Yuan
Gao‡
ab,
Meihua
Yang‡
a,
Cheng
Peng
b,
Xiaohong
Li
a,
Runlan
Cai
a and
Yun
Qi
*a
aInstitute of Medicinal Plant Development, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 100193, P.R. China. E-mail: yunqichai@sohu.com; Fax: (+86) 10 62829207; Tel: (+86) 10 62829207
bPharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, P.R. China
First published on 2nd November 2011
Although anti-zearalenone (ZEN) antibodies have been widely prepared, these antibodies cross-react with α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). To overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate. In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction. After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced. The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively. Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively. An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established. The recovery rates of ZEN in spiked cereal and feed were in the range of 80%–120% with coefficients of variation <15%. The intra-assay variation and inter-assay variation in assay buffer were both <5%. Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.
Immunoassays using specific antibodies, including those targeting ZEN, have been used to detect toxin residues for many years. The key step in the development of an immunoassay is the production of the specific antibody. But the cross-reactivity (CR) of anti-ZEN antibodies with α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) is a serious problem. ZEN and its 5 analogs are structurally identical apart from minor differences at the C7 and C12 positions in the macrocyclic lactone ring (Fig. 1). Therefore it is difficult for an anti-ZEN antibody to distinguish ZEN from its 5 analogs. Anti-ZEN antibodies showed high cross-reactivity (CR) with the 5 analogs (Table 1).
Fig. 1 The chemical structures of zearalenone (ZEN), 7α-hydroxy-zearalenol (α-ZOL), 7β-hydroxy-zearalenol (β-ZOL) and their 11–12 reduced analogs: zearalanone (ZAN), 7α-hydroxy-zearalanol (α-ZAL), and 7β-hydroxy-zearalanol (β-ZAL). *The active hydrogen. |
Substance (active derivative) | Coupling reagent (method) | Type of antibody | Time after immunization | Titer of antibody | Cross reactivity (%) | Reference | ||||
---|---|---|---|---|---|---|---|---|---|---|
α-ZOL | β-ZOL | ZAN | α-ZAL | β-ZAL | ||||||
a DCC: Dicyclohexylcarbodiimide. b M: Monoclonal antibody. c —: Not mentioned in literature. d ND: Not detected. e P: Polyclonal antibody. f EDC: 1-ethyl-3(3-dimethylaminopropyl) carbodiimide. | ||||||||||
ZEN-oxime | DCCa | Mb (IgG2a) | —c | — | 33.7 | 24.5 | NDd | 82.9 | 74 | 19 |
— | — | M (IgG2a, κ) | — | — | 91 | 21 | 138 | 69 | 6 | 20 |
— | — | M | — | — | 69 | <1 | 22 | 42 | <1 | 21 |
ZEN-oxime | DCC | Pe (rabbits) | 11 weeks | 20,480 | 50 | 12 | ND | 5.45 | 2.5 | 17 |
ZEN-oxime | EDCf | P (pigs) | 14 weeks | 3,500 | 100 | 44 | 100 | 53 | 44 | 22 |
— | — | — | — | — | 88–120 | 91–95 | 74–81 | 80–101 | 87–116 | 8 |
ZEN-oxime | EDC | P (rabbits) | — | 1,500 | ND | 18 | 180 | 100 | ND | 23 |
ZEN | Formaldehyde condensation | P (rabbits) | — | — | 0.15 | <0.02 | 31.7 | 0.12 | ND | 5 |
ZEN-oxime | DCC | P (rabbits) | — | — | 37.3 | 7.2 | 59.2 | 3.9 | 5.3 | 24 |
ZEN-oxime | DCC | M (IgG1, κ) | — | — | 121.5 | 65.3 | ND | 21.5 | 18.9 | 25 |
— | — | M | — | — | 96 | 21 | ND | 24 | 5 | 26 |
— | — | M (IgE) | — | — | 102 | 71 | 195 | 139 | 20 | 27 |
— | — | M | 5 weeks | 6,400 | 107 | 29 | ND | 35 | 25 | 28 |
ZEN-oxime | DCC | M | — | — | 26 | 11 | ND | 8 | 10 | 29 |
ZEN-oxime | DCC | P (rabbits) | 10 weeks | 1,500 | 280 | 35 | ND | 22 | 10 | 30 |
5-NH2-ZEN | glutaraldehyde | M (IgG1, λ) | — | 140,000 | 0.9 | <0.1 | 4.0 | <0.1 | <0.1 | 6 |
