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Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20

Production and Characterization of Monoclonal Antibodies against Aflatoxin B1 a

b

c

Mohammad Soukhtanloo , Elham Talebian , Mehdi Golchin , Mojgan d

Mohammadi & Bagher Amirheidari

b

a

Department of Clinical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran b

Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran c

Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran d

Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran Accepted author version posted online: 18 Dec 2013.Published online: 20 May 2014.

To cite this article: Mohammad Soukhtanloo, Elham Talebian, Mehdi Golchin, Mojgan Mohammadi & Bagher Amirheidari (2014) Production and Characterization of Monoclonal Antibodies against Aflatoxin B1, Journal of Immunoassay and Immunochemistry, 35:4, 335-343, DOI: 10.1080/15321819.2013.863207 To link to this article: http://dx.doi.org/10.1080/15321819.2013.863207

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Journal of Immunoassay and Immunochemistry, 35:335–343, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2013.863207

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PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST AFLATOXIN B1

Mohammad Soukhtanloo,1 Elham Talebian,2 Mehdi Golchin,3 Mojgan Mohammadi,4 and Bagher Amirheidari2 1 Department of Clinical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2 Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran 3 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran 4 Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran



In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in food and feed products. After immunization and fusion with myloma cells, two stable clones (A218 and B319 ) were selected. Isotyping showed that these monoclonal antibodies (mAbs) were IgG2b with kappa light chains. The affinity of A218 and B319 clons were 5×1011 M−1 and 6×109 M−1 , respectively. Competitive indirect ELISA results indicated these mAbs had complete (100%) cross-reaction with four major types of aflatoxins: B1, B2, G1, and G2. These mAbs could be used for immunoassay measurement of aflatoxins with high affinity and low detection limits. Keywords aflatoxin B1, monoclonal antibody, ELISA

INTRODUCTION Aflatoxins (AFs) comprise a group of approximately 20 structurally related fungal secondary metabolites. They are primarily produced by the food-borne fungi, Aspergillus flavus and Aspergillus parasiticus, which colonize on a variety of food commodities, including maize, oilseeds, spices, ground nuts, and tree nuts in tropical and subtropical regions. Aflatoxin contamination in food and feed is a serious global health problem, particularly in populations of developing countries in tropical and subtropical areas. In these areas, people are nearly ubiquitously exposed to moderate Address correspondence to Bagher Amirheidari, Faculty of Pharmacy, Kerman University of Medical Sciences, P. O. Box 76175-493, Haft-Bagh Blvd., Kerman 76169-13555, Iran. E-mail: [email protected]

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to high levels of aflatoxin. Employing less sensitive/specific analytical methods for aflatoxin detection is another factor for the poor control of aflatoxin contamination in such areas.[1,2] The four major aflatoxins are known as B1, B2, G1, and G2. Among these, aflatoxin B1 (AFB1) is the most naturally occurring chemical liver carcinogen. It has been included into the first group of human carcinogens by the International Agency for Research on Cancer (IARC). Aflatoxin exposure in food is a significant risk factor for Hepatocellular carcinoma (HCC),[3,4] which is the third leading cause of cancer deaths in worldwide, especially in developing countries.[5] In order to assure the safety and security of foods, the food and feed industry is in constant pursuit of rapid methods that are highly sensitive and help prevent accidental or intentional toxin outbreaks that may occur.[6,7] For screening of these toxins in the food industry and agricultural products, such as pistachio, sensitive and specific analytical tools are a necessity. AFB1-contamination in raw and processed foods can be monitored through several screening and analytical methods that are based on chromatography or antibody platforms. In many developing countries, general methods such as thin-layer chromatography (TLC) and highperformance liquid chromatography (HPLC) are used for detection. These methods are laborious and require expensive instrumentation and extensive clean up of the samples.[8,9] As the chromatographic technologies are not generally applicable for real-time assay in actual fields, they are incompatible with demands of most food industries. For easy and rapid screening of AFB1, antibody-based techniques including enzyme-linked immunosorbent assay (ELISA) and immuno-chromatographic techniques are the most commonly used techniques.[10,11] Antibody-based analyses such as ELISA and immuno-chromatography require highly sensitive and specific antibodies against AFB1 for their accurate detection. Production of mAb against various mycotoxins has been reported in the literature and commercial foreign products are currently available.[12,13] Nevertheless, high costs, variation in sources and qualities, and availability issues are the major drawbacks for imported mAb-based kits. Thus, there is strong motivation to domesticate the production of such key quality control tool for successful marketing, both domestically and internationally, of agricultural products. MATERIALS AND METHODS Materials Aflatoxins B1, B2 , G1 , and G2 , as well as AFB1-BSA conjugate, penicillin G/streptomycin, Bovine Serum Albumin (BSA), Polyethylene

