A sensitivity-enhanced heterologous immunochromatographic assay based on a monoclonal antibody for the rapid detection of histamine in saury samples

Article abstract of DOI:10.1039/c5ra15925c

Histamine (HA) is an essential test item for fishery samples. However, the fast and effective determination of HA is difficult due to its simple structure and small molecular size. In this study, a sensitive and specific monoclonal antibody against p-nitrobenzoylated histamine (NPHA), which can be easily obtained from the reaction of HA and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions, was generated for the first time. Based on this mAb, an immunochromato-graphic assay strip (ICA strip) using a colloidal gold nano particles-antibody (GNPs) probe for rapid detection of HA in saury samples was established. After screening the coating antigens and optimization of analytical parameters, a heterologous coating based ICA strip exhibited the most excellent detection ability with a visual detection limit (VDL) of 6.0 mg kg^-1 in qualitative experiments and a detection limit (by a strip reader) of 1.0 mg kg^-1 in semi-quantitative experiments for saury samples, with no cross-reactivity with HA analogs. Good correlation between the ICA strip with liquid chromatography-tandem mass spectrometry was achieved for spiked and naturally contaminated saury samples. Overall, this method is suitable for screening of HA residue for large scale fish samples in a quick, simple and low-cost manner.

Full text of DOI:10.1039/c5ra15925c

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A sensitivity-enhanced heterologous immunochromatographic assay
based on monoclonal antibody for the rapid detection of histamine in
saury samples
Lin Luo,† JinꢀYi Yang,† ZhiꢀLi Xiao,† DaoꢀPing Zeng, § YongꢀJun Li,† ‡ YuꢀDong
Shen,† YuanꢀMing Sun,†,* HongꢀTao Lei,† Hong Wang,† ZhenꢀLin Xu†,*
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† Guangdong Provincial Key Laboratory of Food Quality and Safety, South China
Agricultural University, Guangzhou 510642, China
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§ Guangzhou Wanlian Biotechnologies Co. Ltd, Guangzhou 510670, China
‡ Zhongshan Quality Supervision & Inspection Institute of Agricultural Products,
Zhongshan, 528403, China
* Corresponding author. No.483, Wushan Road, Tianhe District, Guangzhou, 510642,
China. Tel.: +86 20 8528 3448; Fax: +86 20 8528 0270
Eꢀmail: gzsyming@163.com (Y.M. Sun); jallent@163.com (Z.ꢀL. Xu)
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Abstract: Histamine (HA) is an essential test item for fishery samples. However, the
fast and effective determination of HA is difficult due to its simple structure and small
molecule. In this study, a sensitive and specific monoclonal antibody against
pꢀnitrobenzoylated histamine (NPHA), which can be easily obtained from the reaction
of HA and pꢀnitrobenzoic acid Nꢀhydroxysuccinimide ester (PNBAꢀOSu) under mild
condition, was generated for the first time. Based on this mAb, an immunochromatoꢀ
graphic assay strip (ICA strip) using colloidal gold nano particlesꢀantibody (GNPs)
probe for rapid detection of HA in saury samples was established. After screening the
coating antigens and optimization of analytical parameters, a heterologous coating
based ICA strip exhibited the most excellent detection ability with a visual detection
limit (VDL) of 6.0 mg kg–1 in qualitative experiment and a detection of limit (by strip
reader) of 1.0 mg kg–1 in semiꢀquantitative experiment for fish samples, with no
crossꢀreactivity with HA analogs. Good correlation between the ICA strip with liquid
chromatographyꢀtandem mass spectrometry was achieved for spiked and naturally
contaminated saury samples. Overall, this method is suitable for screening of HA
residue for a large scale of fish samples in a quick, simple and lowꢀcost manner.
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46 1. Introduction
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Histamine (HA) is one of the most important biogenic amines related to human health
for HA poisoning characterized by headache, nausea, vomiting, diarrhea, itching, an
oral burning sensation, red rash, and hypoꢀtension resulting from excessive intake of it.
