Further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG using microplates and fluororeader.
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 beta-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-beta-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 epsilon N-2,4-dinitrophenyl-L-lysine and polystyrene beads, modified by attaching to plates through cylindrical bars and coated with (antihuman IgG gamma-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 gamma-chain) IgG were substituted for microplate wells coated with (anti-2,4-dinitrophenyl group) IgG and modified polystyrene beads coated with (antihuman IgG gamma-chain) IgG, respectively. The fluorescence intensity for bound beta-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 beta-D-galactosidase activity became slightly more time-consuming.
Ishikawa
S
S
Department of Biochemistry, Medical College of Miyazaki, Japan.
Hashida
S
S
Nakamoto
H
H
Tanaka
S
S
Kojima
M
M
Ishikawa
E
E
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000097:blood*; D000936:Antigen-Antibody Complex / Q000032:analysis; D002417:Cattle; D004136:Dinitrobenzenes; D005470:Fluorometry / Q000379:methods; D015481:HTLV-I Antibodies / Q000097:blood*; D007124:Immunoenzyme Techniques / Q000295:instrumentation*; D007074:Immunoglobulin G / Q000097:blood*; D007202:Indicators and Reagents; D008863:Microspheres; D011137:Polystyrenes; D012680:Sensitivity and Specificity
C045618:2,4-dinitrophenyl-IgG; D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; D004136:Dinitrobenzenes; D015481:HTLV-I Antibodies; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011137:Polystyrenes