ELISA detection of semicarbazide based on a fast sample pretreatment method

Article abstract of DOI:10.1039/c3cc42790k

A direct ELISA was established for the fast detection of semicarbazide (SEM) using a novel biotin derivative. Without a tedious extraction procedure, as low as 0.07 μg L^-1 of SEM could be detected reproducibly. This assay has better recovery and accuracy than competitive ELISA. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc42790k

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ELISA detection of semicarbazide based on a fast
sample pretreatment method†
Cite this: DOI: 10.1039/c3cc42790k
Zhiyuan Fang,z Baishan Jiang,z Wei Wu, Zhicheng Xiang, Chuanyan Ouyang,
Received 16th April 2013,
Accepted 23rd May 2013
Tulong Huang, Junhua Chen and Lingwen Zeng*
DOI: 10.1039/c3cc42790k
www.rsc.org/chemcomm
A direct ELISA was established for the fast detection of semicarbazide
The first established method for the detection of SEM
(SEM) using a novel biotin derivative. Without a tedious extraction in food samples was based on liquid chromatography–mass
procedure, as low as 0.07 lg Lꢀ1 of SEM could be detected reproducibly. spectrometry.9 But SEM lacks absorbance in the UV region and
This assay has better recovery and accuracy than competitive ELISA.
possesses a low mass that locates in the range of MS back-
ground noise. Derivatisation and extraction are required for further
Nitrofurazone belongs to the group of nitrofuran antibiotics, which analysis.10 But matrix compounds with similar chemical property
has been widely used as a food additive for the treatment of can also be extracted. In this case, extraction recovery and MS
gastrointestinal infections in cattle, pigs, poultry, fish and shrimps background may be compromised. Furthermore, expensive
etc. It has been reported to cause tumors in experimental animals equipment and well trained operators are a pre-requisite for
and participate in nitrofurazone-induced carcinogenesis.1 Nitro- accurate detection. Therefore, the third-world countries which
furazone and its metabolites are also involved in tumor initiation suffer more from the abuse of illegal drugs need more convenient
through p53 gene damage, and stimulating the proliferation of and cheaper detection methods.
tumor cells.2 Because of its carcinogenicity, the use of nitrofurazone
in food-producing animals has been banned by food regulatory capable of detecting the SEM derivative with 4-carboxybenzaldehyde
authorities, including those in Europe, USA and China. (CBA). Antibody from a strain of hybridoma with high specificity
In a previous study, we prepared monoclonal antibodies
Nitrofurazone is rapidly metabolized in vivo and has a short half- was used to develop a competitive ELISA for the SEM analysis.11
life in edible tissues. The parent drug breaks down in a few hours.3 However, the extraction recovery rate and repeatability were not
The decrease of nitrofurazone is accompanied by the accumulation good. The residual organic extraction solvent also reduced the
of the side chain metabolite semicarbazide. SEM and protein-bound affinity of the antibody, thus compromising the accuracy and
SEM are more stable and can persist in the body for several weeks.4 reproducibility of the test. Therefore, a simpler and more efficient
Thus, the metabolite of nitrofurazone (1a,1b, Fig. S1, ESI†), semi- assay for SEM detection is needed.
carbazide, was used as a marker residue for the monitoring of illegal
To address these limitations, we introduced a ligand–receptor
drug use.5 Recently, it was found that SEM was also produced as a pattern for the purification and sandwich detection of the SEM
breakdown product of azodicarbonamide (ADC) (1c, Fig. S1, ESI†), derivative. Biotin was used as the ligand in this study. Streptavidin
a blowing agent used in the manufacture of plastic packaging (SA) was its natural receptor that can bind biotin with high
materials,6 and a product of the hypochlorite reaction in food affinity (Kd E 10ꢀ14 mol Lꢀ1).12 Thus, using biotin modified
processing.7 ADC may cause allergic reactions in people who CBA, we established a direct ELISA for quantitative analysis of
are sensitive to furazolidone (AZO) compounds, and SEM was small molecule SEM. The derivatisation agent CBA was first
also considered to be a potential carcinogenic agent that may modified with biotin, which was then used for the derivatisation
cause tumor.8 Due to their potential health risks, the use of of SEM. The resultant biotinylated-SEM (1d, Fig. S1, ESI†) had
ADC as a blowing agent in food packaging has been banned in two functional groups: the SEM-CBA moiety and the biotin
the European Union since 2005.
moiety. The SEM-CBA moiety was captured by the antibody that
was coated on the 96-well plate, and the biotin moiety in this
molecule was further captured by a streptavidin–horseradish
peroxidase conjugate (SA–HRP). Thus, the assay is established
with a sandwich format (as shown in Fig. 1).
