usually a deep red,but also dark brown/purple to light orange/yellow,Colloid size can be controlled by Au:Citrate ratios,Anywhere between 1nm-100nm+,Citrate is easily substituted by other more Au-philic molecules(i.e thiols),H,2,O,100,O,C,H,2,AuCl,4,Cit,-,Cit,-,Cit,-,Cit,-,Turkevich,et.al.,Discussions Faraday Soc,.,1951,No.11 55-75.,Procedure of,Colloidal Gold Synthesis,Add 100 mL of 1.0%HAuCl,4,to a 200 mL Erlenmeyer flask on a stirring hot plate.Add a magnetic stir bar and bring the solution to a boil,To the boiling solution,add 1.5 mL of a 1%solution of trisodium citrate dihydrate,Na,3,C,6,H,5,O,7,.2H,2,O,Stop heating when a deep red color is obtained.,Synthesis and p,ropert,ies of different particle size,胶体金粒径,(nm),1%,柠檬酸三钠加入量,(ml),胶体金特性,呈色,max,16,2,橙色,518nm,24.5,1.5,橙色,522nm,41,1,红色,525nm,71.5,0.7,紫红,535nm,TEM Images of Colloidal Au,Average particle size 17nm,Good gold colloids and bad(unstable)gold colloids,劣质金外形不均一，且非球形，有凝集现象，颗粒间变异系数较大,How are the antibodies coupled to the gold?,Conjugation of antibodies to gold particles depends upon three separate but dependent phenomena,:,a).ionic attraction between the negatively charged gold and the positively charged protein,b).hydrophobic attraction between the antibody and the gold surface,c).Sulfur binding(Cysteine and Methionine),Strongest binding suggested to be the sulfur hinge joining the the two F,c,regions,胶体金颗粒带电情况,抗体和金颗粒的耦联（图出处：,IVD Technology,）,SAMPLE GOLD CONJUGATION PROTOCOL,Adjust the gold colloid to the proper pH;,Determined the minimum amount of protein;,Final conjugation,Purification,Adjust the gold colloid to the proper pH,胶体金与蛋白质的结合成功与否，取决于,pH,值，一般只有在蛋白质等电点,(PI),略偏碱的条件下二者才能牢固地结合，因此，标记之前须将胶体金溶液的,pH,值调至待标记蛋白质的等电点略偏碱（如此可是蛋白带负电荷）。,需要提高胶体金的,pH,值时可用,0.1molK,2,CO,3,，需要降低胶体金的,pH,值时可用,0.1N HCl,。测定金溶液的,pH,可能损害,pH,测定计的探头，因此，一般用精密的,pH,试纸测定其,pH,即可,.,蛋白 与胶体金结合的最佳,pH,测定,取若干,1.5ml,的试管，分别加入,1ml,胶体金,用,K,2,CO,3,将,pH,分别调为,3,，,4,，,5,，,6,，,7,，,8,，,9,，,10,；,去,96,孔培养板，按,pH,从高到低分别将上述胶体金分别取,100ul,加入孔中，重复三次；,每孔分别加入,20ul,浓度为,10%,的,NaCl,溶液，混合，室温下放置,10min,；,观察胶体金颜色变化，记录保持红色的最低,pH,Adjust the gold colloid to the proper pH,常用几种蛋白质标记时胶体金所用的,pH,值,Determined the minimum amount of protein,（,1,）光电比色法：制备一系列不同浓度的等体积蛋白质溶液（,1ml,），分别加入,5ml,胶体金中，迅速混匀，然后，各加入,1ml 10%NaCl,溶液，摇匀，静置,5min,后测各管。根据胶体金颗粒的大小，,OD,在,520,580nm,之间测定，以,OD,值为纵坐标，蛋白质用量为横坐标作一曲线，取曲线最先与横轴相接近的那一点处的蛋白质用量为最适稳定量。图,5.2,中,10nm,的胶体金溶液中蛋白质的最适稳定量为,45g/ml,。,（,2,）目测法：以目测法确定胶体金与待标记蛋白质用量比例，将标记的蛋白质逐级稀释后（由,5g,45g,另设对照管），各取等体积顺序加入一系列装有,1ml,胶体金的试管中，,5min,后，在上述各管内分别加入,0.1ml 10%,氯化钠，依表,5-5,顺序进行。,1.Pipette 1ml of colloid into each of a series of 3ml clean plastic tubes.,2.Adjust the antibody(0.1ug/ul)to the proper pH with 100nM K2CO3 or 100mM HC1.,3.Add the antibody to each tube in a series from 0-150ul(ie 0-15ug in steps of 0,1,2,3.15ug).,4.Shake each tube and leave for approximately 5 minutes to conjugate.,6.To each tube add 100ul of 10%NaC1 and agitate for 1 minute.,7.The Tube containing the minimum amount of protein required to stabilize the gold sol is indicated by the one in which the color of the gold sol does not change from red to blue upon the addition of NaCl.,Final conjugation(IgG),Take 100ml of gold sol and adjust to pH9,Adjust the dialyzed and centrifuged antibody solution(0.1ug/ul)to pH9.2,Add the determined amount of antibody solution dropwise to the gold while stirring rapidly,After 5 minutes,add 10ml of filtered 10%BSA at pH9 and stir gently for 10 minutes.,Purification,The gold conjugate must be purified from excess antibody and any small clusters removed before concentrating and stirring.,Spin the gold conjugate at speeds according to gold particle size.,The gold conjugate