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An anti-idiotypic nanobody-phage based real-time immuno-PCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs Jiawen Lei, Peiwu Li, Qi Zhang, Yanru Wang, Zhaowei Zhang, Xiaoxia Ding, and Wen Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5029424 • Publication Date (Web): 02 Oct 2014 Downloaded from http://pubs.acs.org on October 11, 2014
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Analytical Chemistry
An anti-idiotypic nanobody-phage based real-time immunoPCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs § ‡ #
§ ‡ € # £
Jiawen Lei, , , Peiwu Li, *, , , , , Qi Zhang, *, § ‡# £ § ‡ £ Xiaoxia Ding, , , , Wen Zang, , , § ‡
Yanru Wang,
§ , ‡, #
Zhaowei Zhang,
§ , ‡, € , #
Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, P. R. China.
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, P. R. China
€ #
§, €, #
Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan, 430062, P. R. China
Laboratory of Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, 430062, P. R. China Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062, P. R. China
£
ABSTRACT: Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (IPCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05-122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results (R2=0.991) were obtained. In this context, this study provides a novel opportunity to analyse aflatoxins in agri-products.
INTRODUCTION Aflatoxins are a group of extremely toxic and carcinogenic small molecular metabolites produced by certain Aspergillus species, namely A. flavus and A. parasitic.1 To date, more than 20 aflatoxins have been identified,2 and the major aflatoxins of concern are B1, B2, G1, and G23, among which aflatoxin B1 is the most predominant and most toxic.4 Aflatoxins have been implicated as causative agents in human hepatic and extrahepatic carcinogenesis.5 Because of their great harm, the International Agency for Research on Cancer (IARC) has classified aflatoxins B1, B2, G1, and G2 into group I human carcinogens.6 Aflatoxins frequently contaminate a wide range of agrifoods and animal feeds.7 The consumption of these aflatoxincontaminated products caused many outbreaks of acute aflatoxin poisoning during the past decades. In April 2004, one of the largest documented aflatoxicosis outbreaks occurred in rural Kenya, which resulted in 317 cases and 125 deaths. The growing and eating of aflatoxin-contaminated maize on family farms was the main reason of the outbreak.8 From March to June 2011 in Brazil, 60 dogs from 8 different farms died spontaneously after they were fed with cooked corn meal contaminated by high concentrations of aflatoxin B1 (over 1500 ppb).9 Outbreaks of acute aflatoxin poisoning have become a recurrent public health issue. Therefore, to promptly develop a new
and reliable analytical technique for these carcinogenic compounds has become an important requirement of food consumption safety to meet food safety concerns. In recent decades, many methods have been developed for aflatoxin determination, such as high performance liquid chromatography (HPLC),10,11 liquid chromatography–tandem mass spectrometry (LC–MS/MS),12,13 and immunoassay.14,15 Among these methods, immunoassay has been regarded as a valuable supplement to chromatographic techniques due to its advantages in sensitivity, specificity, and cost saving. In recent years, a novel method termed immuno-polymerase chain reaction (IPCR) has been reported for detection of various antigens.16-18 This method is similar to ELISA which detects antigen–antibody reaction; it combines the versatility of ELISA with the sensitivity and signal amplification capability of PCR. As a consequence, IPCR not only leads to higher sensitivity as compared to conventional ELISA, but also reveals a much wider linear range. In typical applications, an antibody linked with a reporter DNA molecule is usually required during IPCR analysis; however, the preparation of antibody–DNA conjugates involves complex covalent coupling chemistry,19 which limits the application of IPCR. The phage display technology was first introduced in 198520 and has been widely used to display peptides or antibody fragments on the surface of the M13 phage.21-24 Recombinant
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phage particles can be easily obtained by simply centrifuging the overnight E.coli culture and amplified in PCR, and therefore the particles are used almost as a 'ready to use' reagent with encoding nucleic acids (single stranded DNA).25 The unique characteristics of the recombinant phage make it an excellent reagent to replace the antibody–DNA conjugate, which can obviate complicated DNA bioconjugation chemistry in conventional IPCR. Phage display mediated IPCR (PDIPCR) has been proven to be a highly sensitive assay for detecting prion protein, nucleocapsid protein of Hantaan virus,25 and hepatitis B virus core antigen.26 Until now, there was no report on PD-IPCR for aflatoxin detection. We have previously reported an anti-idiotypic heavy-chain single-domain antibody for aflatoxin immunoassay by the phage display technology.27 The heavy-chain single-domain antibody is also dubbed as "nanobody"28 due to its much smaller size when compared to traditional antibodies. This socalled anti-idiotypic nanobody can recognize the idiotype of the primary antibody and simulate the reaction of the analyte in ELISA. In this study, we developed real-time PD-IPCR based on an anti-idiotypic nanobody-phage for aflatoxin detection in grains and feedstuffs for the first time.