CR is determined by the antibody specificity, which depends on the antigenic determinants exposed to immune cells. This type of CR, where the antigenic determinants do not distinguish between similar binding sites on ZEN and its analogs, has been termed molecular or antigenic mimicry.4 The crucial step to developing a highly specific antibody is to prepare an appropriate immunogen. Because ZEN is a hapten, its antigenicity is only induced by coupling with a carrier protein. Therefore, the preparation of ZEN-carrier protein conjugates is one of the most important steps in antibody production. Different methods of ZEN-protein conjugate preparation will expose different antigenic determinants. In most previous studies, carboxymethyl oxime was introduced to ZEN first, and then the derivatives were coupled with protein by carbodiimide (Table 1). The Mannich reaction was used solely for conjugate preparation.5 Although the antibody specificity was improved somewhat, the problem of CR with ZEN analogs was not solved by using ZEN itself conjugated with carrier protein directly in the Mannich reaction. Teshima and colleagues6,7 introduced a more complex approach in which a hapten mimic (5-NH2-ZEN) was substituted for the purpose of eliciting an antibody to the compound to be assayed, ZEN. The monoclonal antibody obtained showed low CR values with 5 analogs (≤4%), but the CR value with 5-NH2-ZEN itself was not reported yet. In 2007, Erbs et al.,8 who did not manufacture any of the antibodies, tested 3 commercial immunoaffinity columns (IACs) targeting ZEN, and the CR values for the 5 analogs were ≥74% when they were applied either individually or in a mixture. Therefore, it is necessary to establish an effective and simple method to improve the specificity of anti-ZEN antibody.
A cationic protein that served as an efficient carrier was first reported in 1987.9 There are many reports in which conjugates prepared using a cationic protein as a carrier had good immunological properties.10–12 As an antigen, the characteristics of cationic proteins were different from the native proteins.9,13 Cationic proteins had an increased affinity for antigen-presenting cell membranes due to electrostatic interactions with anionic membrane phospholipids.14 Moreover, cationic carrier proteins could minimize cross-linking and generate stronger immune responses compared to their native forms.15 As an efficient carrier, cationic proteins could induce a rapid, specific immunological response against the antigenic determinants of the hapten coupled to the protein.16 In addition, ZEN contains active hydrogen, which is an essential component for conjugating ZEN to a cationic protein. In this study, we describe an efficient and convenient method to construct ZEN-cationic protein conjugates using a Mannich-type reaction. Based on the immunogen synthesized, we prepared polyclonal antisera and a monoclonal antibody. ZEN in cereal and feed samples was analyzed by the established indirect competitive ELISA, including corn, wheat, barley, oat, coix seed and rodent feed. The results confirmed that the monoclonal antibody obtained was highly specific. The goal of this study was to provide an efficient and simple approach to prepare a highly specific anti-ZEN antibody and significantly improve the specificity of immunoassays.
Fig. 2 A schematic diagram based on position 6-H for the preparation of ZEN-cationic protein conjugates.16 (1)–(2) cationization of protein; (3)–(5) imidization of N-hydroxymethyl amine groups in the cationic protein; (6)–(7) enolation of ZEN; (8)–(9) conjugation of enolated ZEN with the iminium ionized protein. All the reactions were carried out in MES buffer. This reaction can proceed in other directions: the active hydrogen is also available on the other side of the keton group (position 8), position 3′ and 5′ on the benzene ring and the phenolic OH groups. The ZEN conjugates with cationic protein reaction may be a mixture of conjugates with different conjugation sites. |
The preparation of cationic polylysine (cPLL) as another cationic carrier protein was performed according to the procedures mentioned above except that polylysine (PLL) was substituted for nBSA. In addition, PLL can be conjugated with ZEN directly in the next step. It would not affect the assay performance whether PLL was cationized.
