Document 0539
 DOCN  M9480539
 TI    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.
 DT    9410
 AU    Hashinaka K; Hashida S; Saitoh A; Nakata A; Shinagawa H; Oka S; Shimada
       K; Ishikawa E; Department of Biochemistry, Medical College of Miyazaki,
       Japan.
 SO    J Immunol Methods. 1994 Jun 24;172(2):179-87. Unique Identifier :
       AIDSLINE MED/94308543
 AB    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.
 DE    beta-Galactosidase/*CHEMISTRY  Animal  *Antigen-Antibody Complex
       Escherichia coli/*ENZYMOLOGY  HIV Antibodies/*ANALYSIS  IgG/*ANALYSIS
       Immunoenzyme Techniques  Maleimides/CHEMISTRY  Rabbits  Recombinant
       Proteins/CHEMISTRY  Reverse Transcriptase/*CHEMISTRY  Sensitivity and
       Specificity  JOURNAL ARTICLE

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