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glycol (PEG 1500), Hypoxanthine-Aminopterin-Thymidine (HAT) medium, Hypoxanthine-Thymidine (HT) medium, and TiterMax® classic adjuvant were purchased from Sigma-Aldrich Co. (Dorset, UK). Hybridoma fusion and cloning supplement (HFCS) and Isostrip® mouse monoclonal antibody isotyping kits were bought from Roche Applied Science (Indianapolis, IN, USA). Sheep anti-mouse IgG conjugated with horseradish peroxidase (IgG-HRP conjugate) was purchased from GE Healthcare Europe GmbH (Freiburg, Germany). Fetal calf serum (FCS) and L-glutamine and RPMI 1640 medium were purchased from Life Technologies Inc. Gibco/Brl Division (New York, NY, USA). SP2/0-Ag14 myeloma cell line (ATCC number CRL-1581) was obtained from National Cell Bank of Iran (Tehran, Iran). Immunization BSA-Aflatoxin B1 conjugate was prepared by diluting 100 µg of the conjugate in 100 µL sterilized PBS, emulsified with 100 µL of TiterMax classic adjuvant using an emulsifying syringe. This was used to immunize two young male BALB/c mice via two subcutaneous (S.C) injections of 50 µL at 2 sites each time. Same procedure was repeated five more times at two-week intervals with half concentration of conjugate. The serum samples were collected from tail veins periodically. The titers of anti sera were determined using indirect ELISA. The progress of antibody titer was monitored and immunization continued until a strong immune response to AFB1 was perceived. Preparation and Characterization of Monoclonal Antibodies Four days before splenectomy, the mouse with the highest immune response was given a final intravenous adjuvant-free AFB1-BSA boost for lymphocyte activation. Feeder layer cells were prepared two days before fusion. Splenocytes were collected from immunized animal and fused with non-secreting murine SP2/0-Ag14 myeloma cells as described previously.[14] Briefly, 108 splenocytes and 109 SP2/0 cells were mixed (ratio of 1:10) in presence of PEG (MW 1500) in serum free RPMI 1640 medium. Cells were suspended in 2% HAT containing RPMI medium supplemented with 20% fetal calf serum (FCS), penicillin G (100 IU ml−1 ), and streptomycin (100 mg mL−1 ). Subsequently, the cells were seeded in 96 well plates and incubated at 37◦ C in 5% CO2 . Ten days after incubation in HAT selection media, plates were examined microscopically and screened by indirect ELISA for anti-AFB1 production. The contents of positive ones in ELISA were further cloned by limiting dilution to achieve stable anti-AFB1 monoclones. After two further limiting dilutions, two stable clones were selected and cultured for subsequent studies. Protein-G column chromatography used for purification of these mAbs from supernatant of