The preparation of the coating antigen (ZEN-cPLL) was similar to the procedures mentioned above except for the substitution of cPLL for cBSA.
In order to gain massive numbers of mAb, mice were intraperitoneally injected with 500 μL of Freund's incomplete adjuvant. 7 days later, 1 × 105 hybridomas were inoculated into the abdominal cavity of each mouse. 1 week later, the ascites were collected and purified via the octanoic acid-saturated ammonium sulfate precipitation method. The ascites were dialyzed exhaustively against distilled water for 48 h, and finally, the salt-free ascites were lyophilized and stored at −20 °C. The isotype classification of the mAb was identified by the mouse monoclonal antibody isotyping test kit.
Recovery (%) = (amount of ZEN in spiked sample − amount of ZEN in unspiked sample)/added amount of ZEN × 100 |
In order to validate the specificity of the anti-ZEN mAb prepared in our laboratory, we purchased the ZEN competitive ELISA (cELISA) kit from Abraxis Co. which served as a control. The cereal and feed samples were analyzed by icELISA based on the anti-ZEN mAb and Abraxis zearalenone plate kit (AZPK) simultaneously. The procedure of the AZPK was carried out according to its directions. The accuracies of these two methods were ascertained by analysis of the recovery in ZEN standard solution. The assay buffer was fortified by ZEN standard solution at the final concentration of 12.5, 25 and 50 μg L−1.The recovery results were calculated as the ratio of measured level to spiked level (n = 4).
BSA is one of the most commonly used carrier proteins, and it usually yields satisfactory results. A two-step strategy was used to link ZEN to BSA. The first step corresponded to a modification of the BSA carboxylic groups to generate a cationic protein with a high isoelectric point. The carboxylic groups were modified to form aminoethylamide side chains. In the second step, the abundance of primary amines on cBSA made it very receptive in an amino-alkylation reaction, during which amine moieties of cBSA were linked to ZEN through active hydrogen.
The UV absorption spectra of nBSA and cBSA are shown in Fig. 3. The maximum absorbing wavelength in the cBSA UV spectra, which are characteristic of the protein, was slightly blue-shifted from 278 to 276 nm compared with nBSA. This might be caused by the increase in cBSA polarity that is due to the induction of excessive cationic groups and amino-ethylamine groups on nBSA.16 Native-polyacrylamide gel electrophoresis was run, and the protein was not degenerated before electrophoresis indicating that the positive charge of the cationic protein could be maintained in this condition. When the electrode was not reversed, cBSA did not migrate, and nBSA showed a series of bands (data not shown). These results clearly indicated that the cationization was successful.12
Fig. 3 UV spectra of: (a) 1.0 g L−1nBSA and (b) 4.0 g L−1cBSA. |
2 weeks after cell fusion, culture supernatants from each clone were subjected to screening. Positive hybridomas were subcloned 3 times by limiting dilution. The mAb produced from the hybridoma cells was IgM with a κ-type light chain. The titer of the mAb was 20,000. This result indicated that the antibody obtained had a higher affinity when using the ZEN-cBSA as the immunogen. It also suggested that a cationic protein had good antigenicity, as the carrier protein could not only induce a more rapid immune response but also a higher titer of antibody [22,28,30 in Table 1].
