Toxicon 92 (2014) 186e192
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Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin Pingyan Wang a, Guanghui Li a, Junrong Yan a, Yonghong Hu c, Cunzheng Zhang d, Xianjin Liu d, Yakun Wan a, b, * a
The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China Jiangsu Nanobody Engineering and Research Center, Nantong 226010, PR China c College of 543Biotechnology and Pharmaceutical Engineering, Nanjing University of 544Technology, Nanjing 210009, PR China d Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2014 Received in revised form 21 October 2014 Accepted 29 October 2014 Available online 30 October 2014
The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Nanobody Phage-displayed library Cry1Fa Biotin Streptavidin Sandwich immune ELISA
1. Introduction Genetically modified (GM) crops producing insect-resistant Cry proteins from the bacterium, Bacillus thuringiensis (Bt), are revolutionizing agriculture. Genes of Bt toxins have been engineered into major crops, generally these crops have shown considerable economic benefits, and decreased the use of other insecticides (Shelton et al., 2002). A dozen of Cry toxins are commercially applied in agriculture, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D, Cry1E, Cry1Fa, Cry3Aa, Cry3Bb and Cry34/Cry35 (Bravo and Soberon, 2008). Among these Cry toxins, Cry1Fa is constructive to protect crops from the Lepidoptera insects by killing their larvae, which is especially sensitive to Osrinia mubilais Hubner and O. furnacalis Güenee (Tian et al., 2013). However, A main concern with the widespread of Bt crops is their potential to adverse
* Corresponding author. Institute of Life Sciences, Southeast University, Sipailou No.2, Nanjing 210096, PR China. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.toxicon.2014.10.024 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
affects on food safety, human health and non-target organisms including biological control organisms (Romeis et al., 2006). It is important to examine the different potential impacts of Bt crops. The concerns currently being asked of GM crops frequently demand a sensitive and accurate detection method (Dale et al., 2002). In the past decades, numerous detections have been developed for Bt toxins. DNA/proteins of transgenic crops could be conducted by polymerase chain reaction (PCR) and real-time PCR (Asensio et al., 2008). However, they can't determine the expression level of Cry toxins and usually require sophisticated instruments (Randhawa and Singh, 2012). With the development of bioengineering and biotechnology, antibodies against Cry toxins such as single-chain antibody (ScFv), have been developed and used in ELISA and biosensors (Zhang et al., 2012). However, the technique of ScFv proved to be troublesome because of the low yield and instability of these antibodies. Camelids, such as camels, llamas and alpacas, could produce heavy-chain only antibodies, refers to the variable domain of the heavy chain (VHH) or nanobody (Nb), with a molecular mass of approximately 15 kD, which is one kind of the
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smallest known antibodies (Muyldermans, 2013). The decreased size, improved yield and excellent stability of the camelids heavychain fragments form the basis of a new generation of antibodies for the diagnostic application. With these unique features and promising values, many studies have been devoted to their development of the immunoassays (He et al., 2014; Li et al., 2014; Ma et al., 2014). It is known that biotin and streptavidin (SA) show great binding ability (Ka ~ 1013 1015 M1), one SA could specifically bind four molecules of biotin (Culouscou et al., 1993). The SA-biotin set is one of the most widely used conjugation pairs in biotechnological applications and immunoassay (Sun et al., 2014). For immunoassays, these methods often encompass an immune capture of analytes on a solid phase followed by contacting with a labeled, second antibody and sensitive detection of the label. Taking advantage of Biotin-Steptavidin system, we exploited a novel sandwich immunoassay to detect Cry1Fa toxin based on biotechnology of Nb. Sandwich ELISA is the commonly used method for biomarkers detection. The advantage of Sandwich ELISA is that the samples do not need to be purified before analysis, especially for the samples that almost impossible to be purified, such as sera samples. In addition, the assay can be more sensitive than direct or indirect ELISA. Herein, we have obtained Cry1Fa-specific VHHs or nanobodies (Nbs) with high affinity and thermal stability for the first time. The purified anti-Cry1Fa Nbs have been modified with biotin and Horseradish Peroxidase (HRP), respectively. Therefore, a novel sandwich immune ELISA based on biotin-SA interaction was established and it showed good precise of Cry1Fa toxin detection. The immunoassay displayed a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. The immunoassay indicates VHHs derived from camel immunization are promising for sensitive and accurate detection of Cry1Fa toxins. 2. Materials and methods 2.1. Camel immunization To obtain Cry1Fa-specific binding heavy chain-only antibodies (VHHs), a healthy bactrian camel was injected with high purity Cry1Fa (1 mg) mixed with Freund's complete adjuvant (Sigma) for the first time, and Freund's incomplete adjuvant (Sigma) for the following 5 times (Veloso et al., 2014). This camel was provided by “Joint Center for Nanobody Research & Development between SEU and Egens Bio”. After 6 times immunization, 100 mL of blood from the Cry1Fa-immunized camel was collected for experimental use. The camel was still alive and kept on the farm, all camel experiments were performed according to guidelines approved by Southeast University. The peripheral blood lymphocytes (PBLs) were isolated (GE healthcare) and total RNA was purified for library generation. 2.2. Generation of phagemid library The variable heavy repertoires of the heavy chain-only antibodies were amplified by 2-steps nested PCR. In the first PCR, the VH regions were amplified using the cDNA synthesized from mRNA as the template with the primers CALL001 and CALL002 (Conrath et al., 2001). The first PCR fragments have evident bands around 700 bp. From these PCR products, the VHH genes were re-amplified with the VHH-Back and VHH-For primers (Hmila et al., 2008), which contain Pst I and Not I (NEB) restriction enzymes sites, respectively. The final PCR fragments around 400 bp were gelpurified, in company with the phage-display phagemid vector
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pMECS (Vincke et al., 2012), digested with Pst I and Not I, and gelpurified again, then ligated at 16 C with T4 DNA ligase (NEB). The recombinant plasmids were electro-transformed in electrocompetent E. coli TG1 cells, then plated onto 2 yeast extract and tryptone growth medium (2 YT) supplemented with glucose (1% w/v) and ampicillin (100 mg/mL), cultured at 37 C overnight. The size of the library was measured by calculating the number of colonies after gradient dilutions. Generally, 24 colonies were chose to detect the correct insertion rate by PCR. Subsequently, the correct inserted colonies would be preserved in 2 YT with 1% glucose and 50% glycerol, and finally stored at 80 C. 2.3. Phage-displayed bio-panning The library of phage-displayed VHHs was constructed after infecting the bacterial cells with the VCSM13 helper phage (Pardon et al., 2014). The Cry1Fa antigen diluted at 100 mg/mL in 100 mM NaHCO3 was coated on one well of a sterile 96-well plate (Nunc Maxisorp) at 4 C overnight. Next, the well was washed with PBST (PBS with 0.05% Tween-20), then 0.1% casein was added to block residual protein binding sites. Next the antigen well and control well without Cry1Fa were incubated 100 mL phage particles in PBS at room temperature for 1 h. The phage particles eluted from immobilized antigen after each round of bio-panning are subsequently transferred to infect E. coli TG1 cells to further amplify phagemids for the next round of bio-panning (Pardon et al., 2014). Through three consecutive rounds of bio-panning, Cry1Fa specific VHHs expressed on phages' coat proteins were enriched enough for the following screening. 2.4. Periplasmic extract ELISA screening From the bio-panning plates, 95 individual colonies were randomly patched, cultured in Terrific Broth (TB) medium, and the Cry1Fa-specific Nbs were overexpressed with 1 mM isopropyl b-D1-thiogalactopyranoside (IPTG) overnight at 28 C. Cells were collected and the TES buffer (0.5 M sucrose, 0.2 M TriseHCl, pH8.0 and 0.0005 M EDTA) was used to burst the cells and release proteins. A 96-well plate was coated with 2 mg/mL Cry1Fa in 100 mM NaHCO3 (100 mL/well) at 4 C in advance. After washing with PBST, 1% BSA in PBS (200 mL/well) was used to block non-specific binding sites at 25 C for 2 h. Bacterial extracts including soluble Nbs were transferred into coated wells (100 mL/well) and incubated for 1 h. Afterwards, the mouse anti-HA tag antibody (Covance) and antimouse IgG-alkaline phosphatase (Sigma) were added one after another for 1 h, respectively. Finally, the plate was read using an ELISA reader (Bio-rad imark™) at OD405 nm. 2.5. Generation and purification of VHHs Individual Cry1Fa-targeting VHHs were selected on positive clones in PE-ELISA followed by sequence analysis. Nbs were classified into different families derived from great different amino acids sequences especially in CDR3 hypervariable areas. The plasmids of the different families were extracted and electrotransformed into electrocompetent E. coli WK6 cells, which could overexpress Nbs by culturing in TB medium and inducing with 1 mM IPTG. Cells were subjected to the osmosis of TES, and burst to release periplasmic Nbs. Cry1Fa Nbs were produced as C-terminal hexahistidine-tag (His6) proteins, purified via affinity binding with NI-NTA Superflow Sepharose columns (Qiagen). After washing with PBS, the binders were eluted with 500 mM imidazole. Ultimately, the Cry1Fa specific Nbs were ultra-filtrated by Ultra-filtration column (Millipore) to remove imidazole and the final Nbs were concentrated in PBS (pH7.4).