1, 2 However, HA accumulation was commonly observed in tissues of fish and other
seafoods when spoilage by bacteria commenced during storage, which doesn’t
influence the seafood normal appearance and odor initially.3 Therefore, to avoid
serious risk to human health, regulatory levels for HA in fishery products have been
set by many countries and organizations thus far. China has regulated HA in mackerel
and other marine fish should not exceed 1000 mg kg–1 and 300 mg kg–1, respectively.
4 The United States Food and Drug Administration has set 50 mg kg–1 to be maximum
residual level (MRL) for HA in fish tissue. 5 The European Union (EU) 6 has regulated
that the marketing into EU of fish and fishery products belonging to the families of
Coryphaenidae, Scombridae, Clupeidae, and Eugraulidae were authorized after a
systematic check of the compliance to European histamine limits was performed.
Normally, HA content in foods is determined by chromatography analysis which
requires extensive sample cleanup, cost equipment and professional staff and not
7, 8
suitable for highꢀthroughput and onꢀsite analysis.
Antibodyꢀbased immunoassays
are wellꢀestablished and received rapid methods which have been developed for
antibiotics, pesticides and other harmful chemicals rapid screening for their high
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sensitivity, rapidity, low cost and applicability for large numbers of samples.
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However, the production of sensitive and specific antibody against HA is a big
challenge due to the simple structure of HA without sufficient epitome to elicit
sensitive and specific antibody. 13 Mita et al. attempted to produce a specific antibody
against HA through immunizing with rabbit with three conjugates of different HA
derivatives with BSA, but failed. 14 Another strategy is to generate antibodies against
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the
derivatives
of
HA,
such
as
pꢀbenzoquinoneꢀhistamine,15,
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succinylꢀglycinamideꢀhistamine,
then developed immunoassays for HA via a
preꢀderivatization. However, all the reported derivatization of HA suffered from some
drawbacks: long time, low yield, along with side reactions, and unstable of products,
etc.
In this work, 4ꢀ((2ꢀ(1Hꢀimidazolꢀ4ꢀyl) ethyl) carbamoyl) benzoic acid (hapten1, see
Fig. 1)ꢀBSA conjugate was used as an immunogen to prepare a monoclonal antibody
which showed good sensitivity and specificity against a stable HA derivative,
pꢀnitrobenzoylated histamine (NPHA, Fig. 1), which can be easily formed from the
reaction of HA and pꢀnitrobenzoic acid Nꢀhydroxysuccinimide ester (PNBAꢀOSu, Fig.
1) under mild condition. Furthermore, GNPsꢀbased ICA, which is the most stable and
classic ICA 19 and ideally suited as onꢀsite screening tool for large scale of samples
for its lowꢀcost, simplicity and rapid result judgment (within 3~5min), was developed
for HA detection. Several heterologous coating haptens were synthesized and used as
heterologous coating to study their effect on the assay’s sensitivity. In addition,
portable strip reader was introduced when performing result judgment which can
exclude the subjectivity arising from visual assessment. To our knowledge, this is the
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first report of successful qualitative and semiꢀquantitative detection of HA by using
ICA.
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2. Material and Methods
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2.1. Reagent and instrumentals
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Ovalbumin (OVA), bovine serum albumin (BSA) and chloroauric acid
(HAuCl4·4H2O), Sheep antiꢀmouse IgG were provided by Sigma Corporation (St.
Louis, USA). Histamine dihydrochloride, Lꢀhistidine, Lꢀtryptophan, tryptamine
hydrochloride, tyramine hydrochloride, phenethylamine hydrochloride, 4ꢀformyl benꢀ
zoic acid, and benzoic acid were obtained from Heowns Biochem Technologies Co.
Ltd. (Tianjin, China). Trisodium citrate, 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)ꢀcarbodiꢀ
imide hydrochloride (EDC), sodium borohydride, Dimethylformamide (DMF), were
obtained from Aladdin Chemical Technology Co., Ltd. (Shanghai, China).