The synthetic strategy for biotinylated aldehyde derivative 1,
in which the biotin group and the CBA group are linked by a
triethylene glycol group, is shown in Fig. 2 according to reported
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology
and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, Guangzhou 510530, China.
E-mail: zeng6@yahoo.com; Fax: +86 20 32015245; Tel: +86 20 32015312
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc42790k
‡ These authors contributed equally.
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Fig. 1 Schematic presentation of the direct ELISA for SEM detection.
Fig. 3 Standard curve of the assay. Optical densities are obtained from serially diluted
SEM standard solutions (0 mg Lꢀ1, 0.07 mg Lꢀ1, 0.14 mg Lꢀ1, 0.29 mg Lꢀ1, 0.58 mg Lꢀ1
,
1.17 mg Lꢀ1, 2.34 mg Lꢀ1, 4.68 mg Lꢀ1, 9.37 mg Lꢀ1, 18.75 mg Lꢀ1, 37.5 mg Lꢀ1).
be attributed to the following two reasons: the primary amine
group in Tris blocks the free aldehyde group in biotinylated
aldehyde and PBS buffer provides a suitable environment for the
proper functioning of the antibody.
After optimizing the parameters of this assay, a standard
curve was obtained (Fig. 3). It was then applied to curve fitting
using Originpro (Originpro 8.5). The resulting calibration plot
exhibited a well-defined linear relationship between the SEM
concentration and optical density, with a dynamic range of
0.07–40 mg Lꢀ1. The coefficient of variation ranged from 1% to
8%. The mean optical density of the blank was 0.093, and the
Fig. 2 Synthetic strategy for biotinylated aldehyde derivative 1a. Reagents and
conditions: (a) (Boc)2O, DIPEA, DCM; (b) biotin, HATU, DIPEA, DMF; (c) HCl, DCM;
(d) 4-carboxybenz-aldehyde, HATU, DIPEA, DMF.
methods with some modifications.13 Mono-N-Boc protected standard deviation was 0.006. Thus, the limit of detection
amine 3 was first prepared starting from commercially available (LOD) is 0.07 mg Lꢀ1, which is calculated based on the triple
diamine 2 in anhydrous DCM. The mono-Boc protected amine 3 standard deviation from the mean of blanks (mean + 3SD). We
was then coupled to biotin in the presence of the HATU–DIPEA have established a competitive ELISA using the same antibody
system, an effective activating agent used in peptide synthesis. in a previous study. The sensitivity of the present method is
Deprotection of Boc protected biotinylated amide 4 in a HCl– better than that of the competitive ELISA (Fig. S3, ESI†), and is
dioxane system yielded amine 5. Compound 5 was then coupled comparable to those of LC-MS/MS.14
with 4-carboxybenzaldehyde in a HATU–DIPEA system to give
compound 1 with 40.5% overall yield starting from 3 (ESI†).
Recovery is another important parameter for the detection of
SEM. In this study, skimmed milk power purchased at a local
To quantitatively analyze SEM, a standard curve or calibra- supermarket was used as the sample for evaluation of the
tion plot was needed. In the present study, it was obtained by performance. Serially diluted SEM stock solutions were added
performing a calibration experiment over serially diluted SEM to 1 g of milk powder, and the volume was adjusted to 10 mL
standard solutions (SEM spiked ultra-pure water) with a with ultra-pure water. The final SEM concentration ranged
concentration range of 0–70 mg Lꢀ1. SEM standard solution from 0 mg kgꢀ1 to 37.5 mg kgꢀ1. Derivatisation was performed
was first derivatised with biotinylated aldehyde for 4 hours and at 37 1C for 4 hours. After derivatisation, the mixture was
the SEM derivative was analyzed using the present assay.
High backgrounds were observed in our preliminary results. substance. The supernatant was subjected to the present assay.