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2.6. Cross-reactivity assay The specificity of Cry1Fa Nbs was characterized via indirect ELISA. As the Cry1Fa antigen contains a His-tag, 100 mL different toxins Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1Fa and His-tag (2 mg/mL) in 100 mM NaHCO3 were coated on 96-well plate (Nunc Maxisorp), and a blank control was set. After washing and blocking, 100 mL purified Cry1Fa Nbs at the concentration of 2 mg/mL were incubated to detect the specificity. All analysis was repeated 3 times, the signal was read at OD405 nm. 2.7. Thermostability analysis Nb 55, Nb 76 and Nb 84 were diluted to 1 mg/mL in PBS, and placed at 37 C and 70 C for 0, 0.5, 1, 2 and 3 h, respectively. Afterwards, the thermal-treated and untreated Nbs were added into antigen-coated wells for incubating. The rest was performed as indirect ELISA described above. 2.8. Conjugated Nbs with HRP To determine whether the screened Nbs can recognize different antigen epitopes, we used Horseradish Peroxidase (HRP) (Sigmaaldrich) to conjugate the purified Nbs firstly. 1 mg HRP was dissolved in 200 mL ddH2O, and 100 mL freshly prepared 0.1 mol/L NaIO4 was mixed in, standing for 30 min at 4 C, then 200 mL 2.5% glycol was added, and the mixture was placed at room temperature. After 1 h, 1 mg of Nb in PBS was added in and 1 M CB solution (carbonate and bicarbonate solution, pH 9.5) to adjust pH to 9.0, and then was incubated at 4 C overnight. The next day, 20 mL of NaBH4 (5 mg/mL) was added into the mixed solution, placed at 4 C for 3 h. After the above steps are completed, the mixture was transferred to an ultrafiltration tube to replace the buffer with PBS. 2.9. Epitope mapping ELISA plate (Nunc Maxisorp) was coated with 10 mg/mL Nbs (Nb 55, Nb76, Nb84) at 4 C overnight, these three Nbs were coated for three rows and three columns, each Nb was in 3 antigen wells and 3 control wells. After washing and blocking, Cry1Fa (2 mg/mL) in PBS and PBS without Cry1Fa was added to the antigen wells and the control wells, respectively. After 1 h, three different HRP-Nbs (2 mg/ mL) were added to each Nbs coated wells. After extensive washing, the peroxidase reaction was developed by adding 100 mL of tetramethylbenzidine (TMB) substrate solution and incubated for 5 min at room temperature. The enzyme reaction was stopped by the addition of 2 M H2SO4, and the absorbance was read at 450 nm. 2.10. Biotinylation of Nb The VHH genes were subcloned into the vector pBAD17 after digested by Nco I and BstE II restricted enzymes, then cotransformed with pBirA plasmid into E. coli WK6 cells. The cells were cultured in TB medium and induced with 1 mM IPTG to express proteins followed by adding 50 mM D-biotin (Bio Basic Inc). Biotinylated Nbs catalyzed by BirA ligase were purified by Streptavidin Mutein Matrix (Roche) with 6 mM D-biotin elution buffer. The residual D-biotin was removed out by ultra-filtrating with 0.01 M PBS for several times. 2.11. Establishment the immunoassay of the Nbs based on biotin-SA interaction SA were directional coated with high density and even arranged on wells of BeaverNano™ Streptavidin Matrix Coated 96-Well Plate,
