Conjugation of recombinant reverse transcriptase of HIV-1 to beta-D-galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG.
Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to beta-D-galactosidase from Escherichia coli in three different ways. Maleimide groups were introduced into beta-D-galactosidase molecules using N,N'-o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into beta-D-galactosidase molecules, which had been treated with excess of 4,4'-dithiodipyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III). Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl-S-acetylmercaptoacetate. The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-beta-D-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N epsilon-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads with (anti-human IgG gamma chain) IgG. The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.
Hashinaka
K
K
Department of Biochemistry, Medical College of Miyazaki, Japan.
Hashida
S
S
Saitoh
A
A
Nakata
A
A
Shinagawa
H
H
Oka
S
S
Shimada
K
K
Ishikawa
E
E
D000818:Animals; D000936:Antigen-Antibody Complex; D004926:Escherichia coli / Q000201:enzymology*; D015483:HIV Antibodies / Q000032:analysis*; D054303:HIV Reverse Transcriptase; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis*; D008301:Maleimides / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000737:chemistry*
D000936:Antigen-Antibody Complex; D015483:HIV Antibodies; D007074:Immunoglobulin G; D008301:Maleimides; D011994:Recombinant Proteins; C043592:maleimide; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase