Toxicon 80 (2014) 64–72
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Preparation and identification of monoclonal antibody against fumonisin B1 and development of detection by Ic-ELISA Sumei Ling a,1, Jie Pang b,1, Jinjin Yu b, Rongzhi Wang a, Licai Liu a, c, Yanling Ma a, Yuming Zhang b, Ni Jin a, Shihua Wang a, * a
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China School of Food Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China c Environmental Protection Bureau of Qitaihe City, Qitaihe 154600, Heilongjiang, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2013 Received in revised form 11 December 2013 Accepted 19 December 2013 Available online 27 December 2013
Fumonisin B1 (FB1) is one of the mycotoxins produced by Fusarium verticillioides, which was mainly found in corn and related products. FB1 was small molecule with no immunogenicity, so it should be conjugated to carrier proteins such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) to generate immunogenicity. In this study, conjugate FB1-BSA was used to immunize Balb/c mice, and one hybrid cell line 4G5 excreting monoclonal antibody against FB1 was obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mouse. Hybridoma 4G5 was injected into the abdomen of Balb/c mice, and the anti-FB1 mcAb was harvested from ascites and the titer reached 6.4 104 after purification with caprylic/ammonium sulfate precipitation (CA-AS) method. The cross-reactivity results showed that anti-FB1 mcAb was highly specific to fumonisin B1, and the affinity was 2.1 108 L/M. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect FB1 was 1–800 ng/mL with IC50 of 32 ng/mL. The detection limit was 1.0 ng/mL, and the recovery average was 93.75 6.90%. Therefore, the anti-FB1 mcAb excreted by 4G5 can be used to detect fumonisin B1 in corn and related samples. Ó 2014 Published by Elsevier Ltd.
Keywords: Fumonisin B1 Hybridoma Monoclonal antibody Detection ic-ELISA
1. Introduction Fumonisins are mycotoxins produced mainly by Fusarium verticillioides and Fusariumproliferatum (Brera et al., 1998), and fumonisin B1 (FB1) is the most abundant and reaches 70% of the total fumonisins in maize (Orsi et al., 2009). Ingestion of FB1 causes lots of toxicoses in animals, including leukoencephalomalacia, pulmonary edema, and hepatocarcinoma or other liver diseases (Brera et al., 1998). In addition, FB1 in foods has been associated with a high * Corresponding author. Tel./fax: þ86 0591 87984471. E-mail address: [email protected] (S. Wang). 1 These authors contributed equally to this work. 0041-0101/$ – see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.toxicon.2013.12.008
possibility of esophageal cancer (Proctor et al., 2008). Because of the known toxicological evidence, the International Agency for Research on Cancer (IARC) has taken FB1 as a possible carcinogen to humans (Group 2B) (Tardieu et al., 2008). As the above harmful effects, an effective method for FB1 detection is essential and important. To date, many different methods were used to identify FB1, such as HPLC, TLC, GC–MS and LC–MS/MS (Brera et al., 1998). These methods with high sensitivity and specificity can detect fumonisin B1 exactly in food. However, they depend on complex purification steps, expensive instruments and large quantity of raw materials. They are improper for realtime detection in actual fields and incompatible with
S. Ling et al. / Toxicon 80 (2014) 64–72
demands of food industries. In recent years, monoclonal antibody are used widely in the development of diagnostic and research reagents, and becomes one of the most popular methods for food pathogen detection (Brera et al., 1998; Li et al., 2012a,b; Pei et al., 2009). The purpose of the present study was to obtain a monoclonal antibody against FB1 with high specificity and affinity. However, FB1 is a small molecule with no immune response, thus it is necessary to conjugate FB1 to a carrier protein. In the study, FB1 was coupled to BSA (bovine serum albumin) and KLH (keyhole limpet hemocyanin) for immunization and screening respectively, and one positive hybridoma named 4G5 excreting IgG2b monoclonal antibody against FB1 was successfully obtained with affinity of 2.1 108 L/M, and then a competitive indirect ELISA was carried to detect FB1 based on this antibody. The experiments and results were followed. 2. Materials and methods 2.1. Materials Fumonisin (FB1), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), polyethylene glycol 1450 (PEG 1450), Freund’s complete adjuvant, Freund’s incomplete adjuvant, horseradish peroxidase (HRP) conjugated goat anti-mouse IgG, hypoxanthine, aminopterin and thymidine (HAT) medium, hypoxanthine-thymidine (HT) medium, mouse monoclonal antibody isotyping reagent (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA), and RPMI 1640 were purchased from Sigma Chemical Co. (St. Louis, MO, USA.). Female Balb/c mice (8 weeks) were obtained from Wushi Animal Laboratory (Shanghai, China). Myeloma cells SP2/ 0 were stored in our lab. All animal studies were carried out according to institutional guidelines in Fujian Agriculture and Forestry University (China). 2.2. Preparation of complete antigens FB1-BSA and FB1-KLH complete antigens were prepared by glutaraldehyde method (Wang et al., 2011). One microgram BSA was dissolved in 1.0 mL phosphate buffer saline (PBS, pH 7.4) and mixed with 600 mL FB1 standard solution. One milliliter of 0.3% glutaraldehyde was dropped slowly into mixture that contained 0.6 mg FB1 and 1 mg BSA, while shaking and incubated at room temperature for 2 h. Then, glycine (250 mL, 1 mol/L) was added to block unnecessary glutaraldehyde. Finally, the solution was dialyzed against 0.01 mol/L PBS (pH 7.4) at 4 C for 72 h to remove the residual free FB1 and glutaraldehyde. The complete antigen was stored at 20 C for use. The same method was employed to prepare FB1-KLH conjugate. 2.3. Analysis of the conjugates Agarose electrophoresis (1%, v/v, non-denaturing) was carried to determine if the conjugation was successful. Conjugates and carrier proteins (65 mg/mL) were mixed with loading buffer respectively, and then applied to the gel and separated at 200 V for 45 min. The gel was stained with
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coomassie blue R-250 for 2 h and destained with ethanolacetic acid until the band of protein became clear. Fourier transform infrared (FT-IR) transmittance spectroscopy was also carried out as follows. One milligram of BSA, KLH, FB1-BSA or FB1-KLH was milled with KBr respectively to form a very fine powder, and this powder was then compressed into a thin pellet which could be analyzed using the intelligent FT-IR spectrometer. 2.4. Animal immunization and indirect ELISA Animal immunization was preformed by standard procedure using conjugate FB1-BSA as immunogen (Zhou et al., 2009). FB1-BSA (0.2 mL, 100 mg) was emulsified with an equal volume of Freund’s complete adjuvant, and then injected into female Balb/c mice at multiple sites subcutaneously. Subsequently, about 2 weeks intervals, female Balb/c mice were administered by booster injection with FB1-BSA conjugate (0.1 mL, 50 mg) emulsified with an equal volume of Freund’s incomplete adjuvant. After the third immunization, the titer of antiserum was tested by ELISA. Conjugate FB1-KLH as coating antigen was diluted to 0.5 mg/mL and added into the micro-titer plates (100 mL/ well), then incubated overnight at 4 C. Plates coated with FB1-KLH were washed three times with PBS and blocked with PBSM (PBS containing 5% non-fat milk, 200 mL/well) at 37 C for 2 h. After washing for five times with PBST (PBS containing 0.05% Tween-20), the plates were added with the serial diluted anti-serum (100 mL/well) and incubated at 37 C for 1 h. After washing, HPR conjugated goat antimouse IgG (1: 10,000 dilution in PBSM) was added into the reaction wells and incubated at 37 C for 1 h. TMB substrate solution was added into wells for the development of color, and H2SO4 (50 mL, 2 mol/L) was dropped into wells to stop the reaction. The absorbance was measured at 450 nm by Micro-plate Reader. 2.5. Hybridoma generation The immunized mouse with high serum titer was given an intraperitoneal injection with 50 mg FB1-BSA immunogen. After 3 d, the B cells of spleen were isolated, and fused with SP2/0 myeloma cells at the ratio of 1: 10 by polyethylene glycol (PEG, 1450) (Li et al., 2012b; Zhang et al., 2011). Then cells were cultured in HAT medium on 96-well micro-titer plates and incubated with 5% CO2 at 37 C. About 7 d later, HAT medium in micro-titer plates was changed to HT medium. Two days later, the supernatant of culture medium was tested by indirect ELISA, and positive clones were obtained by limiting dilution until the positive percentage reached 100%. 2.6. Chromosome analysis of hybridoma cell Chromosome of hybridoma cell was counted after the treatment of colchicine (Lei et al., 2012). The hybridoma cell line was cultivated in HAT medium at 37 C for 1–2 d, and treated with 0.4 mg/mL colchicine and incubated with 5% CO2 at 37 C for 4–6 h. Hybridoma cells were harvested by centrifugation at 10,000 r/min for 5 min and then resuspended in 15 mL of 0.075 mol/L potassium chloride
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hypotonic solution at 37 C for 20 min. Hybridoma cells were harvested again by centrifugation at 1000 r/min for 10 min, then suspended three times with methanol: acetic acid stationary liquid and kept overnight at 4 C. Centrifugation was preformed to remove supernatant of cell culture and hybridoma cells were resuspended with 0.5–1 mL methanol: acetic acid stationary liquid, then smeared onto the glass slides. After giemsa staining, the number of the chromosome was counted. 2.7. Determination of subtype of the mcAb Mouse Monoclonal Antibody Subtyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit was employed to identify the subclass of the monoclonal antibody against FB1. Different antibody subtyping reagents (1: 1000 dilution in PBS) were dropped into ELISA plates and incubate at 37 C for 1 h. Plates were washed three times with PBST and blocked with PBSM at 37 C for 2 h. Plates were washed three times with PBST, and the supernatant of hybridoma culture was dropped into ELISA plates (100 mL/well). After washing, HRP conjugated goat anti-mouse IgG (1: 10,000 dilution in PBSM) was added into ELISA plates and incubated at 37 C for 1 h, and TMB substrate solution was added into the reaction wells. Then, 50 mL of 2 mol/L H2SO4 was dropped to stop the reaction and the absorbance was measured at 450 nm by Micro-plate Reader. 2.8. Preparation of ascites and purification of mcAb Preparation of mcAb was carried out by the method of introduced ascites in abdomen (Jiang-xiang and QingLing, 1999), and the purified mcAb was analyzed by SDS-PAGE according to the modified method of Laemmli (Laemmli, 1970). Protein concentration was determined by the instruction of Enhanced BCA Protein Assay Kit. 2.9. Cross-reactivity The specificity of monoclonal antibody against FB1 was determined by indirect competitive ELISA. Briefly, CIT, AFB1, DON and T-2 mycotoxins were used as competitive antigens. Fifty microliter of the each standard (0, 0.01, 0.05, 0.1, 1.0, 10, 100, 500, and 1000 ng/mL) were reacted with the anti-FB1 mcAb at 37 C for 30 min, then the above mixture were added into each well (Heussner et al., 2007). The relative cross-reactivity was determined as : % crossreactivity (CR) ¼ [50% inhibitory concentration (FB1)]/ [50% inhibitory concentration (competitor)]. 2.10. Affinity of mcAb To determine the affinity of the purified anti-FB1 mcAb, ic-ELISA was performed. FB1-KLH coating antigen at different dilution (1: 1000, 1: 2000, 1: 4000, and 1: 8000) was dropped into ELISA plates and incubated at 37 C for 2 h. After blocking and washing, anti-FB1 mcAb with serious dilutions (1: 200–1:256,000) were added into the ELISA plates and incubated at 37 C for 1 h. Then, HRP conjugated goat anti-mouse IgG (1: 10,000 dilutions in PBSM) was added into ELISA plates and incubated at 37 C
for 1 h, and the later procedures were the same as the former ic-ELISA. The curve diagram was made and the affinity of the antibody was obtained as described previously (Sheng et al., 2007). 2.11. Standard curve and samples detection FB1-KLH coating antigen at optimum concentration was added into plates, and different concentration of FB1 (0, 0.01, 0.05, 0.1, 1.0, 10, 100, 500, and 1000 ng/mL) mixed with equal volume of anti-FB1 mcAb were dropped into plates (100 mL/well). Then, HRP conjugated goat anti-mouse IgG (1: 10,000 dilution in PBSM) was added into each well. When the reaction stopped, the absorbance was measured at 450 nm. The relationship between concentration of FB1 and inhibition value was analyzed using Microcal Originpro 8.1. The linear range to detect FB1 was defined as the concentration of FB1 toward from 20% to 80% inhibition (Kido et al., 2008). Matrix effect in standard curve was also tested in the study. Methanol was chosen for extraction by the developed ic-ELISA. Matrix effect was minimized by diluting the samples before the ELISA assay (Ono et al., 2000; Quan et al., 2006). In the study, Corn samples without contamination tested by GC–MS (data not show) was analyzed by testing 10-fold–500-fold dilutions. Additionally, matrix interference was determined by comparing a standard curve prepared in PBS with that obtained using diluted matrix. Recovery study was carried out to determine the efficacy of standard curve. Corn and related samples were prepared as described previously (Li et al., 2011) with some modification. One gram of the corn powder samples with non-detectable FB1 by GC–MS were artificially spiked with FB1 at concentrations of 5, 50, 250 and 500 ng/mL, and maintained at 4 C overnight. Then, samples were mixed with 5 mL methanol-water (7: 3, v: v) and incubated at room temperature for 30 min. After that, the suspension was obtained by centrifuging the mixture at 8000 r/min for 20 min and the corn extracts were determined by ic-ELISA. The recovery rate was made according to the standard curve. Corn and related samples (Corn bread, Cornmeal, Corn cake) were collected randomly, and the corn extracts were determined by ic-ELISA. 3. Results 3.1. Preparation and identification of the conjugates FB1 is a small molecular weight mycotoxin and has no immunogenicity. To obtain monoclonal antibody against small non-immunogenic molecules, FB1 should be coupled to carrier protein. In this experiment, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) were chosen as carrier protein. FB1-BSA and FB1-KLH complete antigens were prepared by glutaraldehyde method (Wang et al., 2011). The gel electrophoresis results of conjugates were shown in Figs. 1A and B. The pure carrier proteins and conjugates in non-denaturing agarosegels showed welldefined bands, respectively. The migration velocity of FB1KLH conjugate was faster than that KLH alone (Fig. 1A), and
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Fig. 1. Identification of the conjugates. (A) Non-denaturing agarose electrophoresis of FB1-KLH conjugation. Lane 1, keyhole limpet hemocyanin (KLH). Lane 2, FB1-KLH. (B) Non-denaturing agarose electrophoresis of FB1-BSA. Lane 1, bovine serum albumin (BSA). Lane 2, FB1-BSA. (C) Fourier transform infrared (FT-IR) transmittance spectra of FB1-KLH conjugate. The scan range was 4000–500 cm1 (D) FT-IR transmittance spectra of FB1-BSA conjugate.
the migration velocity of FB1-BSA was also faster than that BSA alone (Fig. 1B). The gel electrophoresis results indicated that FB1 was successfully conjugated to the BSA and KLH carrier protein, respectively. Fourier transform infrared (FT-IR) transmittance spectroscopy was used to detect wavelength and intensity of the absorption of IR radiation (Wang et al., 2011). The characteristic IR groups of the FB1-KLH, FB1-BSA, KLH, BSA, in the range of 4000–500 cm1 were shown in Figs. 1C and D. There were strong absorption from KLH, BSA and FB1, in the regions 3500–3200, 2000–1600 cm1. The characteristic absorption peaks of FB1 could be found in the region 1150–1060 cm1, and the characteristic absorption peaks of KLH, BSA could be found in the region 3000–2850 cm1. The most important was that the absorption peaks of conjugates could be found in the regions of 3500–3200, 3000–2850 cm1, 2000–1600 cm1, 1150–1060 cm1. The results indicated that the functional groups of KLH, BSA and
FB1 were contained in FB1-KLH and FB1-BSA, respectively. Combined to the electrophoresis results, it was shown that FB1 was successfully conjugated to carrier proteins. 3.2. Immunization and determination of anti-serum titer In the study, FB1-BSA conjugate as immune antigen was used to immunize health female Balb/c mice that could generate antibody against FB1. Generally, after three times immunization, the blood samples of female Balb/c mice were taken out to determine antiserum titer by indirect ELISA. In this experiment, FB1-KLH conjugate was used as coating antigen. The titer result showed that the OD value of blood samples from Balb/c mice immunized with FB1BSA was significantly higher than that of control (Fig. 2), indicating that FB1-BSA complete antigen induced generation of antibody against FB1 successfully. The titer of the mouse 1 was higher than that of other two immunized
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coating antigen. The fusion rate reached 96.97%, while positive rate of fusion cells were up to 8.04% (Table 1). After sub-cloning for 3–4 times, three positive clones secreting monoclonal antibodies against FB1 named 4G5, 2E6, and 11F11, respectively were obtained successfully. 3.4. Subtype and chromosome analysis of anti-FB1 mcAb
Fig. 2. Anti-serum titer results of three mice by indirect ELISA. Mouse 1, 2 and 3 were the three mice immunized with FB1-BSA conjugate dissolved in PBS buffer respectively. Control was the mouse immunized with only PBS buffer.
mice (mouse 2 and mouse 3) and the control mouse (only injected with PBS buffer). Therefore, the spleen cell of mouse 1 was chosen to fuse with SP2/0 myeloma cell. Different titers of three mice might attribute to their own immunity. 3.3. Cell fusion and screening of positive clones In this experiment, spleen cells obtained from the Balb/c mouse 1 were fused to SP2/0 myeloma cells by polyethylene glycol (PEG, 1450) at the ratio of 10: 1. During the procedure of fusion, thousands of unfused parental myeloma and spleen cells were died in HAT selective medium, while only hybridoma that spleen cell and myeloma cell fused successfully could grow well in the HAT selective medium. Under microscope, two to six cells aggregations were observed on the bottom of wells after 2d (Fig. 3A). After 4 d, the numbers of fusion cells increased more and more (Fig. 3B). The bottom of 96-well microtiter plate was covered with almost all hybridoma cells at 8 d (Fig. 3C). At the same time, the titer of culture supernatant was determined by indirect ELISA using FB1-KLH conjugate as
The subtype of the monoclonal antibodies against FB1 were determined by Isotyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit, and the results showed that the subtype of 4G5, 2E6, 11F11 positive clones was IgG2b, IgM, IgM, respectively (Fig. 4A). Monoclonal antibody of IgG subtype is stable, so positive clone 4G5 was chosen for the further experiments in the following study. Chromosome analysis was preformed to identify whether spleen cell and SP2/ 0 myeloma cell were successfully fused. The average chromosome number of SP2/0 myeloma cell and spleen cell were 62–70 and 38–40, respectively (Zhou et al., 2009). The chromosome number of positive clone 4G5 was counted as 101 (Fig. 4B), showing that the chromosome number of positive clone 4G5 were obtained from spleen cell and SP2/ 0 myeloma cell. 3.5. Analysis of the purified anti-FB1 mcAb Positive clone 4G5 were cultured and injected into the abdomen of Balb/c mice to produce more monoclonal antibody. The anti-FB1 mcAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CAAS) method. SDS-PAGE was used to analyze the mcAb, and the result showed that the purified anti-FB1 mcAb has a band of heavy chain at 50 kDa and a band of light chain at 27 kDa (Fig. 5A). The indirect ELISA showed that the titer of the purified anti-FB1 mcAb secreted by 4G5 positive clone was above 6.4 104 (Fig. 5B). All these results indicated that the mcAb from 4G5 ascites had a higher titer, and it could be further used to develop an ELISA kit for FB1 detection. 3.6. Specialty and affinity of the anti-FB1 mcAb To further analyze the specificity of the anti-FB1 mcAb secreted by positive clone 4G5, competitive ELISA was performed. The ELISA result showed that 4G5 was highly
Fig. 3. Cells cultured in HAT (Hypoxanthine, aminopterin and thymidine) medium. SP2/0 cell lacking HGPRT enzyme can not grow in HAT medium, and spleen cell which contains HGPRT enzyme also can not grow in vitro. Only hybridoma cells can grow in HAT medium. (A) Hybridoma cells were observed in 2 d, 100. (B) Hybridoma cells were observed in 4 d, 100; (C) Hybridoma cells were observed in 8 d, 40.
S. Ling et al. / Toxicon 80 (2014) 64–72 Table 1 The results of cell fusion and screening. Plates No.
Fusion rate (%)
Positive rate (%)
Positive hybridoma
2 4 11 average
100% (96/96) 94.32% (83/88) 96.59% (85/88) 96.97%
6.25% (6/96) 9.64% (8/83) 8.24% (7/85) 8.04%
2E6 4G5 11F11
specific to FB1 (Fig. 6A). The cross-reactivity of 4G5 indicated that anti-FB1 mcAb had 5% cross reactivity with T-2 toxin, and no cross reactions between FB1 and other mycotoxins (Table 2). The affinity was determined by indirect ELISA. Different concentrations of FB1-KLH coating antigen (296, 148, 74, and 37 ng/mL) was dropped into plate cells respectively, then different dilutions of anti-FB1 mcAb were added. From calculation in Fig. 6B, the affinity of the mcAb was about 2.1 108 L/mol. The affinity result indicated that the anti-FB1 mcAb secreted by 4G5 positive clone was sensitive to FB1. 3.7. Standard curve and samples detection Standard curves were obtained by competitive inhibition ELISA. In the study, 50 mL FB1 (diluted in PBS or matrix)
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of different concentrations were mixed with 50 mL anti-FB1 mcAb, and incubated at 37 C for 30 min. Then, the mixture was dropped into plates which were coated with FB1-KLH. The relationship between concentration of FB1 and inhibition value was analyzed using Microcal Originpro 8.1. The logistic equation was y ¼ 1.33275/(1þ(x/32.24498)^0.43097, with a correlation coefficient (R2) of 0.9968 (Fig. 7A). The linear range to detect FB1 was 1–800 ng/mL, which defined as the concentration of FB1 toward from 20% to 80% inhibition. The concentration of FB1 toward 50% inhibition (IC50) was 32 ng/mL and the detection limit (LOD) was 1.0 ng/mL. The linear equation is y ¼ 20.015x 16.019, with a correlation coefficient (R2) of 0.9929 (Fig. 7B). In the study, methanol-water (7: 3, v: v) was used as extraction solution. A standard curve in matrix prepared by diluted matrix was compared to a standard curve prepared in PBS, and the two curves were superimposed, indicating that the matrix effect was minimized. The result showed that a dilution of 100- fold could reduce the matrix interference, and this dilution was selected for ic-ELISA in the study (Fig. 7A). To test the recovery of ic-ELISA based on the standard curve, corn samples without contamination were spiked with different concentration of FB1 (5, 50, 250 and 500 ng/
Fig. 4. Subtype analysis of monoclonal antibodies against FB1 and Chromosome analysis of positive clone 4G5 hybridoma. (A) The result of subtype of monoclonal antibodies against FB1. The subtypes of anti-FB1 mcAb secreted by 4G5, 2E6, 11F11 were determined by Isotyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit. (B) Chromosomes of 4G5 hybridomas cell.
Fig. 5. SDS-PAGE and titer analysis of the purified anti-FB1 mcAb excreted by 4G5 positive clone. (A) SDS-PAGE of the purified anti-FB1 mcAb. Lanes M, Standard protein marker. Lane 1 and 2, 5 mL and 8 mL of the purified mcAb, respectively. (B) The titer of the purified anti-FB1 mcAb was determined by indirect ELISA.
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Fig. 6. Specialty and affinity of anti-FB1 mcAb. (A) Cross-reactivity of anti-FB1 mcAb to other toxins was determined by ic-ELISA. (B) Affinity was determined by indirect ELISA. Different antigen concentrations (296, 148, 74, and 37 ng/mL) of coating antigen (FB1-KLH) were used, and the affinity was determined by indirect ELISA.
