This article appeared in a journal published by Elsevier.

The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
 Author's personal copy

Talanta 85 (2011) 736–742

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

A high selective immunochromatographic assay for rapid detection of aflatoxin B1

Daohong Zhang a,b,c,1 , Peiwu Li a,b,c,∗,1 , Yang Yang d , Qi Zhang a,∗ , Wen Zhang a,b , Zhi Xiao a,b,c ,
Xiaoxia Ding a,b
a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, 430062, China
b
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Wuhan, 430062, China
c
Quality inspection and Test Center for Oilseeds Products MOA PRC, Wuhan, 430062, China
d
Bureau for Agricultural Food Quality and Safety, Ministry of Agriculture; Beijing 100125, China

a r t i c l e i n f o a b s t r a c t

Article history: To solve the problem of low selectivity of current immunochromatographic assay (ICA) for aflatoxin
Received 20 February 2011 B1 (AFB1 ) alone detection, a novel selective ICA was developed here. With very high selectivity, a new
Received in revised form 21 April 2011 AFB1 monoclonal antibody (MAb) 3G1 was preparaed by immunizing Balb/c mice with aflatoxin B2a -
Accepted 22 April 2011
BSA (AFB2a -BSA) rather than AFB1 -BSA used in other reports and 3G1 possessed the highest selectivity
Available online 29 April 2011
than those used in published ICAs. The ICA with visual detection limit (VDL) of 1 ng mL−1 showed no
cross-reactivity with other aflatoxins. Comparing with previous reports, the ICA here provided the most
Keywords:
powerful guarantee for avoiding false positive results leaded by coexistence of other aflatoxins in samples.
Aflatoxin B1
Selectivity
For validation, naturally contaminated samples including peanut, puer-tea, vegetable oil and feedstuff
Immunochromatographic assay were respectively assayed by ICA and a standard high performance liquid chromatography (HPLC), and
Detection good agreement of results was obtained between two methods. Therefore, the developed ICA could well
meet the selective detection of AFB1 in agro-products.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Aflatoxins are a group of extremely toxic metabolites produced
by some Aspergillus species namely A. flavus, A. parasiticus and the
rare A. nomius, during the growth on foods and/or feeds [1,2].
Although more than 20 aflatoxins have been identified [3], the
major aflatoxins of concern are designated B1 (AFB1 ), B2 , G1 and
G2 , and among them aflatoxin B1 , generally present in the largest
quantity, is the most toxic [4]. AFB1 has been classified as Group I
human carcinogen by the International Agency for Research on Can-
cer [1,5–7]. Aflatoxins, most commonly found in foodstuffs, enter
the food chain mainly by ingestion via the dietary route in humans
and animals. The intake of aflatoxin over a long period of time,
even at very low concentration, may be highly dangerous [8]. There-
fore, legal limits varing from country to country and ranging from
0 to 50 ng mL−1 have been established [9] in many countries for
aflatoxins. Some developing countries like China and Mexico have
set up regulations compatible with those in the United States for
human consumption and for trading [10]. For example, the current
maximum allowed level (MAL) set by United States and China is
20 ng mL−1 in foodstuffs.
∗ Corresponding authors at: Oil Crops Research Institute of Chinese Academy of
Agricultural Sciences, Wuhan 430062, China. Tel.: +86 27 86812943;
fax: +86 27 86812862.
E-mail addresses: peiwuli@oilcrops.cn (P. Li), zhangqi521x@yahoo.cn (Q. Zhang).
1
Both the authors rank the first authors.
Some analytical techniques have been used in the last two
decades for the qualitative and quantitative analysis of aflatoxins
in food, such as Fourier transform near-infrared spectroscopy [11],
thin-layer chromatography (TLC) [12,13], high performance liquid
chromatography (HPLC) [14–16], liquid chromatography–tandem
mass spectrometry (LC–MS) [17], LC–MS/MS [18], enzyme-linked
immunosorbent assay (ELISA) [19–22], ion mobility spectrometry
[23] and so on. Still faster and simpler approaches for aflatoxin
detection are increasingly being requested by the food and feed
industry [24].
The immunochromatographic assay system was devised in
order to determine the concentration of target analyte simply and
rapidly [25], it has many advantages, such as rapid on-site detec-
tion within a few minutes, requiring only a sample extraction step
before use, concentration levels of target analytes can be observed
directly with the naked eyes [25], etc. Because of these advan-
tages, immunochromatographic strip tests have been used in a
wide range of applications. But concerning aflatoxin detection, few
reports on ICA have been found until now. In these reports, to
meet the legislation existing in most countries for MAL of AFB1 ,
three reports [3,26,27] have announced the detection of AFB1 alone.
These reported ICAs offered good sensitivity, but the antibodies
used in these literatures showed considerable cross-reactivities
with other major aflatoxins. Although cross-reactivity can be a
valuable feature, especially when a whole class of relevant ana-
logues have to be determined, it can also pose a big potential
problem from serious false positive results for single analyte analy-
sis, e.g., in most cases, different forms of aflatoxins, including B1 , B2 ,