the coated density is up to 9.3 1013 molecules/cm2. 100 mL of biotinylated Nb 84 (BiNb84, 1 mg/mL) in PBST were coated on a SA coated plate (BeaverNano™) at room temperature for 1 h. After washing and blocking with 5% BSA, the wells were incubated with Cry1Fa with serial dilutions ranged from 1 to 3000 ng/mL for 2 h. Finally, 100 mL of HRP-Nb76 (1 mg/mL) in 5% BSA was added for antigen binding. The rest of the experiment was performed as described above in epitope mapping. 2.12. Assessed by spiked crop samples Cry1Fa toxins in non-transgenic corn and soybean samples were determined by VHH-based ELISA. One gram of the dried and homogenized corn or soybean powder samples were spiked with Cry1Fa toxin at seven concentration levels (0.05, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.00 mg/kg) and then 10 mL of the protein extraction solution (0.1 M PBS containing 0.1% BSA and 0.05% Tween-20) was added. After incubating with gentle shaking at room temperature for 2 h, the suspensions were centrifuged at 10,000 g for 10 min. Then the supernatant was used for sample analysis directly by performing ELISA that described above, each spiked sample was analyzed with three replicates. 3. Results 3.1. VHH library construction To generate VHHs specific binding Cry1Fa, we immunized a healthy Bactrian camel with highly pure and immunogenic Cry1Fa (Fig. 2A). The VHH genes were amplified from the lymphocyte cDNA, and the first PCR fragments have evident bands around 700 bp (Fig. 1B). The 700 bp fragments were gel purified and used as the template in a nested PCR to re-amplify the VHH-only fragments (Fig. 1B). Transforming the ligation of the VHH genes and the pMECS vector into E. coli TG1 cells, and the cells were plated on medium to generate the VHH library. Library titer and correct insertion rate can indicate the quality of the VHH library. The titer of the Cry1Fa-specific VHHs library is 2 109 cfu/mL, which was enough to meet high specificity and sequence diversity. Twentyfour individual colonies were randomly selected and the result of PCR revealed a correct insertion rate of 100% (Fig. 1C). In brief, we have successfully constructed a high quality immunized VHH library belonged to Cry1Fa. 3.2. Phage-displayed bio-panning and PE ELISA screening Phage-displayed Cry1Fa-specific VHHs were enriched after a consecutive of bio-panning. After each round of bio-panning, phage particles were eluted from the well of Cry1Fa, and co-infected TG1 cells together with about 2 1011 helper phages. The infected cells were linear coated on the ampicillin resistant plate. We counted the clones and reckoned the relative enrichment times. The bar graph in Fig. 2B revealed the enrichment folds increased to 20-fold after 3 rounds of panning, while it is possible to select Nbs with diverse sequences. 95 individual colonies were randomly patched to perform PE-ELISA assay. Ten positive clones of anti-Cry1Fa VHH were sequenced (Fig. 2C) and they were classified into 3 families according to the distinct amino acid sequences in CDR3 region. Nb 55, Nb 76 and Nb 84 were chosen as the represents of 3 classes of Cry1Fa Nbs. As shown in Fig. 3, amino acids with blue color stand for the same amino acids and the yellow (in web version) represents the different ones. The amino acid sequences of these three individual Nbs showed great difference in CDR3, they only share a few same amino acids with each other, and the CDR3 size of Nb 55 was longer than that of Nb 76 and Nb 84.
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Fig. 1. Construction of phage-displayed library. (A) SDS-PAGE of the immunogen Cry1Fa toxin reveals molecular mass around 65 kD and high purity. (B) Scheme of VHH genes generation. (C) The first PCR fragments had evident bands around 700 bp (left) and the VHH genes were re-amplified by second PCR (right). (D) The correct insertion rate detected by PCR of 24 individual clones was nearly 100%.
Fig. 2. Cry1Fa-specific Nbs selection. (A) Through three consecutive cycles of bio-panning, the enrichment factor reached to 20. (B) Ten positive clones selected from PE-ELISA.