MATERIALS AND METHODS Reagents. The anti-aflatoxin monoclonal antibody 1C11 (mAb 1C11) was produced in our laboratory as previously described.29 The phage display nanobody V2-5 specific for mAb 1C11 was selected using the phage display technology as previously described27. Aflatoxins B1, B2, G1, and G2 standard solutions, goat anti-mouse immunoglobulin horseradish peroxidase (IgG-HRP), bovine serum albumin (BSA), 3, 3', 5, 5'tetramethylbenzidine (TMB), polyethylene glycol 8000 (PEG 8000), and Tween 20 were purchased from Sigma-Aldrich (St. Louis, MO). The helper phage M13KO7 was purchased from New England Biolabs (Ipswich, MA). Mouse anti-M13 monoclonal antibody-horseradish peroxidase (HRP) was purchased from GE Health Care (Piscataway, NJ). Polystyrene 96-well microtiter plates were from Costar (Corning, Massachusetts, USA). E. coli ER2738 competent cells from the ER2736 lines of E. coli were purchased from Lucigen Corp. (Middleton, WI, USA). Unless otherwise stated, all other inorganic chemicals and organic solvents were of analytical reagent grade or better. Water was obtained from a MilliQ purification system (Millipore). TaqMan probes, iTaqTM Universal Probes Supermix (2×), and the Bio-Rad® iQTM5 real-time PCR system were obtained from Bio-Rad (USA). The sequences of the TaqMan probes and primers were designed using the primer-designing software Primer Expression v3.0 (Applied Biosystems). PDIPCR assay was validated with an Agilent 1100 HPLC system (Agilent Tech, Santa Clara, CA, USA). Phage Preparation. E. coli strains ER2738 containing the phagemid encoding anti-idiotypic nanobody V2-5 were grown in 3 mL SB medium containing 100 µg/mL of ampicillin by overnight shaking at 37°C. Two milliliters of the overnight culture was added into 200 mL SB medium and then incubated at 37°C with vigorous shaking (250 rpm) until OD600 of the medium reached 0.5-0.8. The cells were then infected with helper phages M13K07 by 30 min of incubation without shaking at 37°C. Kanamycin was added to a final concentration of 70 µg/mL, and then the cells were grown overnight with vig-
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orous shaking at 37°C. On the next day, the overnight culture was centrifuged at 10,000 rpm for 15 min and the supernatant was mixed with PEG/NaCl (20% PEG 8000 in 2.5 M NaCl). After 2 h incubation on ice, the phages were precipitated by 30 min of centrifugation at 10,000 rpm. Then, the phage particles were resuspended with 5 mL of suspension buffer (PBS buffer containing protease inhibitor cocktail (Roche Applied Science), 0.02% sodium azide, and 1% BSA). The aliquots were stored at -80°C after determined by phage titration30. Phage-ELISA. The polystyrene microtiter plate was coated with mAb 1C11 at 1 µg/mL in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C. The plate was blocked with PBS (10 mM sodium phosphate buffer containing 137 mM NaCl and 2.68 mM KCl, pH 7.4) containing 3% (w/v) skimmed milk (PBSM) by incubation at 37°C for 1.5 h. The phages were mixed with an equal volume of aflatoxin standards diluted in 10% methanol/PBS (v/v) at various concentrations. One hundred microliters of each