0039-9140/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2011.04.061
 Author's personal copy
D. Zhang et al. / Talanta 85 (2011) 736–742
G1 and G2 , are simultaneously found in contaminated human foods
[28]. The lack of selectivity is considered as one of immunoassay’s
mostly disadvantages when competing with HPLC or LC–MS/MS.
The selectivity of immunoassay, however, is major determined by
the selectivity of antibody.
In this work, to solve the problem mentioned above, we pre-
pared a AFB1 selective MAb. Based on the MAb, we developed a
high selectivity immunochromatographic assay, optimized, evalu-
ated and applied the assay in peanut, puer-tea, vegetable oil and
feedstuff detection for contamination level of AFB1 .
2. Materials and methods
2.1. Materials
2.1.1. Consumables, reagent and equipment
Aflatoxin B1 , B2 , G1 , and G2 standard solutions, 3 ␮g mL−1 in
acetonitrile/water (98:2), were obtained from Supelco Corp. (St.
Louis, MO, USA). Aflatoxin B1 standard (from A. flavus), hydro-
gen tetrachloroaurate (III) hydrate (HAuCl4 ·xH2 O), OVA (agrose
gel electrophoresis grade) and rabbit anti-mouse immunoglobulin
(IgG), Goat anti-mouse immunoglobulin horseradish peroxi-
dase (IgG-HRP), complete and incomplete Freund’s adjuvants,
urea hydrogen peroxide (97%), hypoxanthine/thymidine (HT),
hypoxanthine/aminopterin/thymidine (HAT), 3,3,5,5-tetramethyl
benzidine (TMB), and polyethylene glycol 1450 (PEG 1450, 50%)
were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine
serum albumin (BSA) was purchased from Roche Applied Science
(Indianapolis, IN, USA). RPMI-1640 medium with l-glutamine and
HEPES (free acid, 283.3 g L−1 ) was obtained from HyClone. Fetal
bovine serum (FBS), penicillin (+10,000 U mL−1 ) and streptomycin
(+10,000 ␮g mL−1 ) were from Gibco. Nanogold solution was pre-
pared in our lab [29], and the average diameter of the nanogold
particles was 40 nm according to the correlation between the
diameter sizes of gold particles and the amount of sodium citrate
(1 mL of 1% trisodium citrate was added in a 100 mL of 0.01% gold
chloride solution) [30,31]. Unless otherwise stated, all other inor-
ganic chemicals and organic solvents were of analytical reagent
grade. Water was obtained from a MilliQ purification system (Mil-
lipore). Cell culture plates were from Shanghai Sunub Bio-Tech
Development, Inc. Culture flasks were obtained from Iwaki, Japan.
Nitrocellulose membranes, glass fibers, sample pads, and absorbent
pads were purchased from Millipore Corp. (Bedford, MA, USA).
An XYZ3050 Dispensing Platform, CM4000 Guillotine Cutter, and
LM4000 Batch Laminator (BioDot, Irvine, CA, USA) were used to
prepare test strips. The high-speed freezing centrifuge (CF16RX)
was from Hitachi (Tokyo, Japan). The IC assay was validated with
an Agilent 1100 high performance liquid chromatography system
(Agilent Tech, Santa Clara, CA, USA).
2.1.2. Animal and cells
Female Balb/c mice were purchased from Centers for Disease
Control and Prevention of Hubei province. SP2/0 myeloma cells
were purchased from China Center for Type Culture Collection
(CCTCC).
2.2. Antigen preparation and immunization
2.2.1. Antigen preparation
Immunogen conjugate (aflatoxin B2a -BSA) was synthesized as
previously described [32] with some modifications. Briefly, 4 mg of
AFB1 was dissolved in acetone, and added by 40 ␮L of 10% H2 SO4 .
The mixture was stirred continuously for 4 h at 56 ◦ C, and the prod-
uct was evaporated to dryness followed by addition of 5 mL H2 O.
The mixture was extracted twice by 25 mL of chloroform, then the
737
organic extract phase was washed by 20 mL of H2 O, reserved, evap-
orated to dryness and stripped to a yellow solid. One milligram of
the yellow product was placed in a flask, to which 2 mL of phosphate
buffer (PBS, 0.01 mol L−1 ; pH 7.4) containing 0.5% BSA was added.
After reaction for 30 min at 37 ◦ C, 100 ␮L of 6.5 mM NaBH4 was
added and reacted for another 30 min at 4 ◦ C. Then 50 ␮L of 0.1 N
HCl was added to eliminate excess NaHB4 . Finally, the resultant
product was dialyzed against PBS over 72 h at 4 ◦ C to remove unre-
acted AFB1 and AFB2a , then the dialysis product was scanned by
UV–visible spectroscopy. The coating antigen (aflatoxin B2a -OVA)
was synthesized with the same method.
2.2.2. Immunization
In the initial immunization, 1 mg mL−1 of aflatoxin B2a -BSA con-
jugate in PBS was emulsified with an equal volume of Freund’s
complete adjuvant, the final water-in-oil emulsion was injected
multiple-site subcutaneously into 6-week-old female Balb/c mice.
In subsequent injections, same dosage of AFB2a -BSA was emulsi-
fied with an equal volume of Freund’s incomplete adjuvant. Three
intraperitoneal injections were carried out after the first immuniza-
tion with an interval of 4 weeks. The booster injection three days
before cell fusion was carried out with twofold dosage of antigen
without emulsification with adjuvant.
2.3. Preparation of monoclonal antibody
Fusion between SP2/0 myeloma and spleen cells were per-
formed using hybridoma technique as previously described [33].
Fused cells were suspended in semisolid complete medium (RPMI
1640 medium supplemented with 20% (v/v) FBS, methylcellulose,
HAT, antibiotics and HEPES), and then equally distributed into
nine 6-well plates, incubated in 5% CO2 at 37 ◦ C. Two weeks after
cell fusion, hundreds of cell colonies became macroscopical in the
semisolid medium. The single colony was picked out and trans-
ferred into 24-well plates with liquid complete medium containing
HT. When cell density reached approximately 1/2 of the well, cul-
ture supernatants were assayed by indirect ELISA (in-ELISA) and
indirect competitive ELISA (ic-ELISA). Interested and stable mon-
oclonal cell strains were screened out by repeated confirming
detections. The resulting hybridoma clones were propagated, then
one part of cells were cryopreserved in freezing solution and stored