We speculated that the unreacted aldehyde group in biotinylated All the SEM spiked samples showed positive results and all the
centrifuged at 5000 rpm for 10 min to remove the insoluble
aldehyde covalently linked to free amino groups in the antibody or blank samples showed negative results. The recovery ranged from
bovine serum albumin (BSA) that was coated on the plate. Besides, 96% to 113% (Fig. 4), which were much better than those in
the pH of the derivatisation solution was too low (pH E 5) for the indirect competitive ELISA (20–100%, data not shown). The high
proper functioning of the antibody. Referring to the extraction recovery can be explained by the simplified treatment of the SEM
method used in LC-MS/MS analysis, we employed a chimeric derivative. Without several steps of extraction and clean-up, the
phosphate buffered saline-Tris (hydroxymethyl) aminomethane- biotinylated molecule is directly added to the plate and captured by
HCl buffer (PBS-Tris buffer, pH = 7.5) as the hybridization buffer. antibodies, which means no SEM derivative is lost in this process.
The results were consistent with our hypothesis and low back-
The selectivity of this method is dependent on the specificity
grounds were obtained. Meanwhile, PBS buffer and Tris buffer with of the antibody. Several veterinary drugs, related derivatives and
the same pH were also tested, but neither of their performance was metabolites were tested in this study. The test was conducted
comparable to that of the PBS-Tris buffer. The low background can according to the procedures described above.
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reversibly break the interaction between biotin and streptavidin
without denaturing streptavidin, allowing the collection of the
derivative and re-use of the streptavidin column.16 The limitation of
this method is the complexity of the preparation of the derivatisa-
tion agent. The overall yield is about 40%, which still needs further
improvement.
In conclusion, we have established a direct ELISA for the
detection of small molecule SEM based on our synthesized
biotinylated aldehyde. It not only simplifies and improves the
ELISA based assay but also provides a possibility for the
improvement of the LC-MS/MS performance. The method
reported in this communication is inspiring toward its applica-
tions in other small molecule detection.
Fig. 4 Distribution of the recovery of spiked samples between 0 mg kgꢀ1 and
Financial support was provided by the Ministry of Science
and Technology 973 Project (2013CB967100).
37.5 mg kgꢀ1 (0 mg kgꢀ1, 0.07 mg kgꢀ1, 0.14 mg kgꢀ1, 0.29 mg kgꢀ1, 0.58 mg kgꢀ1
,
1.17 mg kgꢀ1, 2.34 mg kgꢀ1, 4.68 mg kgꢀ1, 9.37 mg kgꢀ1, 18.75 mg kgꢀ1, 37.5 mg kgꢀ1).
Notes and references
Except for nitrofurazone, none of these chemical compounds
had cross-reactivity with this antibody, including those structurally
related to SEM-CBA and the derivatisation agent CBA (Fig. S2, ESI†).
Nitrofurazone has an imidazole group that resembles the benzene
ring in SEM-CBA, which can explain the low cross-reactivity.
We also synthesized and tested biotinylated aldehyde substrates
using one ethylene group and a tetraethylene glycol group as the
linker, respectively. The sensitivity of this method using ethylene as
the linker was lower than that of the others, while the tetraethylene
glycol one had the same sensitivity compared to the triethylene
glycol compound 1 (data not shown). The low sensitivity can
be explained by the steric hindrance between antibody and
streptavidin. These results suggested that the linker has to be
long enough to position the biotin moiety distant from the
reactive aldehyde group.15
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The present assay we developed has the following advantages.
First, it is a novel sandwich like ELISA for the detection of small
molecules. It is important for the monitoring of high risk
chemicals, such as toxins and narcotic drugs. For example, small
molecules with an amine group can first react with the
N-hydroxysuccinimide-activated derivatisation agent that is modified
with biotin. The resulting compound is directly captured by its
antibody. Therefore, small molecules can be detected without
the need of their conjugated hapten. Besides, the sensitivity and
reproducibility within batches can also be improved. Second,
using the biotinylated aldehyde, the pretreatment of the SEM
contaminated sample can be simplified. Without extraction, no
organic extraction solvent is involved in the hybridization buffer,
which renders the assay with high accuracy and reproducibility.
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MS methods as a derivatisation agent. The biotin tagged deriva-
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column, similar to the purification of the his-tagged protein
using a nickel alloy column. A short incubation in hot water will
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