Table 2 The cross-reactivity of anti-FB1 mcAb secreted by 4G5 to other toxins. Toxins
FB1
AFB1
CIT
T-2
Cross-reactivity (%)
100%
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Preparation and identification of monoclonal antibody against fumonisin B1 and development of detection by Ic-ELISA Sumei Ling a,1, Jie Pang b,1, Jinjin Yu b, Rongzhi Wang a, Licai Liu a, c, Yanling Ma a, Yuming Zhang b, Ni Jin a, Shihua Wang a, * a
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China School of Food Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China c Environmental Protection Bureau of Qitaihe City, Qitaihe 154600, Heilongjiang, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2013 Received in revised form 11 December 2013 Accepted 19 December 2013 Available online 27 December 2013
Fumonisin B1 (FB1) is one of the mycotoxins produced by Fusarium verticillioides, which was mainly found in corn and related products. FB1 was small molecule with no immunogenicity, so it should be conjugated to carrier proteins such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin) to generate immunogenicity. In this study, conjugate FB1-BSA was used to immunize Balb/c mice, and one hybrid cell line 4G5 excreting monoclonal antibody against FB1 was obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mouse. Hybridoma 4G5 was injected into the abdomen of Balb/c mice, and the anti-FB1 mcAb was harvested from ascites and the titer reached 6.4 104 after purification with caprylic/ammonium sulfate precipitation (CA-AS) method. The cross-reactivity results showed that anti-FB1 mcAb was highly specific to fumonisin B1, and the affinity was 2.1 108 L/M. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect FB1 was 1–800 ng/mL with IC50 of 32 ng/mL. The detection limit was 1.0 ng/mL, and the recovery average was 93.75 6.90%. Therefore, the anti-FB1 mcAb excreted by 4G5 can be used to detect fumonisin B1 in corn and related samples. Ó 2014 Published by Elsevier Ltd.
Keywords: Fumonisin B1 Hybridoma Monoclonal antibody Detection ic-ELISA
1. Introduction Fumonisins are mycotoxins produced mainly by Fusarium verticillioides and Fusariumproliferatum (Brera et al., 1998), and fumonisin B1 (FB1) is the most abundant and reaches 70% of the total fumonisins in maize (Orsi et al., 2009). Ingestion of FB1 causes lots of toxicoses in animals, including leukoencephalomalacia, pulmonary edema, and hepatocarcinoma or other liver diseases (Brera et al., 1998). In addition, FB1 in foods has been associated with a high * Corresponding author. Tel./fax: þ86 0591 87984471. E-mail address: [email protected] (S. Wang). 1 These authors contributed equally to this work. 0041-0101/$ – see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.toxicon.2013.12.008
possibility of esophageal cancer (Proctor et al., 2008). Because of the known toxicological evidence, the International Agency for Research on Cancer (IARC) has taken FB1 as a possible carcinogen to humans (Group 2B) (Tardieu et al., 2008). As the above harmful effects, an effective method for FB1 detection is essential and important. To date, many different methods were used to identify FB1, such as HPLC, TLC, GC–MS and LC–MS/MS (Brera et al., 1998). These methods with high sensitivity and specificity can detect fumonisin B1 exactly in food. However, they depend on complex purification steps, expensive instruments and large quantity of raw materials. They are improper for realtime detection in actual fields and incompatible with
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demands of food industries. In recent years, monoclonal antibody are used widely in the development of diagnostic and research reagents, and becomes one of the most popular methods for food pathogen detection (Brera et al., 1998; Li et al., 2012a,b; Pei et al., 2009). The purpose of the present study was to obtain a monoclonal antibody against FB1 with high specificity and affinity. However, FB1 is a small molecule with no immune response, thus it is necessary to conjugate FB1 to a carrier protein. In the study, FB1 was coupled to BSA (bovine serum albumin) and KLH (keyhole limpet hemocyanin) for immunization and screening respectively, and one positive hybridoma named 4G5 excreting IgG2b monoclonal antibody against FB1 was successfully obtained with affinity of 2.1 108 L/M, and then a competitive indirect ELISA was carried to detect FB1 based on this antibody. The experiments and results were followed. 2. Materials and methods 2.1. Materials Fumonisin (FB1), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), polyethylene glycol 1450 (PEG 1450), Freund’s complete adjuvant, Freund’s incomplete adjuvant, horseradish peroxidase (HRP) conjugated goat anti-mouse IgG, hypoxanthine, aminopterin and thymidine (HAT) medium, hypoxanthine-thymidine (HT) medium, mouse monoclonal antibody isotyping reagent (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA), and RPMI 1640 were purchased from Sigma Chemical Co. (St. Louis, MO, USA.). Female Balb/c mice (8 weeks) were obtained from Wushi Animal Laboratory (Shanghai, China). Myeloma cells SP2/ 0 were stored in our lab. All animal studies were carried out according to institutional guidelines in Fujian Agriculture and Forestry University (China). 2.2. Preparation of complete antigens FB1-BSA and FB1-KLH complete antigens were prepared by glutaraldehyde method (Wang et al., 2011). One microgram BSA was dissolved in 1.0 mL phosphate buffer saline (PBS, pH 7.4) and mixed with 600 mL FB1 standard solution. One milliliter of 0.3% glutaraldehyde was dropped slowly into mixture that contained 0.6 mg FB1 and 1 mg BSA, while shaking and incubated at room temperature for 2 h. Then, glycine (250 mL, 1 mol/L) was added to block unnecessary glutaraldehyde. Finally, the solution was dialyzed against 0.01 mol/L PBS (pH 7.4) at 4 C for 72 h to remove the residual free FB1 and glutaraldehyde. The complete antigen was stored at 20 C for use. The same method was employed to prepare FB1-KLH conjugate. 2.3. Analysis of the conjugates Agarose electrophoresis (1%, v/v, non-denaturing) was carried to determine if the conjugation was successful. Conjugates and carrier proteins (65 mg/mL) were mixed with loading buffer respectively, and then applied to the gel and separated at 200 V for 45 min. The gel was stained with