in liquid nitrogen, another part of cells were injected intraperi-
toneally into Balb/c mice, which previously had been given 0.5 mL
of Freund’s incomplete adjuvant to produce ascitic fluids. Finally,
the ascitic fluids were purified by the caprylic acid–ammonium
method as described previously [34].
2.4. ELISA procedure
Two formats of ELISA: ic-ELISA and in-ELISA were used in this
study. The procedure of ic-ELISA was as follows: first, microtitre
plates were coated overnight with 2 ␮g mL−1 of AFB2a -OVA in bicar-
bonate coating buffer (0.05 M; pH 9.6) followed by blocking for
1 h at 37 ◦ C with 1% OVA-PBST (PBS containing 0.05% Tween-20
and 1% OVA). Subsequently, the supernatants or aflatoxin-selective
MAb and aflatoxins (50 ␮L/well, respectively) in 0.5% OVA-PBST
(dilution buffer) were added together and incubated for 0.5 h at
37 ◦ C. Next, the plates were incubated with anti-mouse IgG-HRP in
dilution buffer for 45 min at 37 ◦ C. Between each step, the plates
were washed 3-6 times with PBST. Finally, the plates were incu-
bated for 15 min at 37 ◦ C with the enzyme substrate solution (TMB,
100 ␮L/well), then the enzyme catalyzed reaction was stopped by
addition of stop solution (50 ␮L/well). Absorption values at 450 nm
were measured with a microplate reader. The in-ELISA procedure
 Author's personal copy
738 D. Zhang et al. / Talanta 85 (2011) 736–742
was similar to that of ic-ELISA, except that after blocking only MAb
but not MAb and competitors was added.
2.5. Preparation of gold-Ab probe
The probe was prepared as report previously [35,36] with slight
modifications. In briefly, with gentle stirring, 0.6 mg of purified
MAb, in 6 mL of ultrapure water was added dropwise to 100 mL
of pH-adjusted nanogold solution. The mixture was stirred contin-
uously for 30 min, and blocked by 10% (w/v) filtered BSA for 1 h.
Then the reaction solution was centrifuged twice to remove the
uncoordinated protein from the solution. The final pellet was con-
centrated and resuspended in 10 mL of 2 mM borax buffer (pH 9.0)
containing 0.1% (w/v) PEG-20,000 and 0.02% NaN3 , and stored at
4 ◦ C for further experiments.
2.6. Preparation of test strips
Aflatoxin B2a -BSA conjugate and rabbit anti-mouse IgG were
coated respectively on nitrocellulose (NC) membrane as test (T)
line and control (C) line by BioDot XYZ Platform at a proper jet-
ting rate. The distance between T line and C line was 5 mm. The
coated membrane was dried at 37 ◦ C for 10 min. Gold-Ab probe
solution was dispensed onto conjugate pad and dried with the
Vacuum Freeze Drier. The conjugate pad and sample pad were all
made from glass fiber membrane and treated with blocking buffers
beforehand. Then the NC membrane, conjugate pad, sample pad
and absorbent pad were laminated and pasted onto a plastic back-
ing plate. Finally, the plate was cut into 3 mm wide strips and stored
in a test strip tube with desiccant at 4 ◦ C.
2.7. Evaluation of the immunochromatographic assay
Peanut matrix (determined by HPLC to be aflatoxin-free) was
used to evaluate the sensitivity, selectivity, precision and accuracy
of ICA. For sensitivity evaluation, a series concentrations of AFB1
standard in blank peanut extracts (1–30 ng mL−1 ) were assayed by
ICA to find the VDL and threshold level that gave a complete dis-
appearance of color in test line [26]. Selectivity was evaluated by
investigating cross-reactivities of ICA with other major aflatoxins
(B2 , G1 and G2 ) at the concentration of 40 ng mL−1 . Precision and
accuracy were investigated as method described before [37,38].
Finely ground peanut sample was spiked with AFB1 and analyzed
respectively by ICA and a quantitative method (ic-ELISA).
2.8. Application and validation
Naturally contaminated samples (peanut, puer-tea, vegetable
oil and feedstuff) were analyzed using the developed ICA and con-
firmed by HPLC. Samples were separately prepared according to
different methods and samples (see below). In the assay, the sample
pad of the strip was inserted into 96-well microtiter plate contain-
ing test solutions, immediately, due to capillary action, the solution
and released gold-Ab probe moved along on the NC membrane
chromatographically. The results were readout within 15 min.
2.9. Sample treatment
2.9.1. Sample treatment for ICA
Peanut, puer-tea and feedstuff samples were finely ground with
a lab mill and seived through a 20 mesh screen. Then 5 g of the
sample was placed in a 50 mL centrifuge tube, to which 10 mL of
methanol–water (8:2, v/v) was added, extracted by vortex mixing
for 3 min. After standing for 5 min, 1 mL of the clear supernatant
was diluted with 4 mL of water and detected directly by test strips
(100 ␮L/strip) without any further cleanup.
For vegetable oil samples, 5 g of oil was added to a 50 mL
centrifuge tube, to which 10 mL of petroleum ether was added
and thoroughly mixed, then 10 mL of methanol–water (8:2, v/v)
was added and vortex mixed for 3 min. After removing the upper
petroleum ether, 1 mL of lower methanol–water extracting solu-
tion was transferred into a glass vial, diluted with 4 mL of water and
also detected directly by test strips without any further cleanup.
2.9.2. Sample treatment for HPLC analysis
For peanut, puer-tea and feedstuff samples, first, samples were
finely ground and screened as above, then 5 g of the sample was
placed in a 50 mL centrifuge tube and extracted with 10 mL of
methanol–water (8:2, v/v) containing 4% NaCl by ultrasonic in
a 50 ◦ C water bath for 5 min. After centrifugation, 4 mL of clear
extraction supernatant was carefully gathered in a flask. For peanut
extract, 2 mL of petroleum ether was added and thoroughly mixed
to remove fat. For puer-tea and feedstuff extracts, the super-
natants were cleaned up through a multifunction cleanup column
to remove pigment. Then, 3 mL of three resultant solutions were
transferred into glass vials, diluted with 9 mL of water, respectively.