Fig. 3. Alignment of amino acid sequence of Cry1Fa specific binding Nbs. Nbs were classified into three families Nb55, Nb76 and Nb84 based on great diversity in CDR3. In CDR areas, blue stands for the same amino acids and yellow represents the different ones. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.3. Expression and purification of Cry1Fa specific VHHs According to the structure of the recombined vector, Nbs contain an HA-tag and a His6-tag at C-terminal, thus they could be purified through NI-NTA affinity chromatography. After ultrafiltration, the bands of Nbs were revealed by 15% SDS-PAGE around 16 kD, and the purity was greater than 90% (Fig. 4A), though they appeared not
coincide with the mass calculated. The yields of soluble Nbs were all above 10 mg/L, and other characters are showed in Table 1. 3.4. Cross-reactivity assay To ensure the purified Nbs were Cry1Fa specific, equal quantity of different toxins and His-tag was coated for specificity validation.
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Fig. 4. Specificity and thermostability of purified Nbs. (A) Purified soluble Nbs were analyzed by SDS-PAGE nearly 15 kD. (B) Cross-reactivity analysis. Analysis based on ELISA revealed Nb55, Nb76 and Nb84 were specific against Cry1Fa, no other cross-reactivity could be detected. (C) Thermostability assay. Nbs were treated at 37 C and 70 C for 0.5, 1, 2 and 3 h, then the activities tested by ELISA were approximately 100% compared to the untreated Nbs.
ELISA experiment demonstrated great specificity of three Nbs (Nb 55, Nb 76 and Nb 84) to Cry1Fa, no cross-reactivity could be detected with other toxins as well as His-tag (Fig. 4B).
Nb84 can bind to different epitopes on the Cry1Fa, as well as Nb76 and Nb84, but Nb55 and Nb76 appeared to recognize the same epitope. According to results of epitope mapping and specificity, we chose Nb76 and Nb84 to establish the immunoassay.
3.5. Thermostability analysis 3.7. Biotinylation of Nbs The endurance of Nbs to temperature is a key point to their functions. Nb 55, Nb 76 and Nb 84 were diluted to 1 mg/mL in PBS, and placed at 37 C and 70 C for 0, 0.5, 1, 2 and 3 h. Their reactivity with Cry1Fa was analyzed by ELISA. Compared to untreated Nbs, data showed that Nb 55, Nb 76 and Nb 84 could keep nearly 100% of activity after incubated at 37 C and 70 C for different times (Fig. 4C). 3.6. Epitope mapping A coupled enzymatic ELISA requires a first Nb to capture antigen and a second Nb to target its antigen conjugated of HRP for detection. Firstly, we coated enough first Nb (10 mg/mL) on the ELISA plate to capture the Cry1Fa antigen as much as possible. Then the second Nb was added to combine antigen and detection. If the two Nbs recognize two different epitopes, the second Nb could successfully bind to the Cry1Fa on anther epitope, and then the coupled HRP would catalyze the color reaction. When they combine the same epitope of the antigen, no signal or very small signal would be detected. Our results here show that Nb55 and Table 1 Properties of Cryl Fa toxin-specific nanobodies. Nbs
Molecular mass (kD)a
Isoelectric pointa,b
Yield (mg/L)
Nb55 Nb76 Nb84
16.39 15.68 15.63
5.59 6.38 6.02
12.5 11.0 10.5
For establishing a simple, rapid and sensitive immune detection using of Biotin-SA interaction, a biotin domain was added to the Ctermini of Nb. We took the advantage of plasmid pBAD17 that it includes a biotin acceptor domain cloned downstream of the Nb84 sequences. The recombinant plasmid was co-transformed into WK6 cells with the pBirA plasmid encoding biotin protein ligase that catalyzes the ligation between biotin and biotin acceptor domain on pBAD17. By which, the Nb84 realized biotinylation, named BiNb84. The BiNb84 was purified with Streptavidin Mutein Matrix for further study. 3.8. Immunoassay of the Nbs based on biotin-SA interaction On the basis of conventional ELISA, a novel detection based on double Nbs sandwich was established by advantage of the biotinSA interaction, which can directionally detect and amplify signal highly. 0.1 mg/well BiNb84 was coated, antigens were incubated with serial dilutions and 0.1 mg/well HRP-Nb76 were used to develop signals. The results displayed that the signal was stable when the concentrate of antigen was up to 2000 ng/mL (Fig. 5B), the linear range of the direct detection was 1e100 ng/mL with a acceptable correlation coefficient of 0.9898, and the limit of detection was 0.88 ng/mL for Cry1Fa (Fig. 5C). 3.9. Assessed by spriked crop samples
a
Molecular mass includes HA and His6tags. Theoretical isoelectric point and Molecular mass were calculated by the ExPASy ProtParam Tool. b
The recoveries of Cry1Fa from the spiked non-transgenic corn and soybean samples are showed in Table 2. The recoveries ranged
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Fig. 5. Detection of Cry1Fa by sandwich immunoassay. (A) Schematic program of sandwich immunoassay based on biotin-SA interaction. (B) The results displayed that the signal was stable when the concentrate of antigen was up to 2000 ng/mL. (C) Assay standard curve of Cry1Fa toxin. The linear range of detection was 1e100 ng/mL. The linear equation was calculated as Y ¼ 0.0072 X þ 0.1056 with an acceptable correlation coefficient of 0.9898 (R2).