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clons. Briefly, columns were packed and equilibrated with phosphate buffer pH 7.4. The supernatant from each producing clone was loaded and the unbound proteins were removed by several washings. Finally, IgG was eluted by adding 0.2 M Glycine-HCl buffer pH 3. The collected fractions were neutralized with 1 M Tris, pH 10 and dialyzed against phosphate buffer saline. The affinity of purified mAbs determined using Beatty method.[15] Isotyping of the selected mAbs was carried out by mouse monoclonal antibody Isotyping kit (Isostrip) according to the manufacturer instructions. Based on the checkerboard procedure, the Ag concentration of 0.5 µg mL−1 was found to be the best concentration for coating. Purified mAb was employed to set up calibration curve of different concentrations of aflatoxin using indirect ELISA. For determining cross reactivity of mAbs, lyophilized powders of aflatoxin B1 , B2 , G1 , and G2 were dissolved in methanol and diluted in PBS with the concentrations ranging from 10 pg mL−1 to 1000 ng mL−1 and used as a competitor with AFB1 in interaction with mAb added subsequently to each well. The percentage of cross reactivity was determined based on the reduced optical density (OD) in the indirect ELISA. Indirect ELISA The AFB1-BSA conjugate as antigen was diluted in PBS to the concentration of 1 µg/mL. The wells were coated with 100 µL of Ag and incubated at 4◦ C overnight. After washing with PBS, the coated 96 well plate was blocked with 200 µL of blocking buffer (4% w/v skim milk in PBS) in each well, incubated at 37◦ C for 2 hr and washed with PBST (PBS containing 0.05% Tween 20). The different dilutions of serum samples of immunized mice or supernatant of hybridoma cells (100 µL) were added to the blocked wells. After incubation at 37◦ C for 90 min, the plate was washed 3 times with PBST and was added 100 µL sheep anti-mouse-HRP conjugate IgG secondary antibody (diluted 1:3000 containing 0.1% BSA in PBS) onto the well prior to incubation. The plates were again washed three times with PBST and 100 µL of substrate (Tetramethylbenzidine and H2 O2 in citrate buffer at pH 5.0) was added and the reaction was left to proceed for 10 mines at ambient conditions. After stopping the reaction by adding 50 µl of 2M H2 SO4 , the absorbance was measured at 450 nm using a Stat Fax® plate reader (Awareness Technologies, Westport, CT, USA). RESULTS Our results showed that during immunization procedure, the antiAFB1 titer in the sera of experimental animals increased gradually reaching to the maximum on 60th day. Screening by ELISA showed high efficiency of fusion (89%) of the splenocytes and SP2/0 cells. In the next step,

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TABLE 1 Clones selected by in-direct ELISA after fusion for limiting dilution. Only the highest OD from every plate was shown. The positive and negative controls were immunized serum of mice and the supernatant of SP2/0 myloma cells

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OD at 450 nm Clones

AFB1 -BSA

A2 a A4 B3 a C2 D4 E2 F2 G1 Cont+ Cont−

2.715 0.889 2.653 1.232 1.180 1.058 1.363 1.300 2.763 0.435

a Clones

selected for first limiting dilution

20 hybridoma cells were selected out of 600 based on ELISA optical density (OD450 ) results above 1 compared to the supernatant of SP2/0 myloma cells as the negative control (0.45). The ODs ranges from 0.45–2.7 in indirect ELISA. Table 1 contains the details of the results from the wells with highest OD from each plate. Clones B3 and A2 showed higher OD and stability and subjected to two further limiting dilution steps the others ones were unstable. After the first limiting dilution, majority of the clones showed no reactivity, apart from the clones A21 and B31 which were chosen for second limiting dilutions. In this stage, we obtained several positive clones the absorbance of some of the clones are shown in Table 2. Finally, the clones A218 and B319 were selected for further work because they produced the highest titer of Ab. the affinity of A218 and B319 mAbs were 5×1011 M−1 and 6×109 M−1 , respectively. The standard curve of mAbs were shown in Figure 1; showing the detection limit of about 10 ng/ml of AFB1. Cross reactivity of A218 mAb was determined to be 95–100 % against AFB2 , AFG1 , and AFG2 (Table 3). Isotyping tests indicated that both A218 and B319 mAbs were classified as IgG2b and had κ light chain. As pointed out in Table 2, our A218 hybridoma mAb recognized AFB2 , AFG1 , and AFG2 with 95, 100 and 100% of cross reaction, respectively. DISCUSSION In this study, a commercially available AFB1 and BSA conjugate was used as hapten-carrier complex. In several studies, researchers have also utilized this conjugate for immunization step.[16−18] Guan et al. immunized the mice with ovalbumin conjugate of aflatoxin.[19] Wang et al. developed mAb

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TABLE 2 Optical density of the selected clones after the first and second limiting dilution in ELISA. SP2/0 supernatant and 1/2000 diluted immunized mouse sera were used as negative and positive control, respectively

Clones selected after first limiting dilution

Clones selected aftersecond limiting dilution

Clones

OD at 450 nm

Clones

OD at 450 nm

Control (Neg)

0.467

Control (Neg)

0.447

A21 a A22 A27 B31 a B33 B48

2.943 2.706 2.510 3.053 2.173 2.245

A211 A218 b A221 B319 b B340

1.050 3.088 3.085 2.838 1.995

a Clones b Clones

selected for second limiting dilution. finally selected for further experiments and characterization.