Ten milliliters of diluted solutions were respectively loaded on an
immunoaffinity chromatography (IAC) column with a negative-
pressure apparatus. Then, aflatoxins were eluted with 1 mL of
methanol and collected in a tube. The elution solvent was evap-
orated to dryness in a 60 ◦ C water bath under a gentle flow of
nitrogen. The residue was resuspended in 200 ␮L of hexane and
100 ␮L of trifluoroacetic acid and mixed thoroughly. Then, aflatox-
ins were derived for 15 min in 40 ◦ C stoving chest, and evaporated
to dryness at 60 ◦ C under nitrogen. The residue was dissolved in
200 ␮L of acetonitrile–water (15:85, v/v) and injected into the HPLC
autosampler vial for analysis.
Vegetable oil samples were extracted by 10 mL of
methanol–water (8:2, v/v) containing 4% NaCl as described in
the above subsection and the following treatments (clean up,
enrichment and derivation) were as similar as above.
3. Results and discussion
3.1. Characterization and comparison of MAb
At present, two types of legislations for MALs of aflatoxins exist
in most countries: one refers to AFB1 , the other to the total of
the four aflatoxins – AFB1 , AFB2 , AFG1 and AFG2 . Accordingly, for
more exact detection of aflatoxins by immunoassays, two types of
antibodies should be prepared: one refers to AFB1 selective anti-
body, the other to generic antibody that can well recognize the
four aflatoxins simultaneously. The generic MAb had been success-
fully prepared in our group, and research focused on selectivity
MAb against aflatoxin B1 was also carried out by immunizing afla-
toxin B1 -BSA, but we failed to achieve satisfactory MAb due to
the serious cross-reactivity leaded by the very similar structure
between aflatoxins. Then, enlightened by previous reports [32,39]
another kind of immunogen aflatoxin B2a -BSA was synthesized and
used for immunization. Fortunately, after cell fusion and screen-
ing four MAbs were selected out, and MAb 3G1, showed 6.4%,
<0.02% and <0.02% cross-reactivities toward aflatoxin B2 , G1 and
G2 , respectively, was used in the ICA. Dose-response curve of MAb
3G1 for four major aflatoxins was obtained in Fig. 1. The curve
was obtained by plotting inhibition rate (B/B0 ) against logarithm
of analyte concentration (ng mL−1 ), where B was the absorbance at
each concentration of analyte and B0 was the absorbance in the
absence of analyte [40]. The IC50 value (concentration resulting
in half-maximum inhibition) of MAb 3G1 confirmed by ic-ELISA
was 1.6 ng mL−1 , the minimal inhibition concentration (concen-
tration resulting in 90% inhibition) was 0.19 ng mL−1 , the affinity
 Author's personal copy
D. Zhang et al. / Talanta 85 (2011) 736–742
Fig. 1. Dose-response curve of MAb 3G1 for four major aflatoxins (B1 , B2 , G1 and
G2 ). The coeffcients of variation (n = 3) were between 0.4% and 6.9%.
constant, calculated using the method of Beatty et al. [41] was
1.8 × 108 L mol−1 .
As described above, just three ICAs aiming for AFB1 have been
developed so far. Polyclonal antibody (PAb) [26] and MAb have
been used in these reports [3,27]. Cross-reactivities of MAbs used
in the reports and MAb 3G1 were displayed in Table 1 for compar-
ison. It could be conjectured from the data in Table 1 that maybe it
had more advantages for the two reported MAbs in total aflatoxins
detection, otherwise, for AFB1 alone detection, false positive results
leaded by cross-reaction with other aflatoxins would be consider-
able and unavoidable, because several aflatoxins including B1 , B2 ,
G1 and G2 coexist in contaminated samples usually. Therefore, by
comparison, it was believed that due to its high selectivity with
AFB1 , MAb 3G1 was more suitable than reported MAbs for AFB1
alone detection.
3.2. Preparation of gold-Ab probe
3.2.1. Size of nanogold used for production of gold-Ab probe
Several sizes of colloidal gold particles have been tested in pre-
vious reports for conjugation with antibody, e.g., G25 (25 nm gold
particle) [26], G15 [42], G18–20 [35,43,44], etc., but as described in
reports for samll molecular detection [31,45–47], 40 nm colloidal
gold (G40) was the optimal particle size on account of the trade-
off between required visibility and steric hindrance [45]. AFB1 is
(molecular weight = 312) belongs to small molecular. Therefore,
G40 was used in the production of colloidal gold probe, and the
following experiments also indicated that the gold-Ab probe was
stable and the color was obvious and easy to tell.
3.2.2. Optimum pH for gold-Ab conjugation
The optimal pH was ascertained according to the method
described by Xu et al. [48]. The pH of colloidal gold solution was
adjusted by 0.1 M K2 CO3 . First, 1 mL of nanogold solution was added
in 1–10# 1.5-mL Eppendorf tube, respectively, then 1–10 ␮L of
K2 CO3 solution was added correspondingly in 1–10# tube. After
incubation for 5 min, a color shift from red to blue was found in
1–3# tube due to coagulation of nanogold particles. Then, same
Table 1
Cross-reactivities of MAbs used in reported ICAs and 3G1 for major aflatoxins.
Monoclonal antibody Cross-reactivitya (%)
AFB1
3G1 100
[3] 100
[39] 100
a
Cross-reactivity (CR) for different aflatoxins was determined by comparing the IC50 values of analytes and calculated as: %CR = (IC50 AFB1 /IC50 analyte ) × 100.
b
The data were obtained from the original directly.
739
Fig. 2. Effect of addtion volume (␮L) of 0.1 M K2 CO3 on the OD values of supernatants
after centrifugation of nanogold-Ab conjugate.
amount of antibody was added to 4–10# tube, respectively. After
reaction and centrifugation, the titer of supernatant of each tube
was detected by in-ELISA. As shown in Fig. 2, the OD value of
supernatants decreased with 4–6 ␮L of K2 CO3 , and increased with
7–10 ␮L of K2 CO3 . The turning point with the lowest absorption
value was obtained at 6 ␮L of K2 CO3 showing the lowest antibody
amount in 6# supernatant, in another word, the largest amount of
antibody was binded to nanogold particle surface in 6# tube. There-