from 83.33% to 117.17%, with C.V less than 6.0%, suggesting excellent accuracy of for the quantitative detection based on biotin-SA ELISA of Cry1Fa toxin in agricultural samples. 4. Discussion In this report, we demonstrated the generation and characteristics of anti-Cry1Fa VHHs. Anti-Cry1Fa Nbs showed great specificity, high yield and thermal stability, which made Nbs attractive for wide utilization for detecting GM crops and environment residuals. The panning was stopped at the third round of selection, though the enrichment factor reached only 20, it is enough to screen several diversity Nbs and reduce the duplicates. After sequencing, we classified these Nbs into three families according to great diversity at the area of CDR3, and Nb55, Nb76 and Nb84 were chosen as the represents. Soluble VHHs were purified with a yield above 10 mg/L, ignoring the loss during ultrafiltration. By molecular biological technique, Nbs were coupled with biotin and HRP. However, we are not clear whether the interaction between the modified Nbs and Cry1Fa would be minute adjusted. Table 2 Recoveries of Cry1Fa toxin from spiked samples by nanobody-based ELISA. Sample
Spiked Cry1Fa ng/mL
Mean ± SD
Corn
5.00 10.00 20.00 40.00 60.00 80.00 100.00 5.00 10.00 20.00 40.00 60.00 80.00 100.00
4.50 12.83 19.78 39.29 65.40 93.74 102.56 4.17 9.27 19.13 36.49 57.14 82.37 94.64
Soybean
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.12 0.46 0.79 0.79 1.20 3.65 0.17 0.10 0.24 1.12 1.51 1.53 2.46 5.12
Recovery (%)
C.V (%)
90.04 111.49 98.89 98.23 109.00 117.17 102.56 83.33 92.69 95.65 91.23 95.23 102.96 94.64
2.77 3.57 3.99 2.00 1.84 3.90 0.17 2.40 2.62 5.85 4.15 2.68 2.98 5.41
Depend on strong affinity between SA and biotin molecule, we designed a novel sandwich immunoassay to directly detect Cry1Fa. The results showed that the linear range of the detection is 1e100 ng/mL and with a limit of detection of 0.88 ng/mL. By the use of SA-biotin set, biotinylated Nbs (BiNbs) were strong stuck on the plate, but the SA coated on plate was finite, so the detection would not be promoted by increasing addition of BiNbs. If the step can be smartly improved, this type of detection would be more sensitive. As to Cry1Fa, it is the first time to obtain VHHs through camel immunization, which was successfully applied in detection of GM crops in this study. These analyses indicate that the proposed immunoassay based on Cry1Fa-specific VHHs will be a promising tool for extensive research and practical applications. Ethical statement On behalf of, and having obtained permission from all the authors, I declare that: (a) the paper is not currently being considered for publication elsewhere; (b) all authors have been personally and actively involved in substantive work leading to the report, and will hold themselves jointly and individually responsible for its content; (c) all relevant ethical safeguards have been met in relation to animal experimentation. Acknowledgments This work was supported by Jiangsu Nanobody Engineering and Research Center of China (2014-02), Program for New Century Excellent Talents in University (NCET-20130127), Key topics for State Key Laboratory of Materials-Oriented Chemical Engineering (ZK20134), National Natural Science Foundation (grant number 31471692), the Key Technology R & D Program of Jiangsu ProvinceSocial Development (No.BE2014722), National Natural Science Foundation of China (Grant 31271365 and 31471216) and Jiangsu Province Natural Science Foundation (BK2011599). The authors would like to thank Prof. Serge Muyldermans's help for providing
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TG1 cells, WK6 cells, pMECS phagemid and pBAD plasmid. The authors declare that they have no conflict of interest.