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PNBAꢀOSu and NPHA were prepared according to previous work. Nitrocellulose
(NC) membranes, glass–fiber, sample pad, absorbent a pad and release pad were
purchased from Millipore (USA). The HGS 510 dispensing platform, HGS 201
guillotine cutter were supplied by Autokun (Hangzhou, China). Membrane strip
reader (DY6510) was supplied by Wanlian Biotechnologies Co. Ltd. (Guangzhou,
China). The transmission electron microscope (JEMꢀ2100F) was purchased from
JEOL (Japan). The ultraviolet spectrophotometer (UVꢀ3010) was purchased from
Hitachi Corporation (Tokyo, Japan)
2.2. Preparation of HA derivatives and hapten-protein conjugates
Benzoic acid and five HA derivatives (Fig. 2, denoted as hapten1~6) with spacers
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ending in a carboxyl group and were employed as haptens. The synthetic procedures
of hapten1~5 were described in detail at supplemental information (See supplemental
information Fig. S1). Hapten6 is commercial benzoic acid. Via the carboxyl group,
these haptens were conjugated with the amino groups of BSA or OVA by the
carbodiimide method according to previous work with some modifications.18 Briefly,
a hapten (0.1 mmol) and carrier protein (10 mg) were added in the conjugation buffer
(PBS, pH 6.0, containing 5% (v/v) DMF). The obtained solution was cooled to 0 °C at
refrigerator, and EDC (32 mg) was added under agitation. The mixture was kept at
0 °C overnight under agitation, and then the conjugation mixture was dialyzed against
10 mM PBS (4×5 L) at 4 °C for 3 days, and finally stored at –20 °C until used.
2.3 Production of anti-NPHA mAb
20
The production of mAb was performed as previously described.
The animal
experiments were performed according to the Regulation Guideline for Experimental
Animals issued by the Ministry of Science and Technology of China. Briefly, three
BABL/c female mice aged 7 weeks were immunized with the immunogen
(hapten1ꢀBSA) on days 0, 28, 49 and 71. The mouse that exhibited the best titer for
the immunogen was chosen as the donor of spleen cells for hybridoma production.
Through cell fusion technology, the above spleen cells were fused with SP2/0 murine
myeloma cells to form hybridomas. Then hybridomas secreting NPHA specific
antibodies were subcloned five times by limiting dilution. The best
antibodyꢀproducing clone (2G6/S5) was expanded and selected to produce ascetic
antibodies. The obtained ascetics fluids were purified by caprylic acidꢀammonium
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method, 21 and antiꢀNPHA mAb was obtained. Two ELISA formats, indirect ELISA
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(inꢀELISA) and indirect competitive ELISA (icꢀELISA) as previously described
were performed to screen target hybridoma and characterize the mAb’s binding with
coating antigens.
2.4 Preparation of anti-NPHA-mAb-GNPs conjugate
GNPs with an average diameter of 35~40 nm was produced using the sodium citrate
method. 23 Briefly, 100 mL of 0.01% HAuCl4 solution in ultraꢀpurified water was
boiled thoroughly for 1 min, and then 1.5 mL of 1% sodium citrate solution was
rapidly added under continuous agitation, and then the color gradually changed from
light yellow to black, and finally to brilliant red. After the color change, the solution
was boiled for another 3 min, cooled and preserved at 4 °C with 0.05% sodium azide.
Transmission electron microscopy (TEM) was employed to characterize the GNPs.
To conjugate the GNPs with antiꢀNPHAꢀmAb, the optimum mAb amount for labeling
was investigated, and the results was present in Fig S2. As a result, 24 ꢁL of mAb was
added dropwise to 10 mL of GNPs (pH 8.5, adjusted by 0.1 M K2CO3) and kept at
room temperature for 30 min with agitation. Then, 1.2 mL of 10% BSA was added
dropwise to block residual surfaces of the GNPs. The mixure was further incubated
for 30 min at room temperature and centrifuged for 15 min at 10000 rpm, and then the
supernatant was discarded. The precipitate was resuspended in 1 mL of PB (0.05M,
pH 7.4, containing 0.5 % BSA, 1% mycose, 6 % sucrose, and 0.1 % Tweenꢀ20) and
stored at 4 °C until use.
