ISSN 0027-1314, Moscow University Chemistry Bulletin, 2018, Vol. 73, No. 2, pp. 63–68. © Allerton Press, Inc., 2018.
Original Russian Text © M.K. Gubaidullina, A.E. Urusov, A.V. Zherdev, C. Xu, B.B. Dzantiev, 2018, published in Vestnik Moskovskogo Universiteta, Seriya 2: Khimiya, 2018, No. 2,
Immunochromatographic Test Systems using Anti-Species
Antibodies–Colloidal Gold Conjugate: Their Features and Benefits
on the Example of Ochratoxin A Detection
M. K. Gubaidullina
, A. E. Urusov
, A. V. Zherdev
, C. Xu
, and B. B. Dzantiev
Bach Institute of Biochemistry, Federal Research Center “Fundamentals of Biotechnology,”
Russian Academy of Sciences, Moscow, 119071 Russia
Jianggnan University, Wuxi, 214122 China
Received November 23, 2017
Abstract⎯The traditional immunochromatographic assay using a conjugate of gold nanoparticles with spe-
cific ochratoxin A (OTA) antibodies and a new type of assay with indirect labeling using a combination of free
antibodies and a conjugate of gold nanoparticles with anti-species antibodies were compared using the exam-
ple of OTA detection. In the proposed assay, specific antibodies are included in the sample dilution buffer,
which increases the duration of their interaction with the antigen, while a conjugate of anti-species antibodies
with the marker is applied to the test strip. The assay was approbated for OTA detection in maize extracts.
Transition to indirect labeling was shown to reduce the OTA detection limit by two orders of magnitude up to
0.12 ng/mL. The causes of this improvement are discussed. The high sensitivity of immunochromatography with
indirect labeling makes it a promising approach for detection of various antigens with low molecular weight.
Keywords: immunoassay, test-strips, colloidal gold, antibody – nanoparticle complexes, mycotoxins
Immunochromatographic assay (ICA) is widely
used to screen samples for medical diagnostics and
quality control of raw materials and food [1, 2]. The
rapidity and ease of use of ICA are ensured by prelim-
inary application of all assay reagents to the test strip
membranes. Contact with the probe initiates all the nec-
essary immunochemical reactions resulting in the stain-
ing of certain zones of the test strip, and in 10–15 min the
assay results can be visualized or registered with an
instrument. To detect compounds with low molecular
weight, a competitive ICA format is used in which the
antigen contained in the sample reacts with specific
antibodies conjugated to the stained marker, most
often with gold nanoparticles (GNPs), and prevents
the marker from binding to the antigen immobilized in
the assay zone of the test strip.
However, the development of highly sensitive sys-
tems according to this scheme is hampered by an inter-
nal contradiction: it is impossible to simultaneously
ensure sufficient intensity of the assay signal (staining
of the analytical zone) and high sensitivity of the assay
since the signal is weakened at low concentrations of
the detectable antigen. Thus, to improve the reliability
of the assay, the concentration of conjugates of spe-
cific antibodies and GNPs should be as high as possi-
ble, and for highly sensitive detection, the concentra-
tion should be extremely low.
It should be noted that the conjugates used for ICA
have tens to hundreds of antibody molecules per GNP.
Therefore, when the antigen blocks only some of the
antibodies on the surface of a nanoparticle, the conju-
gate retains its binding ability in the analytical zone.
The intensity of staining decreases only when the anti-
bodies are completely or almost completely blocked
(i.e., at high antigen concentrations). In addition, a
polyvalent conjugate is more likely to bind to polyva-
lent antigen sin the analytical zone than to free ana-
lytes in the sample, preventing effective competition.
To solve this problem, we proposed the use of spe-
cific antibodies and a marker as two independent
reagents in the ICA . ICA with indirect labeling
uses free specific antibodies for competitive interac-
tion and conjugates of anti-species antibodies and
GNP to label the specific immune complexes that are
formed.This replacement enables independent varia-
tion in the content of specific antibodies and the
marker, providing optimal conditions for a highly sen-
sitive and reliable assay. This approach allowed us to
reduce the detection limit of aflatoxin B1 by 40 times
The article was translated by the authors.