mixture was added into the plate followed by 1 h incubation at 37°C. The plate was washed 10 times with PBST (PBS containing 0.05% (v/v) Tween 20) and then the bound phages were detected using the anti-M13 phage mAb-HRP with the plate incubated for 45 min at 37°C. Subsequently, freshly prepared TMB solution (3.3 µL of 30% H2O2, 400 µL of 0.6% TMB in DMSO per 25 mL of acetate buffer, pH 5.5) was used for color development. The reaction was stopped after 15 min with 2 M H2SO4. The absorption values at 450 nm were measured with a microplate reader (SpectraMax® M2e, Molecular Devices, USA). Real-Time PD-IPCR. The procedure of real-time PD-IPCR was similar to that of phage ELISA except that the bound phages were eluted by glycine/HCl buffer (0.2 M, pH 2.2, containing 1% BSA)31. After neutralized with 2 M Tris-base solution, the eluted phages were used as DNA template for real-time quantitative PCR (qPCR). qPCR was performed with TaqMan probes using the Bio-Rad® iQTM5 qPCR system. The PCR mixture contained 1×iTaqTM Universal Probes Supermix, a 200 nM concentration of each primer (forward primer: 5'GTGGTAGCACAAACTATG-3'; reverse primer: 5'GGCTGCACAGTAATAAAC-3’), 200 nM TaqMan probes (5'-FAM-CCGATTCACCATCTCCAGAGACA-TAMRA-3'), 5 µL of eluted phages and distilled water in a final volume of 20 µL. The step program for PCR was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10s and 60°C for 30s. The negative control that contained all the qPCR reagents except the DNA template was included to verify the quality of amplification. Sample Analysis. The samples of corn, rice, peanut and feedstuff, were obtained from local farms and markets and finely ground with a laboratory mill. Subsequently, 5 g of each sample was placed in a 50 mL centrifuge tube and extracted by 25 mL of 80% methanol/water (v/v). After incubating at room temperature with shaking at a speed of 250 rpm for 15 min, the mixture was centrifuged at 5000 rpm for 5 min and the supernatant was used for sample analysis after dilution. The matrix effect and recovery percentage were assayed using blank samples. To evaluate the accuracy and validate the method, a comparative study was performed using real-time PD-IPCR and the HPLC reference procedure.32 Statistical Analysis..The standard curve was obtained by plotting the quantification cycle (Cq) value of PD-IPCR versus
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aflatoxin standard concentration (log10) using OriginPro 8.6 (OriginLab Corporation, Northampton, MA, USA) by fourparameter logistic regression, each data point is the average of three independent measurements. The standard curve was used for aflatoxin quantification in grains and feedstuffs. The linear range was calculated by IC20-IC80, and the LOD was calculated by IC10.
The efficiency was a bit low as 84.8% under the phage number from 101-109 pfu/mL; however, the phage eluted form the ELISA plate was only 101-105 pfu/mL, and the relevant efficiency was 94.4% (data not shown). Therefore, the PCR efficiency is good enough for the following detection and quantification.
RESULTS AND DISCUSSION
Figure 1. Schematic diagram of the real-time PD-IPCR.
