Ishikawa E, Ishikawa S, Hashida S, Hashinaka K
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Japan.
J Clin Lab Anal. 1998;12(3):154-61. doi: 10.1002/(SICI)1098-2825(1998)12:3<154::AID-JCLA5>3.0.CO;2-9.
In order to reduce the nonspecific signal of noncompetitive solid phase immunoassays and to improve their sensitivities, the immune complex transfer enzyme immunoassay has been developed. Antigens to be measured were reacted with 2,4-dinitrophenyl-biotinyl-antibody Fab' and antibody Fab'-beta-D-galactosidase conjugate, and antibody IgGs to be measured were reacted with 2,4-dinitrophenyl-antigen and antigen-beta-D-galactosidase conjugate. The immune complexes formed comprising the three components were trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the colored polystyrene beads with epsilonN-2,4-dinitrophenyl-L-lysine, and the eluates were incubated with white polystyrene beads coated with streptavidin for antigens and coated with affinity-purified (anti-human IgG gamma-chain) IgG for antibody IgGs to transfer the immune complexes. By this method, ultrasensitive enzyme immunoassays have been developed for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and reverse transcriptase (RT). The nonspecific signals in the absence of the antigen and the antibody IgGs were reduced 300 to 15,000-fold by the immune complex transfer process, but the amounts of the immune complexes decreased only 1.8 to 3.1-fold by the immune complex transfer. As a result, the sensitivities for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and RT were improved 100 to 5,600-fold by the immune complex transfer. The detection limit of HIV-1 p24 antigen by 20 hr assay of beta-D-galactosidase activity (10 zmol) was 4,000 to 17,000-fold lower than those obtained with currently available commercial kits. The improved sensitivities for antibody IgGs to p17 and RT by 20 hr assay of beta-D-galactosidase activity were 1 x 10(5) to 3 x 10(5)-fold higher than those of Western blotting for p17 and p66 bands. However, the nonspecific signals in the absence of antigens and antibody IgGs were enhanced to various degrees by two factors. In order to transfer the immune complexes more efficiently within shorter periods of time, the colored polystyrene beads were incubated with the white polystyrene beads in the presence of epsilonN-2,4-dinitrophenyl-L-lysine. Such direct contact between solid phases for trapping and transferring of the immune complexes significantly enhanced the nonspecific signals. In addition, the presence of human serum samples containing neither antigens to be measured nor antibody IgGs to be measured also enhanced the nonspecific signals to various extents. Namely, these two factors limited the effect of the immune complex transfer to improve the sensitivity by 20 hr assay of beta-D-galactosidase activity. By 1 hr assay of beta-D-galactosidase activity, the detection limit of HIV-1 p24 antigen using 10 microl of serum samples (0.24 pg/ml) was 40 to 80-fold lower than those obtained with currently available commercial kits using 100 to 200 microl of serum samples (10 to 20 pg/ml) and the detection limits of antibody IgGs to HIV-1 pl7 and RTwere 1 x 10(4) to 3 x 10(4)-fold lower than those by Western blotting for p17 and p66 bands. Finally, the immunoreactions involved in the immune complex transfer enzyme immunoassays--the formation, trapping, and transferring of the immune complexes--will be performed within 15 to 30 min.
为了降低非竞争性固相免疫测定的非特异性信号并提高其灵敏度，已开发出免疫复合物转移酶免疫测定法。待测抗原与2,4-二硝基苯基-生物素化抗体Fab'和抗体Fab'-β-D-半乳糖苷酶结合物反应，待测抗体IgG与2,4-二硝基苯基-抗原和抗原-β-D-半乳糖苷酶结合物反应。形成的包含这三种成分的免疫复合物被捕获到涂有亲和纯化的（抗2,4-二硝基苯基基团）IgG的彩色聚苯乙烯珠上。洗涤后，用εN-2,4-二硝基苯基-L-赖氨酸从彩色聚苯乙烯珠上洗脱免疫复合物，然后将洗脱液与涂有抗生物素蛋白的白色聚苯乙烯珠（用于抗原）和涂有亲和纯化的（抗人IgGγ链）IgG（用于抗体IgG）一起孵育，以转移免疫复合物。通过这种方法，已开发出针对HIV-1 p24抗原以及针对HIV-1 p17和逆转录酶（RT）的抗体IgG的超灵敏酶免疫测定法。在不存在抗原和抗体IgG的情况下，非特异性信号通过免疫复合物转移过程降低了300至15,000倍，但免疫复合物的量仅通过免疫复合物转移降低了1.8至3.1倍。结果，通过免疫复合物转移，HIV-1 p24抗原以及针对HIV-1 p17和RT的抗体IgG的灵敏度提高了100至5,600倍。通过20小时的β-D-半乳糖苷酶活性测定，HIV-1 p24抗原的检测限（10 zmol）比目前市售试剂盒的检测限低4,000至17,000倍。通过20小时的β-D-半乳糖苷酶活性测定，针对p17和RT的抗体IgG的灵敏度提高比针对p17和p66条带的蛋白质印迹法高1×10⁵至3×10⁵倍。然而，在不存在抗原和抗体IgG的情况下，非特异性信号在不同程度上被两个因素增强。为了在更短的时间内更有效地转移免疫复合物，将彩色聚苯乙烯珠与白色聚苯乙烯珠在εN-2,4-二硝基苯基-L-赖氨酸存在下孵育。这种用于捕获和转移免疫复合物的固相之间的直接接触显著增强了非特异性信号。此外，既不包含待测抗原也不包含待测抗体IgG的人血清样品的存在也在不同程度上增强了非特异性信号。也就是说，这两个因素限制了免疫复合物转移通过20小时的β-D-半乳糖苷酶活性测定来提高灵敏度的效果。通过1小时的β-D-半乳糖苷酶活性测定，使用10微升血清样品（0.24 pg/ml）时HIV-1 p24抗原的检测限比使用100至200微升血清样品（10至20 pg/ml）的目前市售试剂盒的检测限低40至80倍，并且针对HIV-1 p17和RT的抗体IgG的检测限比针对p17和p66条带的蛋白质印迹法低1×10⁴至3×10⁴倍。最后，免疫复合物转移酶免疫测定中涉及免疫反应——免疫复合物的形成、捕获和转移——将在15至30分钟内完成。