FIGURE 1 A218 and B319 mAbs standard curves for AFB1 detection using competitive ELISA. A218 clone had higher sensitivity then B319 . This antibody detects as low AFB1 as (2 ng/mL) in-direct ELISA.

against a synthetic AFB1-lysin adduct conjugated with BSA to determine human aflatoxin exposure.[21] Screening by ELISA showed fusion rate of 89% between the splenocytes and SP2/0 cells; which was comparable to the reports of 75.8% and 91.41% 0 achieved in Pei et al.[2 ] and Zhang et al,[4] experiments, respectively. The reading OD450nm for 13 of 600 HAT- grown hybridoma cells was at least 2–3 fold higher than the negative control (OD450 = 0.445). The highest OD from every plate was shown in Table 1. Nonetheless, after first limiting dilution, only two hybridomas remained Ab-positive (A21 and B31 ) and stable which were selected for second limiting dilution. Although the other clones grew well but the ELISA results of their supernatants showed gradual

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TABLE 3 Cross reaction analysis of A218 mAb was done with major types of aflatoxins. Because of the similar furofuran moiety in aflatoxins the cross reaction of this mAb was great A218 mAb

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Major Type of aflatoxin

IC50 (ng/ml)

CR (%)

B1

1.53

100

B2

1.61

95

G1

1.53

100

G2

1.53

100

decrease in antibody production so that final ODs were not significantly different from negative controls, this could be due to chromosomal instability. A218 mAb showed a higher affinity to AFB1 compared to the B319 mAb using non-competitive ELISA (5 × 1011 M−1 vs 6 × 109 M−1 ). The affinity of A218 was (5 × 1011 M−1 ) much higher than affinities reported in literature for anti aflatoxin mAbs, that could be used in immunoassay methods. The affinity of our clone (A218 ) is much higher than affinities reported in literature for anti aflatoxin mAbs. In Guan et al study affinity of antibody was 1.74 ×109 M−1 , that is 287 fold smaller than ours results.[19] Although there are cases where a handful of clones have finally been reported such as the work of Devi et al who generated 10 clones, in frequent other reports just one or two clones have finally been selected for full characterization.[16] Won-Ki et al.[17] and Wang et al.[21] finally finished their research with only one monoclone recognizing AFB1 specifically. Kolosava et al. finally proceeded with two mAbs as their final products submitted for characterization analysis.[18] Won-Ki et al. determined their constructed mAb as IgG1 (λ).[17] In Pei et al., mAb was characterized as IgG2a with λ light chain.[20] The isotype of mAb constructed by Guan et al. was found to be IgG2a .[19] The cross reaction analysis exhibited that A218 mAb shows low specificity for AFB1. Most aflatoxins share similar functional groups. A typical chemical structure of aflatoxins is known to be a heterocyclic ring structure (Table 3).

