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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,*,实验五 1.酶联免疫吸附试验ELISA)2.补体结合反响,1.酶联免疫吸附试验ELISA),免疫标记技术,定义：,将抗原-抗体反响与标记技术相结合，用放射性同位素、酶或荧光素标记的抗体或抗原，通过检测标记物，间接测定未知的抗原或抗体。,分类：,放射免疫测定法：131I，32P,免疫荧光技术：FITC，PE,免疫酶测定法：HRP，AP,免疫酶测定法,(,E,nzyme,I,mmuno,A,ssay,EIA),用酶标记的抗原或抗体进行的抗原抗体反响。,辣根过氧化物酶(HRP)，碱性磷酸酶(AP),特点：,高度特异性：保持抗原抗体反响的特异性,高灵敏度：酶催化底物显色的高效性，pg水平微量检测,定性定量检测：反响液颜色深浅或光密度值,分类：,酶联免疫吸附试验：可溶性抗原或抗体,酶免疫组化法：组织中或细胞外表的抗原。,酶联免疫吸附试验,(,E,nzyme-,L,inked,I,mmuno,S,orbent,A,ssay,ELISA),用酶标记抗原或抗体检测液体血液、细胞液中未知抗体或抗原的方法。,1.定义,2.分类,间接法Indirect ELISA：,将抗原吸附于固相载体，酶标记二抗,检测液相中未知抗体,双抗体夹心法Sandwich ELISA)：,将抗体吸附于固相载体，酶标记一抗,检测液相中的可溶性抗原,竞争法Competitive ELISA)：,将抗体或抗原吸附于固相载体，酶标记抗原或抗体,检测液相中的可溶性抗原或抗体,酶联免疫吸附试验,(,E,nzyme-,L,inked,I,mmuno,S,orbent,A,ssay,ELISA),3.原理以间接性ELISA为例：,显色,实验内容：,间接法测定溶血素 定性检测,实验材料,脱纤维绵羊红细胞 抗原,兔抗绵羊红细胞抗体溶血素 待测兔血清,HRP标记羊抗兔IgG 二抗,包被缓冲液：0.05M pH9.6碳酸盐缓冲液,洗涤液：含0.05%Tween 20的0.01M pH7.4 PBS,底物缓冲液：pH5.0磷酸盐柠檬酸缓冲液,底物溶液：OPD 10mg，底物缓冲液25ml，30%H2O2 40L,酶标板，酶标仪,实验方法,1.抗原的处理：,脱纤维绵羊血,加3mL N.S，混匀,2000rpm，5min,加3mL N.S，混匀,2000rpm，5min,取2mL压积红细胞,参加2mL蒸馏水，震荡破碎红细胞,用包被缓冲液稀释成1：400备用,2.包被抗原：,取酶标板，参加100L/孔的抗原稀释液,4，湿盒内，18h,取出包被好抗原的酶标板4孔/组，甩干孔内液体,以洗涤液200L/孔洗涤3次，3min/次,3.加待测血清,标记各孔，参加相应液体100L/孔,1,2,3,4,阴性,空白,阳性,待测,4.加二抗：,37，湿盒内，30min,甩干孔内液体,以洗涤液200L/孔洗涤3次，3min/次,各孔参加酶标二抗100L/孔,37，湿盒内，30min,5.显色：,甩干孔内液体,以洗涤液200L/孔洗涤3次，3min/次,参加底物溶液100L/孔,避光显色，10min,6.观察结果,实验方法,预期结果,阳性：显色为黄色,阴性：无色,1 2 3 4,阴性,阴性,阳性,阳性,本卷须知,洗涤彻底，防止交叉污染。,按顺序加样，孔底无气泡。,包被和温育在湿盒内进行。,2.补体结合反响Complement fixation Test,CFT,概念：,补体结合反响：,是指在补体的参与下，以绵羊红细胞SRBC和溶血素抗SRBC的抗体作为指示系统的抗原抗体反响。,补反中包括两个系统五种成分：,待检系统：Ag或Ab、待检Ab或Ag及仅供一组Ag-Ab系统反响的定量补体。,指示系统：绵羊红细胞和溶血素。,原理：,待测系统,抗原+未知血清,1,2,补体,1,2,Step 1,抗原抗体复合物的形成,Step 2,补体的结合和耗竭,指示系统,SRBC+抗SRBC抗体,Step 3,SRBC的裂解,1,2,1：No Ab；,2：Ab,Ag,待测血清,Ab,阳性,No Ab,阴性,Ag,Ag,Ag,抗原：伤寒菌裂解产物,待测血清：56，30min,补体：豚鼠血清,2%绵羊红细胞,溶血素,材料：,方法：,1,2,3,4,5,试验管,血清,对照管,抗原,对照管,补体,对照管,SRBC,对照管,待测血清,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.4mL,0.8mL,抗原,补体,N.S,摇匀，37，水浴15min,溶血素,SRBC,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,0.2mL,摇匀，37，水浴15min,1份/2人,预期结果：,1管内液体混浊呈浅红色为不溶血，管内液体透明呈红色为溶血。,溶血,不溶血,2.,试验管,血清对照管,抗原对照管,补体对照管,SRBC对照管,？,溶血,溶血,溶血,不溶血,绵羊红细胞用前应轻晃混匀，不可剧烈震荡。,各种试剂的吸管不可混用。,本卷须知：,0C4F7IaMdPgSkVnZq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t*w-z1D4G7JbMeQhTkWoZr$u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2E5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp#s&v)y0C3F6IaLdPgSjVnYq!t*w-A1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-A1D5G8JbNeQ!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfRiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t*w-z1D4G7JbMeQhTkWoZr$u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*2E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMdPhSkWnZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZv(y+B3E6I9LcOgRjUmYp!s&w)z1C4F7JaMdPhSkWnZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZry0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhSkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-E5H8KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%0C4F7IaMdPgSkVnZq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfQiU
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