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M9480539.TXT
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1994-08-20
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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
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).