2.5 Development of the GNPs-based ICA strip for HA
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An ICA strip was assembled by five parts including PVC plate, three pads (sample,
release, and absorbent pads) and one nitrocellulose (NC) membrane (Fig. 1). The
coating antigen and sheep antiꢀmouse IgG were dispensed onto NC as the test (T) and
control (C) line, respectively. The antiꢀNPHAꢀmAbꢀGNPs conjugate was dispensed
by Biostrip dispenser HGS510 onto glass fiber pad to form the release pad. Then the
release pad was dried over night at 37 °C. The sample pad was saturated with PB
(0.05 M, pH 7.5, containing 0.1% PEG 20000, 0.1% Tweenꢀ20, 1% mycose, 6%
sucrose) and dried at 37 °C overnight prior to use. For the absorbent pad, it was used
directly without treatment. The assembly procedure was as follows: the NC
membrane was pasted on the center of PVC plate, then the absorbent pad was pasted
on it by overꢀcrossing 1 mm on the top of the NC membrane, next, the release pad
was pasted on it by overꢀcrossing 1 mm on the bottom of the NC membrane, and then
the sample pad was pasted by overꢀcrossing 2 mm on the bottom of the release pad.
At last, the whole assembled ICA strip was cut into 3 mm wide strips using HGS 201
guillotine cutter and stored in sealed plastic bags containing desiccant until use. Fig.1
shows the schematic illustration of the ICA strip.
2.6 Sample preparation and test procedure
Fish samples were purchased from local market in Guangzhou, China, and the
preparation steps were as previously described with some modifications. 18 Briefly, an
equivalent weight of distill water (d.w.) was added to the muscles and homogenized,
and then d.w. (8 mL) and the homogenate (2 g) were mixed and shaken vigorously for
1 min. The mixture was centrifuged at 3000 g for 10 min, and the supernatant (sample
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extracts) was and subjected to the following derivatization steps: 200 ꢁL of standard
solution or sample extracts, 200 ꢁL of borate buffer (pH 9.0) and 200 ꢁL of
PNBAꢀOSu solution (4 mg mL–1 in acetonitrile (ACN))were mixed in a 1.5 mL
conical tube and vortexed for 10 s. The reaction mixture was kept at room temperature
for 20 min, and 100 mg of NaCl was added to separate the organic layer (ACN) from
the aqueous layer. The organic layer was then diluted 20ꢀfold in working solution
(0.05 M PBS, pH 7.4). A 70 ꢁL of the above diluted solution was added to sample
pad. The result was judged by both naked eyes and DY6510 strip reader within 3~5
min. As illustrated in Fig. 1, if the sample solution has the target analyte, the target
analyte will compete the binding sites of mAbꢀGNPs with the coating antigen on T
line, then the T line will be weak or not appeared, indicating the test result was
weakly positive or positive; if no target analyte was contained in the sample solution,
mAbꢀGNPs will fully bind to the coating antigen on T line, and the T line will display
the strongest color, demonstrating the result was negative. While the C line must
appear to indicate the test is valid.
2.7 Evaluation and validation of ICA strip
HA free fish matrices solution confirmed by previously proposed LCꢀMS/MS, 18 was
used to dilute HA standard stock solution (10 mg mL–1 in distill water) to form a serial
concentrations (0~2000 ꢁg L–1) of HA containing matrices solutions. Then, these
solutions were subjected to the above derivatization procedure and assayed by ICA
strip to evaluate the sensitivity of the ICA strip for fish samples. Structure analogs
(histidine, tyramine, phenethylamine and serotonins) at the concentration of 1, 5, 500
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mg L–1 were used to test the specificity of the ICA strip. HA spiked fish samples and
naturally contaminated fish samples were also assayed by the proposed ICA strip and
confirmed by reference method (LCꢀMS/MS) to evaluate the accuracy and reliability
of the ICA strip.