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The anti-AFB1 monoclonal antibody generated by Delmulle et al showed cross reactivity for AFB2 (76%), AFG1 (55%) and AFG2 (6%).[22] Studies by Chu and Ueno showed an antibody against AFB1 which had approximately 10% of cross reactions for AFB2 and AFG1 .[23] On the other hand, Hefle et al.[24] and Devi et al.[16] achieved monoclones which recognized all four aflatoxins with 100% cross reactivity the same as our antibodies. Higher sensitivity toward AFG1 than AFB2 seemed to be because AFB1 and AFG1 have a similar furofuran moiety, which differs from AFB2 contributing to slightly different three-dimensional structures. Therefore, the immunodominant determinant in epitope might be ascribed to the furofuran moiety of AFB1. We also coated the wells using BSA, rather than AFB1-BSA conjugate, in Indirect ELISA and there was no reaction (OD = 0.23) which indicates that the antibodies just recognized aflatoxin epitopes. A218 mAb can be employed for total aflatoxin detection rather than specific AFB1 recognition because of its relative specificity towards major aflatoxin derivatives. This of course would have its own benefits especially wherever the aim of antibody usage is total aflatoxin removal from a liquid foodstuff but not discriminative analysis of aflatoxin types. Another application could be in areas where regulations concern the total aflatoxin levels rather than specific type limits. This research was beneficial in domesticating the production of antiaflatoxin monoclonal antibodies, could be used for development of relevant immunoassay kits. As we shown in Figure 1, the A218 mAb was able to detect as low AFB1 as (2 ng/mL) using in-direct ELISA, which could be improved for designing of immunoassays for aflatoxin in food and feed. And these mAbs used in other devices immunoassay (such as microtiter plate, reader-based immunoassay), lateral flow strip, immunosensor, and portable rapid tester.[25] FUNDING This work was taken from a pharmacy student thesis at Kerman University of Medical Sciences (Thesis No 643) and was financially supported by a grant (No. 91-228) from the Vice President for Research, Kerman University of Medical Sciences, Kerman, Iran. REFERENCES 1. Kiyota, T.; Hamada, R.; Sakamoto, K.; Iwashita, K.; Yamada, O.; Mikami, S. Aflatoxin Non-productivity of Aspergillus Oryzae caused by Loss of Function in the aflJ Gene Product. J. Biosci. Bioeng . 2011, 111(5), 512–517. 2. Basappa, S. Aflatoxins: Formation, Analysis and Control. Alpha Science International Ltd.: Oxford, 2009; pp. 37–66. 3. Wild, C.P.; Gong, Y.Y. Mycotoxins and Human Disease: A Largely Ignored Global Health Issue. Carcinogenesis 2010, 31(1), 71–82.