Fig. 4 icELISA competition curves. a. icELISA competition curve for the binding of ZEN to anti-ZEN mAb. b. icELISA competition curves for the binding of 5 ZEN analogs to anti-ZEN mAb. c. icELISA competition curve for the binding of ZEN to anti-ZEN polyclonal antisera. d. icELISA competition curves for the binding of 5 ZEN analogs to anti-ZEN polyclonal antisera. |
Compound | Polyclonal antisera | Monoclonal antibody | ||
---|---|---|---|---|
IC50 (μg L−1) | Cross-reactivitya (%) | IC50 (μg L−1) | Cross-reactivitya (%) | |
a The percentage of cross-reactivity is defined as the ratio of IC50 value for ZEN to that for competitors. | ||||
ZEN | 233.35 | 100 | 55.72 | 100 |
α-ZOL | 7430.19 | 3.14 | 8542.80 | 0.65 |
β-ZOL | 11912.42 | 1.96 | 5956.62 | 0.94 |
ZAN | 3435.58 | 6.79 | 3767.04 | 1.48 |
α-ZAL | 10375.28 | 2.25 | 8830.80 | 0.63 |
β-ZAL | 4130.48 | 5.65 | 6063.17 | 0.92 |
Sample | Spiked ZEN (μg L−1) | Indirect competitive ELISA | AZPK | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Detected ZEN (μg L−1) | Mean detected ZEN (μg L−1) | Recoveryb (%) | CV (%) | Detected ZEN (μg L−1) | Mean detected ZEN (μg L−1) | Recoveryb (%) | CV (%) | ||||||
a The unspiked sample which did not contain ZEN and its 5 analogues. b All unspiked samples contained some background level of ZEN and this had to be subtracted from the level measured for spiked samples in order to calculate the recovery. | |||||||||||||
Corn | 0a | 19.73 | 19.73 | 20.67 | 20.04 | - | 2.71 | 33.78 | 37.35 | 34.76 | 35.30 | — | 5.22 |
12.5 | 29.54 | 30.94 | 30.47 | 30.32 | 82.20 | 2.36 | 73.31 | 78.76 | 84.62 | 78.90 | 348.76 | 7.17 | |
25 | 41.56 | 54.96 | 48.54 | 48.36 | 113.26 | 13.86 | 116.03 | 97.67 | 106.46 | 106.72 | 285.68 | 8.60 | |
50 | 66.22 | 79.78 | 70.46 | 72.15 | 104.23 | 9.61 | 194.49 | 186.30 | 231.04 | 203.94 | 337.28 | 11.68 | |
Wheat | 0 | 6.65 | 6.45 | 6.35 | 6.48 | — | 2.38 | 2.28 | 2.52 | 2.74 | 2.51 | — | 9.29 |
12.5 | 19.12 | 18.25 | 17.97 | 18.45 | 95.67 | 3.25 | 48.35 | 49.76 | 48.35 | 48.82 | 370.50 | 1.66 | |
25 | 34.50 | 26.09 | 28.19 | 29.59 | 92.41 | 14.79 | 40.13 | 40.13 | 40.70 | 40.32 | 151.23 | 0.83 | |
50 | 65.20 | 58.48 | 56.70 | 60.13 | 107.27 | 7.46 | 188.99 | 203.05 | 188.99 | 193.68 | 382.33 | 4.19 | |
Barley | 0 | 6.25 | 6.16 | 5.97 | 6.13 | — | 2.36 | 4.66 | 4.66 | 4.73 | 4.69 | — | 0.83 |
12.5 | 16.63 | 16.89 | 20.03 | 17.85 | 93.76 | 10.62 | 387.28 | 381.76 | 387.28 | 385.44 | 3045.99 | 0.83 | |
25 | 30.47 | 29.08 | 29.54 | 29.69 | 94.26 | 2.38 | 345.28 | 335.51 | 340.36 | 340.38 | 1342.77 | 1.43 | |
50 | 54.96 | 64.19 | 57.58 | 58.91 | 105.57 | 8.08 | 244.69 | 255.45 | 251.81 | 250.65 | 491.92 | 2.18 | |
Oat | 0 | 13.80 | 14.24 | 12.97 | 13.67 | — | 4.71 | 16.72 | 18.23 | 17.46 | 17.47 | — | 4.30 |
12.5 | 25.68 | 26.09 | 27.33 | 26.37 | 101.57 | 3.25 | 79.90 | 77.64 | 79.90 | 79.14 | 493.40 | 1.65 | |