3. Results and discussion
3.1 Characterization of anti-NPHA mAb
Titer curves of the antiꢀNPHA mAb against six coating antigens were achieved by
inꢀELISA, and then doseꢀresponse curves for NPHA against theses effective coating
antigens (titer>1.0×103) were obtained by icꢀELISA (Fig. S4A). As shown in Fig.
S4A, the titer, which was defined as antibody dilution factor when the A450 value was
at about 1.0, under homologous coating format (hapen1ꢀOVA) was the highest
(5.12×106). However, the titers under heterologus coating format exhibited degree of
heterology dependent decrease compared to the titer under homologous coating
format. From the viewpoint of geometry, hapten1, hapten2 and hapten3 share the
same main structure, a 4ꢀethylꢀ1Hꢀimidazole and a benzene ring, their difference only
exists in the linking bond between benzene ring and 4ꢀethylꢀ1Hꢀimidazole: the linking
bond is amido bond (ꢀCONHꢀ) for hapten1, CꢀN double bond (ꢀHC=Nꢀ) for hapten2,
CꢀN single bond (ꢀHCꢀNHꢀ) for hapten3. Since amido bond (ꢀCONHꢀ) and
CꢀN double bond (ꢀHC=Nꢀ) are planar rigid bond, while CꢀN single bond (ꢀHCꢀNHꢀ)
is a rotatable bond, therefore the structure of hapten3 is more flexible than hapten1
and hapten2, thus hapten3 was considered to be of higher degree of heterology with
hapten1 than that of hapten2. Accordingly, higher titer (2.56×106 versus 6.4×105)
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using hapten2ꢀOVA coating than using hapten3ꢀOVA was obtained. Meanwhile, the
antiꢀmAb NPHA shows no reactivity with hapten5ꢀOVA and hapten6ꢀOVA, and much
lower titer (8×103) was achieved using hapten4ꢀOVA coating (Fig. S4A). Thus, it
could be concluded that the benzene ring contained in hapten1, 2, 3 is conducive for
the antibody recognition, while the acylated HA moiety (Fig. 1 red part in
hapten1ꢀOVA) is a more critical part for antibody recognition, which can be further
confirmed by the results of doseꢀresponse icꢀELISA curves (Fig. S4B). As shown in
Fig. S4B, the highest sensitivity was attained by using hapten3ꢀOVA as coating
antigen with a IC50 of 2.7 ng mL–1, indicating the mAb has the lowest affinity to
hapten3ꢀOVA. However, a relative higher IC50 (3.1 ng mL–1) was obtained by using
hapten4ꢀOVA, which demonstrates that altering the acylated HA moiety of hapten1
imposes a more significant effect on antibody affinity decrease than altering benzene
ring moiety. In conclusion, hapten1, 2, 3, 4ꢀOVA were effective coating antigens for
development of ELISA method for histamine based on the mAb, and the sensitivity of
the corresponding ELISA method on these coating antigens decreased in the
following order: hapten3ꢀOVA, hapten4ꢀOVA, hapten2ꢀOVA, hapten1ꢀOVA.
Furthermore, the specificity of the antiꢀNPHA mAb was also tested by icꢀELISA
under hapten4ꢀOVA coating, and the results demonstrated that the antiꢀNPHA mAb
was highly specific to NPHA and can’t recognize histamine and pꢀnitrobenzoic acid
(Fig S4C).