fore, the pH value (about 5.5) resulting from 6 ␮L of 0.1 M K2 CO3 in
per milliliter of colloidal gold solution was considered the optimal
value for conjugation.
3.3. Principle of immunochromatographic assay
The ICA is based on the competitive theory. As illustrated in
Fig. 3, when detection, liquid sample is applied to the sample pad
and quickly diffuses into the conjugate pad [49]. The gold-Ab probe
is solubilized and moves upward with the sample flow chromato-
graphically across the membrane. For negative sample solution,
when the probe passes over the test line (T), on which aflatoxin
B2a -BSA conjugate is immobilized, the colloidal gold-Ab will be cap-
tured by AFB2a -BSA. Then, due to capillary action, excess gold-Ab
will move continuously to the control region (C) and be captured
by goat anti-rabbit IgG. Therefore, for a negative sample, two red
bands will appear due to the accumulation of red colored gold-Ab
probe [50].
On contrast, for positive sample solutions containing analyte,
the antibody will react first with the analyte during moving, and
as the mixture of free probe, probe-analyte and free analyte passes
over the test line, the analyte competes with AFB2a -BSA for the
limited number of antibody binding sites. Consequently, less gold-
Ab will be remained at the test line. So, the intensity of the color in
test zone is inversely proportional to the analyte concentration in
the sample.
3.4. Optimization of the strip test
3.4.1. Optimum concentration of immunoreagents
The concentrations of immunoreagents were screened accord-
ing to the criterias: completely release of gold-Ab probe from
AFB2 AFG1 AFG2
6.4 <0.02 <0.02
76b 55 6
26 61 24
 Author's personal copy
740 D. Zhang et al. / Talanta 85 (2011) 736–742
Fig. 3. Schematic description for principle of competitive assay in test strip format. C and T standed for control line and test line, respectively.
conjugate pad and no background color just owning to gold-Ab
particles did not finish moving upward with the sample flow
across the membrane within 15 min; the appearance of clear,
red color on the test line for negative samples; best sensitivity;
minimum immunoreagent consumption. Based on these criterias,
the concentrations of immunoreagents were screened similar to
a “checkerboard titration” in ELISA. Finally, gold-Ab conjugate
containing 6 ␮g mL−1 of antibody was selected as the optimal
probe. The optimum combination of 8 ␮L cm−1 of gold-Ab probe
(0.06 mg mL−1 ), 0.75 ␮L cm−1 of aflatoxin B2a -BSA (0.5 mg mL−1 )
and 0.5 ␮L cm−1 of rabbit anti-mouse IgG (0.5 mg mL−1 ) were dis-
pensed on conjugate pad, T line and C line, respectively.
3.4.2. Optimum conditions for sample tests
Acetone, ethyl acetate, methanol as organic solvents in extrac-
tion solution were tested, although the two former obviously
enhanced the color intensity of both T and C lines, the sensitivity of
assay was also decreased. Besides, compared with methanol, ace-
tone and ethyl acetate had stronger irritant smell. So, methanol as a
most common extraction solvent was used in the following exper-
iments. Different methanol contents (60–90%) were studied for
the maximum extraction efficiency. Results showed that with the
increase of methanol content, the extraction efficiency increased,
but meanwhile, to obtain the same methanol content (e.g., 10%) in
final test solution, the extract dilution factor had to be increased
which also decreased the final aflatoxin concentration in test
solution. Besides, the emulsification degree to high-concentration
oil-bearing samples (e.g., peanut) increased when methanol con-
tent was higher than 80%. NaCl was added in the extraction solution
in many studies to decrease emulsification of samples [51]. Results
showed that the emulsification had little effect on ICA due to its
Fig. 4. Methanol and matrix effects on the color intensity of test strip. (a) Effect of methanol content, the percentage (0–80%) standed for the methanol concentration; (b)
effect of extract dilution factor, the number (2–10) standed for the dilution factor.
chromatography separation, but NaCl could slightly inhibite the
color intensity of test line. Therefore, methanol–water (8:2, v/v)
solution without any extra ion was used as the extraction solution.
Then, optimum dilution factor of extract was evaluated.
Methanol and matrix were the two major aspects that would
affect the color intensity of test strip. To investigate the effect of
methanol, extraction solution (methanol–water) but not extract
was diluted and tested as blank solution. As shown in Fig. 4a,
high methanol content (≥50%) resulted in inefficient release of the
gold-Ab probe from conjugate pads. While, this effect became not
evident when methanol concentration was decreased to 40%. So,
taking no account of matrix effect, the minimum extract dilution
factor of 2 times could be used. However, as shown in Fig. 4b, the
color of test line was weak due to the more remarkable influence
from matrix until the dilution factor was no less than 5. For positive
samples, the final aflatoxin concentration in test solution would
reduce with the increase of extract dilution factor. Therefore, 5
times diluted extract was used as the final test solution of ICA.
3.5. Evaluation of immunochromatographic assay
3.5.1. Sensitivity and selectivity
The VDL of ICA could be defined as the minimum analyte con-
centration producing color on test line significantly weaker than
that of negative control strip [50,52]. As deduced from Fig. 5, the
VDL of the strip test for AFB1 was 1 ng mL−1 and the threshold level
was 30 ng mL−1 . Selectivity evaluation results showed that com-
pared with negative control no inhibition to color intensity was
found for 40 ng mL−1 of AFB2 , AFG1 and AFG2 , respectively. There-
fore, the assay showed high selectivity which to a great extent was
attributed to the MAb 3G1.
 Author's personal copy