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4. Zhang, D.; Li, P.; Zhang, Q.; Zhang, W.; Huang, Y.; Ding, X.; Jiang, J. Production of Ultrasensitive Generic Monoclonal Antibodies against Major Aflatoxins using a Modified Two-Step Screening Procedure. Anal Chim Acta. 2009, 636(1), 63–69. 5. Ferlay, J.; Shin, H.R.; Bray, F.; Forman, D.; Mathers, C.; Parkin, D.M. Estimates of Worldwide Burden of Cancer in 2008: GLOBOCAN 2008. Int. J. cancer 2010, 127 (12): 2893–2917. 6. Jaimez, J.; Fente, C.A.; Vazquez, B.I.; Franco, C.M.; Cepeda, A.; Mahuzier, G.; Prognon, P. Application of the Assay of Aflatoxins by Liquid Chromatography with Fluorescence Detection in Food Analysis. J. Chromatogr. A 2000, 882 (1–2), 1–10. 7. Blesa, J.; Soriano, J.M.; Molto, J.C.; Marin, R.; Manes, J. Determination of Aflatoxins in Peanuts by Matrix Solid-Phase Dispersion and Liquid Chromatography. J. Chromatogr. A. 2003, 1011(1–2), 49–54. 8. Richard, J. Some Major Mycotoxins and Their Mycotoxicoses—An Overview. Int. J. Food Microbiol. 2007, 119(1–2), 3–10. 9. Barug, D.; Bhatnagar, D.; Egmond, H. The Mycotoxin Factbook: Food & Feed Topics. Wageningen Academic Publishers: Wageningen, The Netherlands, 2006; pp. 100–150. 10. Lee, N.J.; Rachmawati, S.A. rapid ELISA for Screening Aflatoxin B1 in Animal Feed and Feed Ingredients in Indonesia. Food Agric Immunol. 2006, 17 (2), 1–14. 11. Stroka, J.; Anklam, E.; Jorissen, U.; Gilbert, J. Immunoaffinity Column Cleanup with Liquid Chromatography using Post-Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study. J. AOAC Int. 2000, 83(2), 320–340. 12. Wang, J.; Liu, B.-H.; Hsu, Y.-T.; Yu, F.-Y. Sensitive Competitive Direct Enzyme-Linked Immunosorbent Assay and Gold Nanoparticle Immunochromatographic Strip for Detecting Aflatoxin M1 in Milk. Food Control. 2011, 22(6), 964–969. 13. Garden, S.R.; Strachan, N.J.C. Novel Colorimetric Immunoassay for the Detection of Aflatoxin B1. Analytica Chimica Acta 2001, 444(2), 187–191. 14. Soukhtanloo, M.; Ansari, M.; Paknejad, M.; Parizadeh, M.R.; Rasaee, M.J. Preparation and Characterization of Monoclonal Antibody against Melatonin. Hybridoma (Larchmt) 2008, 27 (3), 205–209. 15. Beatty, J.D.; Beatty, B.G.; Vlahos, W.G. Measurement of Monoclonal Antibody Affinity by NonCompetitive Enzyme Immunoassay. J. Immunol. Meth. 1987, 100(1–2), 173–179. 16. Devi, K.T.; Mayo, M.A.; Reddy, K.L.; Delfosse, P.; Reddy, G.; Reddy, S.V.; Reddy, D.V. Production and Characterization of Monoclonal Antibodies for Aflatoxin B1. Lett. Appl. Microbiol. 1999, 29(5), 284–288. 17. Won-Ki, M.; Dae-Hyuk, K.; Kyungmoon, P.; Yong-Cheol, P.; Jin-Ho, S. Characterisation of Monoclonal Antibody against Aflatoxin B1 Produced in Hybridoma 2C12 and its Single-Chain Variable Fragment Expressed in Recombinant Escherichia coli. Food Chem. 2011, 126(3), 1316–1323. 18. Kolosova, A.Y.; Shim, W.B.; Yang, Z.Y.; Eremin, S.A.; Chung, D.H. Direct Competitive ELISA based on a Monoclonal Antibody for Detection of Aflatoxin B1. Stabilization of ELISA kit Components and Application to Grain Samples. Anal. Bioanal. Chem. 2006, 384(1), 286–294. 19. Guan, D.; Li, P.; Zhang, Q.; Zhang, W.; Zhang, D.; Jiang, J. An Ultra-Sensitive Monoclonal AntibodyBased Competitive Enzyme Immunoassay for Aflatoxin M1 in Milk and Infant Milk products. Food Chem. 2011, 125(4), 1359–1364. 20. Pei, S.C.; Zhang, Y.Y.; Eremin, S.A.; Lee, W.J. Detection of Aflatoxin M1 in Milk Products from China by ELISA using Monoclonal Antibodies. Food Control. 2009, 20(12), 1080–1085. 21. Wang, J.-S.; Abubaker, S.; He, X.; Sun, G.; Strickland, P.T.; Groopman, J.D. Development of Aflatoxin B1 -lysin Adduct Monoclonal Antibody for Human Exposure Studies. Appl. Environ. Microbiol. 2001, 67 (6), 2712–2717. 22. Delmulle, B.S.; De Saeger, S.M.; Sibanda, L.; Barna-Vetro, I.; Van Peteghem, C.H. Development of an Immunoassay-Based Lateral Flow Dipstick for the Rapid Detection of Aflatoxin B1 in pig feed. J. Agric. Food Chem. 2005, 53(9), 3364–3368. 23. Chu, F.S.; Uneo, I. Production of Antibody against Aflatoxin B1 . Appl. Environ Microbiol. 1997, 33(5), 1125–1128. 24. Hefle, S.L.; Chu, F.S. Production and Characterization of Monoclonal Antibodies Cross-Reactive with Major Aflatoxins. Food Agric. Immunol. 1990, 2(4), 181–188. 25. Li, P.; Zhang, Q.; Zhang, W. Immunoassays for Aflatoxins. Trends Analyt. Chem. 2009, 28(9), 1115–1126.

Production and characterization of monoclonal antibodies against aflatoxin B1.

In this article, we embarked on production of mouse monoclonal antibodies against aflatoxin B1 which is the most commonly occurring fungal toxin in fo...
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