3.2 Characterization of GNPs
GNPs of various sizes have been used to conjugate with antibody to prepare the ICA
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probe in previous reports. However, according to several ICAs for small molecular
compound detection, diameter of 40 nm GNPs was the optimal particle size for its
tradeꢀoff between steric hindrance and required visibility. 24, 25, 26 As shown in Fig. S3,
the average diameter of the prepared GNPs was about 35~40 nm, which approximates
the optimal particle size. Meanwhile, the GNPs solution was stable by labeling with
optimal amount of antiꢀNPHA mAb and the immunoprobes present a clear, stable and
equably color on T with C line of NC membrane in the strip assay. These results
evidenced that the prepared GNPs was effective and could be applied in the following
experiments
3.3 Screening the coating antigen for ICA strip
Since immunoassays for small molecular compound (monoꢀepitope) are performed
based on competitive format, impairing the binding ability between antibody and
competitor antigen (coating antigen) usually improved assay sensitivity. Therefore,
four coating antigens, hapten1, 2, 3, 4ꢀOVA, which showed reactivity with anitꢀNPHA
mAb in ELISA were coated on the T line, respectively, to study their effect on the
sensitivity of the ICA strip. Consequently, hapten3,4ꢀOVA were failed to develop a
ICA strip, because faint color line (hapten3ꢀOVA) or no visible line (hapten4ꢀOVA)
were appeared in T line for negative simple (data not shown). However, for T line
coated with hapten1ꢀOVA or hapten2ꢀOVA, clear red color, which is of equably
intensity to that on C line, was appeared for negative simple. After optimization the
coating concentration (hapten1ꢀOVA 0.5 mg mL–1, hapten2ꢀOVA 2 mg mL–1), serial
concentrations of NPHA standard solutions (0~400 ng mL–1 in PBS) were tested the
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sensitivity under hapten1ꢀOVA and hapten2ꢀOVA coating. As shown in Fig. 3, the
sensitivity under hapten2ꢀOVA coating (heterologous) improved about 8ꢀfold
compared to that under hapten1ꢀOVA coating (homologous). These results indicated
that heterolougs coating was also effective in enhancing sensitivity of ICA strip, while
some coating haptens (hapten3, hapten4) having higher degree of heterology with
immunogen hapten, which could be applied in developing highly sensitive ELISA can
be ineffective for ICA strip, as the antibody’s binding ability may decline due to the
steric hindrance after conjugated with GNPs.
3.4 Optimization of analytical parameters for ICA test
As an antibodyꢀantigen reaction based immunoassay, ICA was largely influenced by
ionic strength, pH and organic solvents concentration in test solution. Thus PBS of
3ꢀ
different PO4 concentrations (0.01~0.1M) and pH values (6.2~10.0) containing
different concentrations of Tweenꢀ20 (0~0.1%) were used as working solution to
prepare a series of concentrations of NPHA standard solutions from NPHA stock
solution (1 mg mL–1 in methanol), respectively. Then, theses standard solutions were
tested by the ICA strip. As shown in Table S1, Tweenꢀ20 contained in working
solution can decrease the sensitivity of ICA strip. Furthermore, it was found PBS
(0.05M, pH7.4) results in the most sensitive ICA test (Table S2, Table S3). Thus, PBS
(0.05M, pH7.4) was chosen as the working solution. To evaluate the organic solvent
susceptibility of the ICA strip, the working solution (PBS, 0.05M, pH7.4) containing
different concentrations of ACN (0~20%, v/v) was used to dilute NPHA stock
solution (1 mg mL–1 in methanol) to form a series concentration of NPHA standard
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solutions, and then the obtained NPHA standard solutions were tested by the ICA strip.
As shown in Table S4, the sensitivity of the ICA strip was decreased significantly as
the concentration of ACN increased, and the maximum ACN tolerance level for the
ICA strip was 5% (Table. S4). Thus, the upper layer of derivatization reaction mixture
was required to be diluted 20ꢀfold by PBS (0.05M, pH7.4) for the ICA test.
3.5 Sensitivity and specificity of ICA
As shown in Fig. 4, the red color of T line became weaker as the concentration of HA
increased. When HA concentration exceeded 600 ng mL–1(corresponding to 6.0 mg
kg–1 in fish sample according to the extraction procedure in sample preparation), the T
line completely disappeared, thus, 600 ng/mL (corresponding to 6.0 mg/kg in fish
sample according to the extraction procedure in sample preparation) was confirmed to
be visual detection limit (VDL) of the ICA strip. Meanwhile, color intensity of T line
–1
(AT) and C line (AC) were measured by DY 6510 strip reader. The AT AC ratio of
negative and positive samples were designated as B0 and Bx. Calibration curve
constructed by plotting the B B0–1 against logarithm of HA concentration (Fig.S5) . As
the color intensity of T line was significantly different (B B0–1≈0.5) with that of blank
sample (HA free matrices solution), when HA concentration was at 100 ng mL–1(1 mg
kg–1 in fish sample). Thus, 1 mg kg–1 was confirmed to be limit of detection (LOD)
for the ICA strip in this study.
