Hashida S, Hashinaka K, Nishikata I, Saito A, Takamizawa A, Shinagawa H, Ishikawa E
Department of Biochemistry, Miyazaki Medical College, Japan.
J Clin Lab Anal. 1996;10(5):302-7. doi: 10.1002/(SICI)1098-2825(1996)10:5<302::AID-JCLA11>3.0.CO;2-0.
Previously, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV-1 was developed. The immune complex comprising 2,4-dinitrophenyl-biotinyl-bovine serum albumin-rabbit anti-p24 Fab' conjugate, p24 antigen, and rabbit anti-p24 Fab' -beta-D-galactosidase conjugate was trapped onto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, was eluted with epsilon N-2, 4-dintrophenyl-L-lysine, and was transferred to polystyrene beads coated with streptavidin. beta-D-Galactosidase activity bound to the streptavidin-coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound beta-D-galactosidase activity had to be assayed for a long time (20 h), and the nonspecific signal was observed in 5% serum samples from subjects with low risk of HIV infection. In the present study, the assay time for bound beta-D-galactosidase activity was shortened to 2.5 h by using 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and affinity-purified rabbit anti-p24 Fab' -beta-D-galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at -20 degrees C, and this increase was prevented without prolongation of the assay time for bound beta-D-galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate for affinity-purified rabbit anti-p24 Fab'beta-D-galactosidase conjugate.
此前，已开发出一种用于检测HIV-1 p24抗原的超灵敏酶免疫测定法（免疫复合物转移酶免疫测定法）。包含2,4-二硝基苯基-生物素化-牛血清白蛋白-兔抗p24 Fab'缀合物、p24抗原和兔抗p24 Fab'-β-D-半乳糖苷酶缀合物的免疫复合物被捕获到包被有亲和纯化（抗2,4-二硝基苯基基团）IgG的聚苯乙烯珠上，用ε-N-2,4-二硝基苯基-L-赖氨酸洗脱，并转移到包被有链霉亲和素的聚苯乙烯珠上。通过荧光法测定与包被链霉亲和素的聚苯乙烯珠结合的β-D-半乳糖苷酶活性。该测定法具有高度敏感性。然而，结合的β-D-半乳糖苷酶活性必须长时间测定（20小时），并且在HIV感染低风险受试者的5%血清样本中观察到非特异性信号。在本研究中，通过使用2,4-二硝基苯基-生物素化-牛血清白蛋白-亲和纯化的兔抗p24 Fab'缀合物和亲和纯化的兔抗p24 Fab'-β-D-半乳糖苷酶缀合物，将结合的β-D-半乳糖苷酶活性的测定时间缩短至2.5小时。此外，发现非特异性信号随着血清样本在-20℃下储存时间的延长而增加，通过用单克隆小鼠抗p24 Fab'-β-D-半乳糖苷酶缀合物替代亲和纯化的兔抗p24 Fab'-β-D-半乳糖苷酶缀合物，在不延长结合的β-D-半乳糖苷酶活性测定时间且不损失敏感性的情况下，可防止这种增加。