Principle of Real-Time PD-IPCR. The principle of realtime PD-IPCR is shown in Figure 1. Anti-aflatoxin mAb 1C11 was first immobilized on the solid surface of a polystyrene microtiter plate. The phage particles could compete with aflatoxins in the microtiter plate wells because the anti-idiotypic nanobody V2-5 has the same function as aflatoxins in reacting with mAb 1C11. After the reaction, the bound phages were eluted and used as the DNA template for real-time PCR. Specific primers of the nanobody encoding sequence were designed for TaqMan real-time PCR, which provided a possibility to develop multiplex immunoassays by sequencedependent amplification31. Efficiency Assessment of Real-Time PD-IPCR. The efficiency of real-time PCR was assessed in the following procedure: The phage particles were diluted 10-fold to serial dilutions (0-1010 pfu/mL) with distilled water, and then 5 µL of each dilution was applied to real-time PCR amplification. The curves of the amplification data (relative fluorescence units) are shown in Figure 2A. The Cq value defined as the number of cycles required for the fluorescent signal to cross the threshold is inversely proportional to the number of phage particles (Figure 2B). We obtained similar Cq values when the number of phage particles was less than 101, but could not get a Cq value when the number was more than 109. In other words, real-time PCR was able to detect phage particles from 101 to 109. The plot of the Cq values against the number of phages (log10) showed good correlation with the R2 value of 0.997. The efficiency (E) of real-time PCR was calculated on the slope of the standard curve using the following formula: 26 E (%) = [101/-slope-1]×100
Figure 2. Determination of real-time PCR efficiency. (A) Realtime PCR amplification of 10-fold serial dilutions of phage particles with distilled water was performed in three replications. (B) The standard curve of the real-time PCR amplification of phage particles.
The Cq value of real-time PD-IPCR and the percent inhibition of phage ELISA against serial concentrations (log10) of aflatoxin B1 were analysed using OriginPro 8.6 by fourparameter logistic regression. The sigmoid curves of the two methods are shown in Figure 3, which illustrates that the limit of detection (LOD) of real-time PD-IPCR (0.02 ng/mL) is four times lower than phage ELISA (0.08 ng/mL). Moreover, the major potential advantage of real-time PD-IPCR is the feasibility to develop a multiplex detection platform where individual TaqMan probes can be designed for the unique DNA sequence of a specific phage, providing a possibility to detect multiple analytes simultaneously.
(1)
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To assess the influence of the methanol concentration, a series of aflatoxin B1 concentrations was diluted in 5%, 10%, 20%, and 40% methanol/PBS (v/v). As shown in Figure 5, the error bar illustrate that the data points of the standard curve under 40% methanol was unstable, which means the reproducibility was poor; and the LOD of the standard cruve under 10% (LOD=0.02 ng/mL) was 5 times lower than that under 20% methanol (LOD=0.1 ng/mL). Therefore, 40% and 20% methanol were not suitable for the following test. Though the standard curve under 5% and 10% methanol/PBS exhibited similar linear range and LOD, we selected 10% methanol in order to obtain a higher sensitivity. Therefore, the sample extract was diluted to a final concentration of 10% methanol. Figure 3. LOD of phage-ELISA and real-time PD-IPCR for aflatoxin. The Cq value of real-time PD-IPCR and the percent inhibition of phage ELISA were plotted against serial concentrations (log10) of aflatoxin B1 by four-parameter logistic regression. Each data point is the average of three independent measurements.
Figure 5. Performance of the real-time PD-IPCR under 5%, 10%, 20%, and 40% methanol/PBS. Each data point is the average of three independent measurements. Figure 4. Cross reactivity of real-time PD-IPCR against aflatoxin B1, B2, G1, and G2.