Since the MRLs for HA in fish varied from 50 mg kg–1 to 1000 mg kg–1, it seems that
the proposed ICA strip is much too sensitive. However, it is necessary to develop a
screening method having a LOD lower than MRLs. For one thing, to make the
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working range of the method suitable for the varied MRLs set by different countries
or districts, only suitable dilution factors are needed to be introduced in sample
pretreatment; for another, larger dilution factor is required for sample pretreatment to
remove martrices effect, when analyzing samples having more complex martrices.
Several HA analogs were used to investigate the specificity of the ICA strip, while the
color intensity of T line were almost identical with that of negative control (distill
water), when these competitors at 1, 5, 500 mg L–1, indicating that the ICA strip was
highly specific for HA with negligible crossꢀreactivity to these analogs (Table 1).
3.6 Analysis of HA in saury samples
Saury, a kind of fish belonging to family of Scombridae, which was reported to be
potential histamine poisoning source, 2 is commonly consumed in Guangzhou, China.
Therefore, saury was chosen as a model matrices for the proposed ICA strip and a
saury sample which initially contains 0.23 mg kg–1 of HA (confirmed by LCꢀMS/MS)
was spiked with HA at the level of 2.0, 5.0 and 10.0 mg kg–1, and then the nonꢀspiked
and spiked samples were tested by the proposed ICA strip (Fig 5) and LCꢀMS/MS,
respectively to evaluate the accuracy of the test strip. As shown Table 2, the results of
the ICA strip correlate well with that of LCꢀMS/MS. Another saury sample was
immediately pretreated (washing and removal of inedible part) after purchased from
local supermarket, and its muscle was stored at 4°C for using as naturally
contaminated sample. Then, it was subjected to HA analysis every two days by the
proposed ICA strip and LCꢀMS/MS. As shown in Table 3, HA content in this saury
was sharply increased to 224.5 mg kg–1 in the sixth days, which even surpasses the
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regulated level set by FDA (50 mg kg–1).
As a small molecular amine compound having strong polarity, HA is highly aqueous
soluble, which is seriously adverse to the sample purification prior to analysis, since
routine organic solvents cannot extract it from aqueous solution, thus, cation
ionꢀexchange, which is a laborꢀintensive and timeꢀconsuming operation, was often
involved in the sample purification for some instrumental methods for HA detection.
27, 28
Although a derivatization step was required for the proposed ICA strip test, the
whole sample preparation was simple and can be completed within 35 min, and all the
used equipments can be portable, which benefit from a moderately polar compound,
NPHA, was formed after HA interacted with PNBAꢀOSu (derivatizing reagent); since
the NPHA is of bad waterꢀsolubility and good solubility in ACN, thus the formed
NPHA was contained in the upper layer (ACN layer) and separated from most of
interfering substances and can be tested directly via a preꢀdilution by working solution
after a certain amount of NaCl was added to the derivatization mixture solution
(ACNꢀborate buffer mixtures). Thus, the proposed ICA strip can be used as an
important tool to achieve rapid detection of HA in fish samples.
4. Conclusion
In this work, a stable hybridoma cell line generating mAb against NPHA was
developed, which can be an unlimited supply of mAb for NPHA. Then, a sensitivity
enhanced heterologous ICA based on this mAb for rapid HA detection was established
and applied in assaying HA spiked saury samples and naturally contaminated saury
samples. The ICA showed a VDL of 6.0 mg kg–1 and a LOD of 1.0 mg kg–1 (by strip
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reader) for saury samples, with no crossꢀreactivity with other HA related compounds.