D. Zhang et al. / Talanta 85 (2011) 736–742 741

Table 2
Results of ic-ELISA and ICA for spiked samples.

Spiked level (ng mL−1 ) ELISA (n = 4) ICA (n = 20)

−1 a b
Dilution factor Mean (ng mL ) SD RC (%) Dilution factor Visual result FN rated (%)

8.0 10 8.6 0.067 107 5 +c 10

20.0 10 22.8 0.181 114 5 + 0
80.0 10 78.9 0.343 98.6 5 + 0
a
SD, standard deviation.
b
RC%, recovery percentage.
c
+, positive.
d
FN rate, false negative rate was calculated as: %FN = (fn/tp) × 100, where fn are false negative test samples; tp are truly positive test samples.

3.5.2. Precision and accuracy Table 3

Detection results of HPLC and ICA for AFB1 in peanut, puer-tea, vegetable oil and
Spiking samples were analyzed by ICA in duplicate and con-
feedstuff samples.
firmed by ic-ELISA. By comparison, the results of ICA were in
conformity with those of the quantitative method ELISA (Table 2). Samples HPLC (ng g−1 ± SD) ICAa (n = 6)
10% false negative (fn) result was found in 20 times repeated tests Peanuts
for the first fortified concentration due to its close to the VDL 1 NDb −, −, −, −, −, −c
(1 ng mL−1 ) after dilution for 5 times (1.6 ng mL−1 ), but consistent 2 10.32 ± 0.12 +, +, +, +, +, ±e
3 17.45 ± 0.23 +, +, +, +, +, +d
results were obtained for other spiking levels with twenty replica-
4 14.77 ± 0.32 +, +, +, +, +, +
tions, showing good reproducibility and strip-to-strip performance. 5 0.46 ± 0.09 −, −, −, −, −, −
6 14.21 ± 1.22 +, +, +, +, +, +
7 30.80 ± 1.69 +, +, +, +, +, +
3.6. Application and validation
8 ND −, −, −, −, −, −
9 15.99 ± 0.95 +, +, +, +, +, +
In this work, 37 samples including peanut, puer-tea, feedstuff 10 11.62 ± 0.41 +, +, +, +, +, +
and vegetable oil were detected by the developed ICA with HPLC 11 13.19 ± 0.35 +, +, +, +, +, +
for reference. AFB1 contents in samples were diluted 10 times in test 12 0.08 ± 0.02 −, −, −, −, −, −
13 12.49 ± 0.66 +, +, +, +, +, +
solutions for ICA. Results (Table 3) showed that when contamina-
14 8.62 ± 0.65 −, −, −, −, −, −
tion level was around 10 ng mL−1 (e.g., 10.32 ng mL−1 ), inconsistent 15 0.56 ± 0.38 −, −, −, −, −, −
results appeared [e.g., six repeats with five (+) and one (±)] which 16 11.02 ± 0.31 +, +, +, +, +, +
caused mainly by reproducibility of the assay. But when toxin level 17 4.67 ± 0.85 −, −, −, −, −, −
was obviously higher or lower than 10 ng mL−1 , the results of ICA 18 12.49 ± 0.97 +, +, +, +, +, +
19 26.33 ± 1.14 +, +, +, +, +, +
were in good agreement with those of HPLC. Maybe the detectable
concentrion by ICA in real samples could be further decreased, Puer-tea
1 13.11 ± 0.86 +, +, +, +, +, +
e.g., by enrichment and concentration using an immuno-affinity
2 4.98 ± 0.54 −, −, −, −, −, −
colum. Besides, results of HPLC (data not shown) showed that 62.2% 3 59.30 ± 2.13 +, +, +, +, +, +
(23/37) of the detected samples were also contaminated by AFB2 , 4 15.40 ± 0.87 +, +, +, +, +, +
10.8% (4/37) by AFG1 and 10.8% (4/37) by AFG2 . However, the coex- 5 12.64 ± 0.46 +, +, +, +, +, +
istence of other aflatoxins had no effect on result judgement for Vegetable oil
AFB1 contamination, e.g., the content of AFG2 in 2# puer-tea sample 1 0.76 ± 0.12 −, −, −, −, −, −
was up to 59.8 ng mL−1 , but the result of ICA expressed absolutely 2 0.27 ± 0.07 −, −, −, −, −, −
0.37 ± 0.11 −, −, −, −, −, −
negative (6/6), because the concentration of AFB1 was 4.98 ng mL−1 . 3
4 0.52 ± 0.17 −, −, −, −, −, −
5 0.46 ± 0.08 −, −, −, −, −, −
6 0.45 ± 0.10 −, −, −, −, −, −
7 0.40 ± 0.04 −, −, −, −, −, −

Feedstuff
1 1.76 ± 0.08 −, −, −, −, −, −
2 0.91 ± 0.05 −, −, −, −, −, −
3 0.76 ± 0.12 −, −, −, −, −, −
4 0.03 ± 0.02 −, −, −, −, −, −
5 0.76 ± 0.14 −, −, −, −, −, −
6 0.67 ± 0.09 −, −, −, −, −, −
a
The AFB1 original concentrations were diluted 10 times in ICA.
b
ND, not detectable.
c
−, Negative: AFB1 final concentration in test solution was less than 1 ng mL−1 .
d
+, Positive: AFB1 final concentration in test solution was higher than 1 ng mL−1 .
e
±, Positive/negative.