25 | 42.21 | 44.92 | 43.54 | 43.56 | 119.55 | 3.10 | 156.83 | 159.10 | 161.40 | 159.11 | 566.55 | 1.43 | |
50 | 57.58 | 62.23 | 61.27 | 60.36 | 93.39 | 4.07 | 251.81 | 278.42 | 255.45 | 261.89 | 488.84 | 5.51 | |
Coix seed | 0 | 365.36 | 327.73 | 312.80 | 335.30 | — | 8.07 | 340.36 | 335.51 | 335.51 | 337.13 | — | 0.83 |
12.5 | 354.19 | 343.36 | 348.70 | 348.76 | 107.65 | 1.55 | 466.70 | 447.03 | 466.71 | 460.14 | 984.10 | 2.47 | |
25 | 382.78 | 338.07 | 371.10 | 363.98 | 114.69 | 6.37 | 546.49 | 570.53 | 587.14 | 568.05 | 923.70 | 3.60 | |
50 | 420.15 | 371.08 | 388.80 | 393.33 | 116.06 | 6.32 | 707.54 | 738.67 | 697.47 | 714.56 | 754.86 | 3.00 | |
Rodent feed | 0 | 232.90 | 222.30 | 215.50 | 223.57 | — | 3.92 | 290.66 | 299.12 | 278.42 | 289.40 | — | 3.60 |
12.5 | 247.82 | 225.78 | 232.90 | 235.50 | 95.46 | 4.78 | 570.53 | 554.39 | 595.63 | 573.52 | 2272.92 | 3.62 | |
25 | 276.28 | 247.83 | 229.30 | 251.14 | 110.27 | 9.42 | 494.27 | 578.78 | 554.39 | 542.48 | 1012.31 | 8.02 | |
50 | 298.58 | 272.02 | 276.30 | 282.29 | 117.45 | 5.05 | 793.61 | 760.17 | 760.17 | 771.32 | 963.84 | 2.50 |
Level (μg L−1) | Indirect competitive ELISA | AZPK | ||||||
---|---|---|---|---|---|---|---|---|
n | Measured amounta (μg L−1) | CVb (%) | R c (%) | n | Measured amounta (μg L−1) | CVb (%) | R c (%) | |
a Mean ± SD. b CV: coefficient of variation= SD/mean *100. c R: recovery. | ||||||||
12.5 | 4 | 10.75 ± 0.010 | 2.17 | 85.96 | 4 | 14.19 ± 0.004 | 1.08 | 113.50 |
25 | 4 | 24.12 ± 0.005 | 1.20 | 96.49 | 4 | 23.23 ± 0.005 | 1.54 | 92.93 |
50 | 4 | 52.40 ± 0.004 | 1.06 | 104.80 | 4 | 52.61 ± 0.002 | 0.60 | 105.21 |
In the optimum conditions, the LOD of icELISA based on anti-ZEN mAb was 1.56 μg L−1. The repeatability test (Table 5) was performed by comparing the OD ratios of 6 replicates in the same plate (intra-assay repeatability) or triplicates at different days (inter-assay repeatability). The intra-assay CV of 6 tests ranged from 1.60% to 3.63%, with a median value of 2.42%. The inter-assay CV of 3 tests was between 4.20% and 4.90%, with a median value of 4.46%. These data showed that the assay was repeatable and yielded a low and acceptable variation.
Level (μg L−1) | Intra-assaya | Inter-assayb | ||||
---|---|---|---|---|---|---|
n | OD value c | CVd (%) | n | OD valuec | CVd (%) | |
a Intra-assay variation was calculated from 6 replicates on a single day. b Inter-assay variation was calculated from triplicates on 5 different days. c Mean ± SD. d CV: coefficient of variation. | ||||||
25 | 6 | 0.277 ± 0.006 | 2.13 | 3 | 0.283 ± 0.012 | 4.20 |
50 | 6 | 0.262 ± 0.004 | 1.50 | 3 | 0.267 ± 0.011 | 4.27 |
100 | 6 | 0.204 ± 0.007 | 3.63 | 3 | 0.211 ± 0.010 | 4.90 |
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1an15487g |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2012 |