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coomassie blue R-250 for 2 h and destained with ethanolacetic acid until the band of protein became clear. Fourier transform infrared (FT-IR) transmittance spectroscopy was also carried out as follows. One milligram of BSA, KLH, FB1-BSA or FB1-KLH was milled with KBr respectively to form a very fine powder, and this powder was then compressed into a thin pellet which could be analyzed using the intelligent FT-IR spectrometer. 2.4. Animal immunization and indirect ELISA Animal immunization was preformed by standard procedure using conjugate FB1-BSA as immunogen (Zhou et al., 2009). FB1-BSA (0.2 mL, 100 mg) was emulsified with an equal volume of Freund’s complete adjuvant, and then injected into female Balb/c mice at multiple sites subcutaneously. Subsequently, about 2 weeks intervals, female Balb/c mice were administered by booster injection with FB1-BSA conjugate (0.1 mL, 50 mg) emulsified with an equal volume of Freund’s incomplete adjuvant. After the third immunization, the titer of antiserum was tested by ELISA. Conjugate FB1-KLH as coating antigen was diluted to 0.5 mg/mL and added into the micro-titer plates (100 mL/ well), then incubated overnight at 4 C. Plates coated with FB1-KLH were washed three times with PBS and blocked with PBSM (PBS containing 5% non-fat milk, 200 mL/well) at 37 C for 2 h. After washing for five times with PBST (PBS containing 0.05% Tween-20), the plates were added with the serial diluted anti-serum (100 mL/well) and incubated at 37 C for 1 h. After washing, HPR conjugated goat antimouse IgG (1: 10,000 dilution in PBSM) was added into the reaction wells and incubated at 37 C for 1 h. TMB substrate solution was added into wells for the development of color, and H2SO4 (50 mL, 2 mol/L) was dropped into wells to stop the reaction. The absorbance was measured at 450 nm by Micro-plate Reader. 2.5. Hybridoma generation The immunized mouse with high serum titer was given an intraperitoneal injection with 50 mg FB1-BSA immunogen. After 3 d, the B cells of spleen were isolated, and fused with SP2/0 myeloma cells at the ratio of 1: 10 by polyethylene glycol (PEG, 1450) (Li et al., 2012b; Zhang et al., 2011). Then cells were cultured in HAT medium on 96-well micro-titer plates and incubated with 5% CO2 at 37 C. About 7 d later, HAT medium in micro-titer plates was changed to HT medium. Two days later, the supernatant of culture medium was tested by indirect ELISA, and positive clones were obtained by limiting dilution until the positive percentage reached 100%. 2.6. Chromosome analysis of hybridoma cell Chromosome of hybridoma cell was counted after the treatment of colchicine (Lei et al., 2012). The hybridoma cell line was cultivated in HAT medium at 37 C for 1–2 d, and treated with 0.4 mg/mL colchicine and incubated with 5% CO2 at 37 C for 4–6 h. Hybridoma cells were harvested by centrifugation at 10,000 r/min for 5 min and then resuspended in 15 mL of 0.075 mol/L potassium chloride
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hypotonic solution at 37 C for 20 min. Hybridoma cells were harvested again by centrifugation at 1000 r/min for 10 min, then suspended three times with methanol: acetic acid stationary liquid and kept overnight at 4 C. Centrifugation was preformed to remove supernatant of cell culture and hybridoma cells were resuspended with 0.5–1 mL methanol: acetic acid stationary liquid, then smeared onto the glass slides. After giemsa staining, the number of the chromosome was counted. 2.7. Determination of subtype of the mcAb Mouse Monoclonal Antibody Subtyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit was employed to identify the subclass of the monoclonal antibody against FB1. Different antibody subtyping reagents (1: 1000 dilution in PBS) were dropped into ELISA plates and incubate at 37 C for 1 h. Plates were washed three times with PBST and blocked with PBSM at 37 C for 2 h. Plates were washed three times with PBST, and the supernatant of hybridoma culture was dropped into ELISA plates (100 mL/well). After washing, HRP conjugated goat anti-mouse IgG (1: 10,000 dilution in PBSM) was added into ELISA plates and incubated at 37 C for 1 h, and TMB substrate solution was added into the reaction wells. Then, 50 mL of 2 mol/L H2SO4 was dropped to stop the reaction and the absorbance was measured at 450 nm by Micro-plate Reader. 2.8. Preparation of ascites and purification of mcAb Preparation of mcAb was carried out by the method of introduced ascites in abdomen (Jiang-xiang and QingLing, 1999), and the purified mcAb was analyzed by SDS-PAGE according to the modified method of Laemmli (Laemmli, 1970). Protein concentration was determined by the instruction of Enhanced BCA Protein Assay Kit. 2.9. Cross-reactivity The specificity of monoclonal antibody against FB1 was determined by indirect competitive ELISA. Briefly, CIT, AFB1, DON and T-2 mycotoxins were used as competitive antigens. Fifty microliter of the each standard (0, 0.01, 0.05, 0.1, 1.0, 10, 100, 500, and 1000 ng/mL) were reacted with the anti-FB1 mcAb at 37 C for 30 min, then the above mixture were added into each well (Heussner et al., 2007). The relative cross-reactivity was determined as : % crossreactivity (CR) ¼ [50% inhibitory concentration (FB1)]/ [50% inhibitory concentration (competitor)]. 2.10. Affinity of mcAb To determine the affinity of the purified anti-FB1 mcAb, ic-ELISA was performed. FB1-KLH coating antigen at different dilution (1: 1000, 1: 2000, 1: 4000, and 1: 8000) was dropped into ELISA plates and incubated at 37 C for 2 h. After blocking and washing, anti-FB1 mcAb with serious dilutions (1: 200–1:256,000) were added into the ELISA plates and incubated at 37 C for 1 h. Then, HRP conjugated goat anti-mouse IgG (1: 10,000 dilutions in PBSM) was added into ELISA plates and incubated at 37 C