Therefore, the developed ICA demonstrated good practical applica-

bility in real samples with little risk of false positive results.

4. Conclusions

In this work, a high selectivity MAb based immunochromato-

Fig. 5. Visual detection limit for AFB1 . The tests were run at room temperature by
spiking blank sample extracts with AFB1 standard solutions at the concentrations of graphic assay for aflatoxin B1 detection had been developed. AFB1
1, 2, 5, 10, 20, 30 ng mL−1 , respectively, and the results were obtained within 15 min. selective MAb 3G1 was successfully screened out by immunizing
 Author's personal copy
742 D. Zhang et al. / Talanta 85 (2011) 736–742
aflatoxin B2a -BSA, and by comparison, it was more suitable for AFB1
alone detection than those used in reported ICAs. The ICA was opti-
mized and evaluated. The VDL and threshold level of optimized
ICA for AFB1 were 1 and 30 ng mL−1 , respectively. In the assay of
AFB1 in 37 samples, the visual evaluation results of strip test were
in good agreement with those of HPLC. Considering the evaluation
and application results and the advantages of rapid, low cost and
convenient, the developed ICA was well suited for on-site screening
of aflatoxin B1 in agro-products such as peanuts, puer-tea, feedstuff
and vegetable oil.
Acknowledgments
This work was supported by the Key Project of Ministry of Agri-
culture (2011-G5, 2010-G1), the Industry Technology System of
Rapeseed in China (nycytx-005), the Project of National Science &
Technology Pillar Plan (2010BAD01B07, 2011BAD02D02) and Spe-
cial Foundation of President of the Chinese Agricultural Academy
of Sciences.
References
[1] M. Ardic, Y. Karakaya, M. Atasever, H. Durmaz, Food Chem. Toxicol. 46 (2008)
1596–1599.
[2] D.H. Zhang, P.W. Li, Q. Zhang, W. Zhang, Y.L. Huang, X.X. Ding, J. Jiang, Anal.
Chim. Acta 636 (2009) 63–69.
[3] B.S. Delmulle, S.M.D.G. De Saeger, L. Sibanda, I. Barna-Vetro, C.H. Van Peteghem,
J. Agric. Food Chem. 53 (2005) 3364–3368.
[4] S.J. Daly, G.J. Keating, P.P. Dillon, B.M. Manning, R. O’Kennedy, H.A. Lee, M.R.
Morgan, J. Agric. Food Chem. 48 (2000) 5097–5104.
[5] B.F. Huang, Z. Han, Z.X. Cai, Y.J. Wu, Y.P. Ren, Anal. Chim. Acta 662 (2010)
62–68.
[6] P.W. Li, Q. Zhang, W. Zhang, Trends Anal. Chem. 28 (2009) 1115–1126.
[7] Y. Tan, X. Chu, G.L. Shen, R.Q. Yu, Anal. Biochem. 387 (2009) 82–86.
[8] S. Piermarini, L. Micheli, N.H.S. Ammida, G. Palleschi, D. Moscone, Biosens.
Bioelectron. 22 (2007) 1434–1440.
[9] A.Y. Kolosova, W.B. Shim, Z.Y. Yang, S.A. Eremin, D.H. Chung, Anal. Bioanal.
Chem. 384 (2006) 286–294.
[10] Z.X. Liu, J.X. Gao, J.J. Yu, J. Stored Prod. Res. 42 (2006) 468–479.
[11] S. Tripathi, H.N. Mishra, Food Control 20 (2009) 840–846.
[12] R.W. Beaver, D.M. Wilson, M.W. Trucksess, J. AOAC 73 (1990) 579–581.
[13] J. Stroka, R. van Otterdijk, E. Anklam, J. Chromatogr. A 904 (2000) 251–256.
[14] J. Gomez-Catalan, E. Pique, G. Falco, N. Borrego, M. Rodamilans, J.M. Llobet,
Phytochem. Anal. 16 (2005) 196–204.
[15] A.C. Manetta, L. Di Giuseppe, M. Giammarco, I. Fusaro, A. Simonella, A. Gra-
menzi, A. Formigoni, J. Chromatogr. A 1083 (2005) 219–222.
[16] Y. Wang, T. Chai, G. Lu, C. Quan, H. Duan, M. Yao, B.A. Zucker, G. Schlenker,
Environ. Res. 107 (2008) 139–144.
[17] L.E. Edinboro, H.T. Karnes, J. Chromatogr. A 1083 (2005) 127–132.
[18] C. Cervino, S. Asam, D. Knopp, M. Rychlik, R. Niessner, J. Agric. Food Chem. 56
