Further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-htlv-i igg using microplates and fluororeader

✍ Scribed by Dr. Setsuko Ishikawa; Seiichi Hashida; Hidetaka Nakamoto; Satoshi Tanaka; Masaaki Kojima; Eiji Ishikawa

• Publisher: John Wiley and Sons
• Year: 1995
• Tongue: English
• Weight: 643 KB
• Volume: 9
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860090310

✦ Synopsis

Abstract

In a previously reported ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti‐HTLV‐I IgG, polystyrene beads were handled with tweezers, and bound b̃‐D‐galactosidase activity was measured with a fluorometer. The use of tweezers was causative of false‐positivity by carryover, and testing many samples was difficult. Recently, these drawbacks have been minimized using microplates and a fluororeader. However, tweezers were still required in the initial and final steps. In the present study, the immune complex, comprising 2,4‐dinitrophenyl‐antigen, anti‐HTLV‐I IgG, and antigen‐b̃‐D‐galactosidase conjugate, was formed in and trapped onto microplate wells coated with (anti‐2,4‐dinitrophenyl group) IgG. Subsequently, the microplate wells were incubated with ϵN‐2,4‐dinitrophenyl‐L‐lysine and polystyrene beads, modified by attaching to plates through cylindrical bars and coated with (antihuman IgG γgM‐chain) IgG, to transfer the immune complex from the microplate wells to the modified polystyrene beads. Alternatively, modified polystyrene beads coated with (anti‐2,4‐dinitrophenyl group) IgG and microplate wells coated with (antihuman IgG γgM‐chain) IgG were substituted for microplate wells coated with (anti‐2,4‐dinitrophenyl group) IgG and modified polystyrene beads coated with (antihuman IgG γgM‐chain) IgG, respectively. The fluorescence intensity for bound b̃‐D‐galactosidase activity was quickly measured with a fluororeader. Thus the modified polystyrene beads were transferred from wells to wells more quickly and easily without tweezers, eliminating false‐positivity due to carryover, and it became easy to test many samples with high sensitivity and reliability, although the assay of bound b̃‐D‐galactosidase activity became slightly more time‐consuming.©1995 wiley‐Liss, inc.