for 1 h, and the later procedures were the same as the former ic-ELISA. The curve diagram was made and the affinity of the antibody was obtained as described previously (Sheng et al., 2007). 2.11. Standard curve and samples detection FB1-KLH coating antigen at optimum concentration was added into plates, and different concentration of FB1 (0, 0.01, 0.05, 0.1, 1.0, 10, 100, 500, and 1000 ng/mL) mixed with equal volume of anti-FB1 mcAb were dropped into plates (100 mL/well). Then, HRP conjugated goat anti-mouse IgG (1: 10,000 dilution in PBSM) was added into each well. When the reaction stopped, the absorbance was measured at 450 nm. The relationship between concentration of FB1 and inhibition value was analyzed using Microcal Originpro 8.1. The linear range to detect FB1 was defined as the concentration of FB1 toward from 20% to 80% inhibition (Kido et al., 2008). Matrix effect in standard curve was also tested in the study. Methanol was chosen for extraction by the developed ic-ELISA. Matrix effect was minimized by diluting the samples before the ELISA assay (Ono et al., 2000; Quan et al., 2006). In the study, Corn samples without contamination tested by GC–MS (data not show) was analyzed by testing 10-fold–500-fold dilutions. Additionally, matrix interference was determined by comparing a standard curve prepared in PBS with that obtained using diluted matrix. Recovery study was carried out to determine the efficacy of standard curve. Corn and related samples were prepared as described previously (Li et al., 2011) with some modification. One gram of the corn powder samples with non-detectable FB1 by GC–MS were artificially spiked with FB1 at concentrations of 5, 50, 250 and 500 ng/mL, and maintained at 4 C overnight. Then, samples were mixed with 5 mL methanol-water (7: 3, v: v) and incubated at room temperature for 30 min. After that, the suspension was obtained by centrifuging the mixture at 8000 r/min for 20 min and the corn extracts were determined by ic-ELISA. The recovery rate was made according to the standard curve. Corn and related samples (Corn bread, Cornmeal, Corn cake) were collected randomly, and the corn extracts were determined by ic-ELISA. 3. Results 3.1. Preparation and identification of the conjugates FB1 is a small molecular weight mycotoxin and has no immunogenicity. To obtain monoclonal antibody against small non-immunogenic molecules, FB1 should be coupled to carrier protein. In this experiment, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) were chosen as carrier protein. FB1-BSA and FB1-KLH complete antigens were prepared by glutaraldehyde method (Wang et al., 2011). The gel electrophoresis results of conjugates were shown in Figs. 1A and B. The pure carrier proteins and conjugates in non-denaturing agarosegels showed welldefined bands, respectively. The migration velocity of FB1KLH conjugate was faster than that KLH alone (Fig. 1A), and
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Fig. 1. Identification of the conjugates. (A) Non-denaturing agarose electrophoresis of FB1-KLH conjugation. Lane 1, keyhole limpet hemocyanin (KLH). Lane 2, FB1-KLH. (B) Non-denaturing agarose electrophoresis of FB1-BSA. Lane 1, bovine serum albumin (BSA). Lane 2, FB1-BSA. (C) Fourier transform infrared (FT-IR) transmittance spectra of FB1-KLH conjugate. The scan range was 4000–500 cm1 (D) FT-IR transmittance spectra of FB1-BSA conjugate.
the migration velocity of FB1-BSA was also faster than that BSA alone (Fig. 1B). The gel electrophoresis results indicated that FB1 was successfully conjugated to the BSA and KLH carrier protein, respectively. Fourier transform infrared (FT-IR) transmittance spectroscopy was used to detect wavelength and intensity of the absorption of IR radiation (Wang et al., 2011). The characteristic IR groups of the FB1-KLH, FB1-BSA, KLH, BSA, in the range of 4000–500 cm1 were shown in Figs. 1C and D. There were strong absorption from KLH, BSA and FB1, in the regions 3500–3200, 2000–1600 cm1. The characteristic absorption peaks of FB1 could be found in the region 1150–1060 cm1, and the characteristic absorption peaks of KLH, BSA could be found in the region 3000–2850 cm1. The most important was that the absorption peaks of conjugates could be found in the regions of 3500–3200, 3000–2850 cm1, 2000–1600 cm1, 1150–1060 cm1. The results indicated that the functional groups of KLH, BSA and
FB1 were contained in FB1-KLH and FB1-BSA, respectively. Combined to the electrophoresis results, it was shown that FB1 was successfully conjugated to carrier proteins. 3.2. Immunization and determination of anti-serum titer In the study, FB1-BSA conjugate as immune antigen was used to immunize health female Balb/c mice that could generate antibody against FB1. Generally, after three times immunization, the blood samples of female Balb/c mice were taken out to determine antiserum titer by indirect ELISA. In this experiment, FB1-KLH conjugate was used as coating antigen. The titer result showed that the OD value of blood samples from Balb/c mice immunized with FB1BSA was significantly higher than that of control (Fig. 2), indicating that FB1-BSA complete antigen induced generation of antibody against FB1 successfully. The titer of the mouse 1 was higher than that of other two immunized
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coating antigen. The fusion rate reached 96.97%, while positive rate of fusion cells were up to 8.04% (Table 1). After sub-cloning for 3–4 times, three positive clones secreting monoclonal antibodies against FB1 named 4G5, 2E6, and 11F11, respectively were obtained successfully. 3.4. Subtype and chromosome analysis of anti-FB1 mcAb
Fig. 2. Anti-serum titer results of three mice by indirect ELISA. Mouse 1, 2 and 3 were the three mice immunized with FB1-BSA conjugate dissolved in PBS buffer respectively. Control was the mouse immunized with only PBS buffer.