(2008) 1873–1879.
[19] D. Bhattacharya, R. Bhattacharya, T.K. Dhar, J. Immunol. Methods 230 (1999)
71–86.
[20] N.A. Lee, S. Wang, R.D. Allan, I.R. Kennedy, J. Agric. Food Chem. 52 (2004)
2746–2755.
[21] P.W. Li, Q. Zhang, W. Zhang, J.Y. Zhang, X.M. Chen, J. Jiang, L.H. Xie, D.H. Zhang,
Food Chem. 115 (2009) 313–317.
[22] Z. Zheng, C.W. Humphrey, R.S. King, J.L. Richard, Mycopathologia 159 (2005)
255–263.
[23] A. Sheibani, M. Tabrizchi, H.S. Ghaziaskar, Talanta 75 (2008) 233–238.
[24] A. Molinelli, K. Grossalber, R. Krska, Anal. Bioanal. Chem. 395 (2009) 1309–1316.
[25] R. Tanaka, T. Yuhi, N. Nagatani, T. Endo, K. Kerman, Y. Takamura, E. Tamiya,
Anal. Bioanal. Chem. 385 (2006) 1414–1420.
[26] X.L. Sun, X.L. Zhao, J. Tang, X.H. Gu, J. Zhou, F.S. Chu, Food Control 17 (2006)
256–262.
[27] W.B. Shim, Z.Y. Yang, J.S. Kim, J.Y. Kim, S.J. Kang, G.J. Woo, Y.C. Chung, S.A.
Eremin, D.H. Chung, J. Microbiol. Biotechnol. 17 (2007) 1629–1637.
[28] J. Hashemi, G.A. Kram, N. Alizadeh, Talanta 75 (2008) 1075–1081.
[29] D.H. Zhang, P.W. Li, Q. Zhang, W. Zhang, Biosens. Bioelectron. 26 (2011)
2877–2882.
[30] S. Wang, C. Zhang, Y. Zhang, Methods Mol. Biol.: Biosens. Biodetect. 504 (2009)
237–252.
[31] J. Zhu, W.C. Chen, Y.T. Lu, G.H. Cheng, Environ. Pollut. 156 (2008) 136–142.
[32] H.P. Lau, P.K. Gaur, F.S. Chu, J. Food Saf. 3 (1981) 1–13.
[33] K.O.A. Yu, J.S. Im, P.A. Illarionov, R.M. Ndonye, A.R. Howell, G.S. Besra, S.A.
Porcelli, J. Immunol. Methods 323 (2007) 11–23.
[34] C. Russo, L. Callegaro, E. Lanza, S. Ferrone, J. Immunol. Methods 65 (1983)
269–271.
[35] X.L. Wang, K. Li, D.S. Shi, X. Jin, N. Xiong, F.H. Peng, D.P. Peng, D.R. Bi, J. Chro-
matogr. B Analyt. Technol. Biomed. Life Sci. 847 (2007) 289–295.
[36] B.H. Liu, Z.J. Tsao, J.J. Wang, F.Y. Yu, Anal. Chem. 80 (2008) 7029–7035.
[37] A. Molinelli, K. Grossalber, M. Führer, S. Baumgartner, M. Sulyok, R. Krska, J.
Agric. Food Chem. 56 (2008) 2589–2594.
[38] J.H. Cho, S.H. Paek, Biotechnol. Bioeng. 75 (2001) 725–732.
[39] P.K. Gaur, H.P. Lau, J.J. Pestka, F.S. Chu, Appl. Environ. Microbiol. 41 (1981)
478–482.
[40] L. Anfossi, M. Calderara, C. Baggiani, C. Giovannoli, E. Arletti, G. Giraudi, J. Agric.
Food Chem. 56 (2008) 1852–1857.
[41] J.D. Beatty, G.B. Barbara, G.V. William, J. Immunol. Methods 100 (1987)
173–179.
[42] Y. Zhu, L. Li, Z.H. Wang, Y.Q. Chen, Z.M. Zhao, L. Zhu, X.P. Wu, Y.P. Wan, F.Y. He,
J.Z. Shen, J. Agric. Food Chem. 56 (2008) 5469–5474.
[43] Y.P. Xu, L.Q. Liu, Q.S. Li, C.F. Peng, W. Chen, C.L. Xu, Biomed. Chromatogr. 23
(2009) 308–314.
[44] X.L. Wang, K. Li, D.S. Shi, N. Xiong, X.E. Jin, J.D. Yi, D.R. Bi, J. Agric. Food Chem.
55 (2007) 2072–2078.
[45] Y.R. Guo, S.Y. Liu, W.J. Gui, G.N. Zhu, Anal. Biochem. 389 (2009) 32–39.
[46] P. Zhou, Y. Lu, J. Zhu, J. Hong, B. Li, J. Zhou, D. Gong, A. Montoya, J. Agric. Food
Chem. 52 (2004) 4355–4359.
[47] R. Verheijen, P. Stouten, G. Cazemier, W. Haasnoot, Analyst 123 (1998)
2437–2441.
[48] Y. Xu, Z.B. Huang, Q.H. He, S.Z. Deng, L.S. Li, Y.P. Li, Food Chem. 119 (2010)
834–839.
[49] L.Q. Liu, C.F. Peng, Z.Y. Jin, C.L. Xu, Biomed. Chromatogr. 21 (2007) 861–866.
[50] D.W. Li, S. Wei, H. Yang, Y. Li, A.P. Deng, Biosens. Bioelectron. 24 (2009)
2277–2280.
[51] H.Z. Senyuva, D. Cimen, J. Gilbert, J. AOAC Int. 92 (2009) 1128–1135.
[52] Y. Zhou, F.G. Pan, Y.S. Li, Y.Y. Zhang, J.H. Zhang, S.Y. Lu, H.L. Ren, Z.S. Liu, Biosens.
Bioelectron. 24 (2009) 2744–2747.