Cross Reactivity. As aforementioned, aflatoxin B1 is generally the most predominant aflatoxin among the major aflatoxins. However, four aflatoxins (B1, B2, G1, and G2) always occurred simultaneously in agri-products. Therefore, the crossreactivity against aflatoxin B1, B2, G1, and G2 was tested and the results were shown in Figure 4, which demonstrated that the lowest cross-reactivity toward aflatoxin G2 (13.5%) was obtained. However, this deficiency does not constrain the use of real-time PD-IPCR for detection of total aflatoxins since aflatoxin G2 is the least toxic among the four aflatoxins and is not widely detected in agri-products.33 Solvent and Matrix Effects. Aflatoxins are often extracted from samples by high concentrations of methanol because of their low solubility in water.7 As a consequence, the activity of the antibody can be easily interfered by sample extracts containing high concentrations of methanol and the sample matrix. Dilution is commonly used to reduce these interferences; however, it needs to be noted that dilution by definition reduces sensitivity.34,35
Figure 6. Calibration curves of real-time PD-IPCR produced in 10% methanol/PBS, corn, rice, peanut, and feedstuff matrix. Each data point is the average of three independent measurements.
The sample extract (corn, rice, peanut, and feedstuff) containing 80% methanol was diluted 8 times to reach the methanol concentration of 10%. The aflatoxin B1 calibration curves produced in both 10% methanol/PBS and 8-fold dilution of the
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sample extract were compared to evaluate the matrix effects. As expected, matrix effects were observed in the test samples (date not shown). In order to eliminate the matrix effects, BSA or Tween was added to the diluted extract until the matrix effects of corn, rice, and feedstuff were eliminated by adding 3% BSA while those of peanut were eliminated by adding 0.05% Tween 20. By these measures, no significant differences were observed between the calibration curves produced in 10% methanol/PBS and the four-sample matrix (Figure 6). In subsequent sample analysis, 8-fold dilution of the sample extract by PBS containing 3% BSA (corn, rice, and feedstuff matrix) or 0.05% Tween (peanut matrix) was used.
clusion, real-time PD-IPCR developed in this study can be applied for determination of aflatoxins according to the maximum tolerable limits in most countries (20 µg/kg). Table 1. Recovery analysis of aflatoxin by real-time PDIPCR Spiked level
Aflatoxin recovered
Within assay
(μg/kg) 2
(n=3)a
Mean ± SD
Average recovery (%)
CV (%)
2.03±0.35
101.64
17.38
20
18.19±2.59
90.95
14.24
100c
93.86±6.51
93.86
6.94
Between assay
2
1.86±0.35
92.78
18.91
(n=5)b
20
18.42±2.57
92.12
13.95
100
92.96±4.32
92.96
4.64
Within assay
2
1.85±0.13
92.35
7.02
(n=3) a
20
17.64±0.91
88.19
5.14
100
120.58±7.68
120.58
6.37
Between assay
2
1.79±0.14
89.56
7.74
(n=5) b
20
17.26±0.73
86.32
4.25
100
122.16±6.74
122.16
5.52
Within assay
2
1.82±0.13
90.93
7.15
(n=3) a
20
15.41±1.53
77.05
9.92
100
85.84±6.89
85.84
8.03
Between assay
2
1.94±0.29
96.98
15.17
(n=5) b
20
17.04±1.62
85.18
9.53
100
97.09±19.98
97.09
20.58
2
2.36±0.24
117.90
10.26
20
18.86±1.66
94.30
8.83
100
95.75±15.07
95.75
15.74
2
2.23±0.28
111.28
12.44
20
18.61±1.90
93.05
10.19
100
110.00±19.00
110.00
17.27
Sample type Corn
Rice
Figure 7. Correlation of results obtained by both real-time PDIPCR and reference HPLC method.