mice (mouse 2 and mouse 3) and the control mouse (only injected with PBS buffer). Therefore, the spleen cell of mouse 1 was chosen to fuse with SP2/0 myeloma cell. Different titers of three mice might attribute to their own immunity. 3.3. Cell fusion and screening of positive clones In this experiment, spleen cells obtained from the Balb/c mouse 1 were fused to SP2/0 myeloma cells by polyethylene glycol (PEG, 1450) at the ratio of 10: 1. During the procedure of fusion, thousands of unfused parental myeloma and spleen cells were died in HAT selective medium, while only hybridoma that spleen cell and myeloma cell fused successfully could grow well in the HAT selective medium. Under microscope, two to six cells aggregations were observed on the bottom of wells after 2d (Fig. 3A). After 4 d, the numbers of fusion cells increased more and more (Fig. 3B). The bottom of 96-well microtiter plate was covered with almost all hybridoma cells at 8 d (Fig. 3C). At the same time, the titer of culture supernatant was determined by indirect ELISA using FB1-KLH conjugate as
The subtype of the monoclonal antibodies against FB1 were determined by Isotyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit, and the results showed that the subtype of 4G5, 2E6, 11F11 positive clones was IgG2b, IgM, IgM, respectively (Fig. 4A). Monoclonal antibody of IgG subtype is stable, so positive clone 4G5 was chosen for the further experiments in the following study. Chromosome analysis was preformed to identify whether spleen cell and SP2/ 0 myeloma cell were successfully fused. The average chromosome number of SP2/0 myeloma cell and spleen cell were 62–70 and 38–40, respectively (Zhou et al., 2009). The chromosome number of positive clone 4G5 was counted as 101 (Fig. 4B), showing that the chromosome number of positive clone 4G5 were obtained from spleen cell and SP2/ 0 myeloma cell. 3.5. Analysis of the purified anti-FB1 mcAb Positive clone 4G5 were cultured and injected into the abdomen of Balb/c mice to produce more monoclonal antibody. The anti-FB1 mcAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CAAS) method. SDS-PAGE was used to analyze the mcAb, and the result showed that the purified anti-FB1 mcAb has a band of heavy chain at 50 kDa and a band of light chain at 27 kDa (Fig. 5A). The indirect ELISA showed that the titer of the purified anti-FB1 mcAb secreted by 4G5 positive clone was above 6.4 104 (Fig. 5B). All these results indicated that the mcAb from 4G5 ascites had a higher titer, and it could be further used to develop an ELISA kit for FB1 detection. 3.6. Specialty and affinity of the anti-FB1 mcAb To further analyze the specificity of the anti-FB1 mcAb secreted by positive clone 4G5, competitive ELISA was performed. The ELISA result showed that 4G5 was highly
Fig. 3. Cells cultured in HAT (Hypoxanthine, aminopterin and thymidine) medium. SP2/0 cell lacking HGPRT enzyme can not grow in HAT medium, and spleen cell which contains HGPRT enzyme also can not grow in vitro. Only hybridoma cells can grow in HAT medium. (A) Hybridoma cells were observed in 2 d, 100. (B) Hybridoma cells were observed in 4 d, 100; (C) Hybridoma cells were observed in 8 d, 40.
S. Ling et al. / Toxicon 80 (2014) 64–72 Table 1 The results of cell fusion and screening. Plates No.
Fusion rate (%)
Positive rate (%)
Positive hybridoma
2 4 11 average
100% (96/96) 94.32% (83/88) 96.59% (85/88) 96.97%
6.25% (6/96) 9.64% (8/83) 8.24% (7/85) 8.04%
2E6 4G5 11F11
specific to FB1 (Fig. 6A). The cross-reactivity of 4G5 indicated that anti-FB1 mcAb had 5% cross reactivity with T-2 toxin, and no cross reactions between FB1 and other mycotoxins (Table 2). The affinity was determined by indirect ELISA. Different concentrations of FB1-KLH coating antigen (296, 148, 74, and 37 ng/mL) was dropped into plate cells respectively, then different dilutions of anti-FB1 mcAb were added. From calculation in Fig. 6B, the affinity of the mcAb was about 2.1 108 L/mol. The affinity result indicated that the anti-FB1 mcAb secreted by 4G5 positive clone was sensitive to FB1. 3.7. Standard curve and samples detection Standard curves were obtained by competitive inhibition ELISA. In the study, 50 mL FB1 (diluted in PBS or matrix)
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of different concentrations were mixed with 50 mL anti-FB1 mcAb, and incubated at 37 C for 30 min. Then, the mixture was dropped into plates which were coated with FB1-KLH. The relationship between concentration of FB1 and inhibition value was analyzed using Microcal Originpro 8.1. The logistic equation was y ¼ 1.33275/(1þ(x/32.24498)^0.43097, with a correlation coefficient (R2) of 0.9968 (Fig. 7A). The linear range to detect FB1 was 1–800 ng/mL, which defined as the concentration of FB1 toward from 20% to 80% inhibition. The concentration of FB1 toward 50% inhibition (IC50) was 32 ng/mL and the detection limit (LOD) was 1.0 ng/mL. The linear equation is y ¼ 20.015x 16.019, with a correlation coefficient (R2) of 0.9929 (Fig. 7B). In the study, methanol-water (7: 3, v: v) was used as extraction solution. A standard curve in matrix prepared by diluted matrix was compared to a standard curve prepared in PBS, and the two curves were superimposed, indicating that the matrix effect was minimized. The result showed that a dilution of 100- fold could reduce the matrix interference, and this dilution was selected for ic-ELISA in the study (Fig. 7A). To test the recovery of ic-ELISA based on the standard curve, corn samples without contamination were spiked with different concentration of FB1 (5, 50, 250 and 500 ng/
Fig. 4. Subtype analysis of monoclonal antibodies against FB1 and Chromosome analysis of positive clone 4G5 hybridoma. (A) The result of subtype of monoclonal antibodies against FB1. The subtypes of anti-FB1 mcAb secreted by 4G5, 2E6, 11F11 were determined by Isotyping (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) Kit. (B) Chromosomes of 4G5 hybridomas cell.
Fig. 5. SDS-PAGE and titer analysis of the purified anti-FB1 mcAb excreted by 4G5 positive clone. (A) SDS-PAGE of the purified anti-FB1 mcAb. Lanes M, Standard protein marker. Lane 1 and 2, 5 mL and 8 mL of the purified mcAb, respectively. (B) The titer of the purified anti-FB1 mcAb was determined by indirect ELISA.
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Fig. 6. Specialty and affinity of anti-FB1 mcAb. (A) Cross-reactivity of anti-FB1 mcAb to other toxins was determined by ic-ELISA. (B) Affinity was determined by indirect ELISA. Different antigen concentrations (296, 148, 74, and 37 ng/mL) of coating antigen (FB1-KLH) were used, and the affinity was determined by indirect ELISA.
Table 2 The cross-reactivity of anti-FB1 mcAb secreted by 4G5 to other toxins. Toxins
FB1
AFB1
CIT
T-2
Cross-reactivity (%)
100%
