使用2,4-二硝基苯基抗HIV-1 p24 IgG和间接固定化（抗2,4-二硝基苯基基团）Fab的HIV-1 p24抗原超敏免疫复合物转移酶免疫测定法，血清干扰较少
Ultrasensitive immune complex transfer enzyme immunoassay of HIV-1 p24 antigen with less serum interference using 2,4-dinitrophenyl-anti-HIV-1 p24 IgG and indirectly immobilized (anti-2,4-dinitrophenyl group) Fab.
作者信息
Ishikawa S, Hashida S, Hashinaka K, Saito A, Takamizawa A, Shinagawa H, Ishikawa E
机构信息
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Japan.
出版信息
J Clin Lab Anal. 1999;13(3):126-32. doi: 10.1002/(sici)1098-2825(1999)13:3<126::aid-jcla7>3.0.co;2-3.
In the immune complex transfer enzyme immunoassay previously reported, the immune complex consisting of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-HIV-1 p24 Fab' conjugate, HIV-1 p24 antigen and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate was trapped on polystyrene beads coated directly with affinity-purified (anti-2,4-dinitrophenyl group) IgG and was transferred to polystyrene beads coated with biotinyl-bovine serum albumin and streptavidin. The serum volume used was limited to 10 microL due to serious serum interference, and the detection limit of HIV-1 p24 antigen was 240 fg/mL serum. In the present study, HIV-1 p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-affinity-purified rabbit anti-HIV-1 p24 IgG and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate in the presence of excess nonspecific rabbit IgG. The immune complex of the three components formed was trapped on polystyrene beads coated successively with biotinyl-bovine serum albumin, streptavidin and biotinyl-affinity-purified (anti-2,4-dinitrophenyl group) Fab'. After washing, the immune complex was eluted from the polystyrene beads with excess epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads coated with affinity-purified goat (antirabbit IgG) IgG. The serum volume used was increased to 90 microL with only slight serum interference, and the detection limit of HIV-1 p24 antigen was lowered 9-fold to 26 fg/mL serum.
在先前报道的免疫复合物转移酶免疫测定中，由2,4 - 二硝基苯基 - 生物素化 - 牛血清白蛋白 - 亲和纯化兔抗HIV - 1 p24 Fab'缀合物、HIV - 1 p24抗原和单克隆小鼠抗HIV - 1 p24 Fab'-β - D - 半乳糖苷酶缀合物组成的免疫复合物被捕获在直接包被有亲和纯化（抗2,4 - 二硝基苯基基团）IgG的聚苯乙烯珠上，并转移至包被有生物素化 - 牛血清白蛋白和链霉亲和素的聚苯乙烯珠上。由于严重的血清干扰，所用血清体积限制为10微升，HIV - 1 p24抗原的检测限为240 fg/mL血清。在本研究中，HIV - 1 p24抗原在过量非特异性兔IgG存在的情况下，与2,4 - 二硝基苯基 - 亲和纯化兔抗HIV - 1 p24 IgG和单克隆小鼠抗HIV - 1 p24 Fab'-β - D - 半乳糖苷酶缀合物同时孵育。形成的三种成分的免疫复合物被捕获在依次包被有生物素化 - 牛血清白蛋白、链霉亲和素和生物素化 - 亲和纯化（抗2,4 - 二硝基苯基基团）Fab'的聚苯乙烯珠上。洗涤后，用过量的εN - 2,4 - 二硝基苯基 - L - 赖氨酸从聚苯乙烯珠上洗脱免疫复合物，并转移至包被有亲和纯化山羊（抗兔IgG）IgG的聚苯乙烯珠上。所用血清体积增加至90微升，仅伴有轻微的血清干扰，HIV - 1 p24抗原的检测限降低9倍至26 fg/mL血清。
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