Method Validation with Agricultural Samples. The recovery of the established real-time PD-IPCR method was assessed by spiking blank samples (determined by HPLC as aflatoxin-free) with aflatoxin B1. The samples of corn, rice, peanut, and feedstuff obtained from the local market were spiked with known concentrations (2, 20, and 100 µg/kg) of aflatoxin B1. The assays were carried out in three replicates on the same day for within-assay precision evaluation and in five different days for between-assay precision evaluation. The recoveries of the assays were 77.05-122.16% with relative standard deviations (RSD) of 4.25-20.58% (Table 1). The above results demonstrated reliable reproducibility of realtime PD-IPCR in analysing samples. A total of 30 naturally contaminated samples gathered from local farms were analysed by both real-time PD-IPCR and HPLC methods. The standard curve for real-time PD-IPCR was established using mixed aflatoxins, which was used as the standard to detect total aflatoxins (aflatoxin B1, B2, G1, and G2). The mixed aflatoxin standard was prepared with aflatoxin B1, B2, G1, and G2 at a ratio of 1.0:0.1:0.3:0.03 according to the proportion of four naturally occurring aflatoxins.36 Concordant results (Table 2) were obtained with both methods, and the linear regression analysis (Figure 7) yielded good correlation (R2 = 0.991) between the two methods. As compared with our previous research,27 real-time PDIPCR reveals higher sensitivity (IC50 = 5.6 µg/kg) and a wider linear range (1.97-30.75 µg/kg) than nanobody-based ELISA (IC50 = 13.8 µg/kg, linear range from 10 to 20 µg/kg). In con-
Peanut
Feedstuff Within assay (n=3) a
Between assay (n=5)
b
a
The assays are carried out in 3 replicates on the same day.
b
The assays are carried out on 5 different days
c Samples spiked with 100µg/kg aflatoxin were extracted and diluted 20 times in order to make the final concentration included in the linear range
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Table 2. Comparison of results obtained using real-time PD-IPCR and reference HPLC method in corn and peanuts Number
Sample
HPLC (µg/kg)
real-time PD-IPCR (µg/kg)
1
Corn
1.77
NDa
27.20
19.36
2
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in agreement with the reference HPLC method. This study demonstrated that phage-displayed anti-idiotypic nanobody could be an excellent reagent to develop a novel real-time PDIPCR assay with high sensitivity for detection of small molecules. To the best of our knowledge, this is the first time that the anti-idiotypic nanobody based real-time PD-IPCR assay has been applied in detection of aflatoxins in agricultural samples. Besides, taking advantages of the unique characteristics of the recombinant phage, there is great potential to develop a multiplexed detection platform for simultaneously detecting multiple analytes in agri-products.
3
6.66
5.89
4
62.38
48.34
5
1.52
ND
AUTHOR INFORMATION
6
5.52
4.97
Corresponding Author
7
6.19
3.88
8
7.27
5.23
9
82.76
65.81
*Tel.: +86 27 86812943; Fax: +86 27 86812862; E-mail address:
[email protected] (P. Li);
[email protected] (Q. Zhang).
10
1.63
ND
11
48.13
37.93
12
18.13
17.25
13
77.07
65.58
14
57.75
35.39
15
16.70
15.45
79.91
62.37
17
30.34
24.91
18
5.84
4.11
19
20.16
14.85
20
226.22
197.56
21
14.71
12.36
22
308.08
246.70
23
165.27
154.58
16
Peanut
24
9.93
7.59
25
184.22
169.23
26
19.93
13.00
27
33.90
29.87
28
11.25
6.31
29
264.34
237.36
30
94.26
92.90
a
ND: not detectable.
CONCLUSION In this report, a novel anti-idiotypic nanobody-phage based real-time PD-IPCR method for aflatoxin detection was developed. This newly developed assay can integrate the versatility of ELISA with the sensitivity and signal amplification capability of PCR. The limit of detection of real-time PD-IPCR was 0.02 ng/mL, which was four times lower than phage ELISA. The spike-and-recovery test result indicated that real-time PDIPCR was a suitable method for detection of aflatoxins in corn, rice, peanuts, and feedstuffs. Real-time PD-IPCR analysis also revealed higher sensitivity and a wider linear range when compared with our previous research. The test data were
ACKNOWLEDGMENT The research is supported by the Project of National Science & Technology Pillar Plan (2012BAB19B09), the Special Fund for Agro-scientific Research in the Public Interest (201203094), and the National Natural Science Foundation of China (31171702).
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