Moreover, the results obtained from the ICA strip was in agreement with that from
LCꢀMS/MS. Thus, the ICA strip was suited as a tool for rapid HA onꢀsite screening,
and ICA may also be developed for other biogenic amines by referring to the
strategies involved in this study. In addition, the heterologous coating should be
adopted as a potential general strategy for improving sensitivity when developing ICA
based detection method for small molecules.
Acknowledgement
This work was supported by the National Basic Research Program of China (973
Program, 2012CB720803), the Excellent Young Teachers Program on Higher
Education of Guangdong Province (awarded to Z.ꢀL. Xu, Y920/4026), and the
Guangdong Provincial Project of Science and Technology (2012A020100002).
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FIGURE CAPTIONS
Fig. 1. Schematic diagram of the immunochromatographic assay for HA detection.
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Fig. 2. Schematic diagram of coating antigens, hapten1ꢀOVA conjugate was
homologous coating antigen; hapten2, 3, 4, 5, 6ꢀOVA conjugates were used as
heterologous coating antigens.
Fig. 3. The NPHA sensing results of ICA strip under hapten1ꢀOVA (left) and
hapten2ꢀOVA coating (right). The concentration of NPHA standard solutions (diluted
from stocking solution by PBS) were 0, 20, 50, 100, 200, 400 ng mL–1 from left to
right.
Fig. 4. Sensitivity of ICA for fish samples. The test results of a serial concentrations
of HA standard solution (prepared by HA free matrices solution). From left to right,
the HA concentration was 0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, 1000, 2000
ng mL–1.
Fig. 5. Results of spiked fish sample assayed by ICA strip. 0.23 mg kg–1 was
confirmed to be the initial HA concentration of the fish sample by LCꢀMS/MS. Then
the fish sample were spiked with 2.0, 5.0, 10.0 mg kg–1 of HA.
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Figure 1
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Figure 2
O
O
O
O
N
N
N
N
N
HN
HN
OVA
N
OVA
HN
HN
HN
OVA
HN
H
H
Coating antigen2
(hapten2-OVA)
Coating antigen1
(hapten1-OVA)
Coating antigen3
(hapten3-OVA)
O
O
H
H
N
O
N
N
N
NH OVA
N
H
OVA
O
HN
HN
HN
OVA
Coating antigen5
(hapten5-OVA)
Coating antigen6
(hapten6-OVA)
Coating antigen4
(hapten4-OVA)
440
441
442
443
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Figure 3
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Figure 4
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Figure 5
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Table 1. Cross reactivity (CR) with histamine related compounds by the ICA strip
compounds
structure
Concentration (mg/L)
CRa(%)
0
1b
1
1
5
0
500
0
Histamine
Histidine
0.03
0.97
100
0.98
0.95
<0.01
Trytamine
1
0.98
0.95
0.93
<0.01
Tryamine
1
1
0.98
0.96
0.98
0.96
0.95
0.96
<0.01
<0.01
Phenethylamine
485
486
487
488
489
490
491
492
493
494
495
a CR, crossꢀreactivity. The percentage of CR was calculated by the following equation:
CR (%) = [IC50 (histamine, mmol/L)/IC50 (crossꢀreactant, mmol/L)] × 100.
b Recorded B B0–1 by strip reader
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Table 2. Results of spiked fish sample analyzed by ICA strip and LCꢀMS/MS (n=3)
Spiked
ICA a
LCꢀMS/MS
(mg kg–1)
0.23
(mg kg–1)
(mg/kg)
b
0
2
－
2.0
6.2
2.30
5
5.27
10
11.8
10.30
499
500
501
502
503
504
505
506
507
508
509
510
511
a HA concentration was extrapolated from the calibration curve
b HA concentration lower than the LOD
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Table 3. Results of monitoring HA formation in saury by ICA strip and LCꢀMS/MS (n=3)
Time
ICAa
LCꢀMS/MS
(mg kg–1)
0.76
(mg kg–1)
(days)
b
0
2
4
6
－
5.2
14.5
260.0
4.62
13.71
224.52
513
514
515
516
517
518
519
a HA concentration was extrapolated from the calibration curve
b HA concentration lower than the LOD
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