Conflict of interest We declared that we have no conflict of interest.
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Li, H., Sun, Y., Elseviers, J., Muyldermans, S., Liu, S., Wan, Y., 2014. A nanobody-based electrochemiluminescent immunosensor for sensitive detection of human procalcitonin. Analyst 139, 3718e3721. Ma, L., Sun, Y., Kang, X., Wan, Y., 2014. Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin. Biosens. Bioelectron. 61, 165e171. Muyldermans, S., 2013. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775e797. Pardon, E., Laeremans, T., Triest, S., Rasmussen, S.G., Wohlkonig, A., Ruf, A., Muyldermans, S., Hol, W.G., Kobilka, B.K., Steyaert, J., 2014. A general protocol for the generation of nanobodies for structural biology. Nat. Protoc. 9, 674e693. Randhawa, G.J., Singh, M., 2012. Multiplex, construct-specific, and real-time PCRbased analytical methods for Bt rice with cry1Ac gene. J. AOAC Int. 95, 186e194. Romeis, J., Meissle, M., Bigler, F., 2006. Transgenic crops expressing bacillus thuringiensis toxins and biological control. Nat. Biotechnol. 24, 63e71. Shelton, A.M., Zhao, J.Z., Roush, R.T., 2002. Economic, ecological, food safety, and social consequences of the deployment of bt transgenic plants. Annu. Rev. Entomol. 47, 845e881. Sun, Q., Qian, J., Tian, H., Duan, L., Zhang, W., 2014. Rational design of biotinylated probes: fluorescent turn-on detection of (strept)avidin and bioimaging in cancer cells. Chem. Commun. 50, 8518e8521. Tian, J.C., Wang, X.P., Long, L.P., Romeis, J., Naranjo, S.E., Hellmich, R.L., Wang, P., Earle, E.D., Shelton, A.M., 2013. Bt crops producing Cry1Ac, Cry2Ab and Cry1Fa do not harm the green lacewing, Chrysoperla rufilabris. PloS One 8, e60125. Veloso, A.J., Chow, A.M., Ganesh, H.V., Li, N., Dhar, D., Wu, D.C., Mikhaylichenko, S., Brown, I.R., Kerman, K., 2014. Electrochemical immunosensors for effective evaluation of amyloid-beta modulators on oligomeric and fibrillar aggregation processes. Anal. Chem. 86, 4901e4909. Vincke, C., Gutierrez, C., Wernery, U., Devoogdt, N., Hassanzadeh-Ghassabeh, G., Muyldermans, S., 2012. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol. Biol. 907, 145e176. Zhang, X., Liu, Y., Zhang, C., Wang, Y., Xu, C., Liu, X., 2012. Rapid isolation of singlechain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin. Ecotoxicol. Environ. Saf. 81, 84e90.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin Pingyan Wang a, Guanghui Li a, Junrong Yan a, Yonghong Hu c, Cunzheng Zhang d, Xianjin Liu d, Yakun Wan a, b, * a
The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, PR China Jiangsu Nanobody Engineering and Research Center, Nantong 226010, PR China c College of 543Biotechnology and Pharmaceutical Engineering, Nanjing University of 544Technology, Nanjing 210009, PR China d Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2014 Received in revised form 21 October 2014 Accepted 29 October 2014 Available online 30 October 2014
The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Nanobody Phage-displayed library Cry1Fa Biotin Streptavidin Sandwich immune ELISA
1. Introduction Genetically modified (GM) crops producing insect-resistant Cry proteins from the bacterium, Bacillus thuringiensis (Bt), are revolutionizing agriculture. Genes of Bt toxins have been engineered into major crops, generally these crops have shown considerable economic benefits, and decreased the use of other insecticides (Shelton et al., 2002). A dozen of Cry toxins are commercially applied in agriculture, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D, Cry1E, Cry1Fa, Cry3Aa, Cry3Bb and Cry34/Cry35 (Bravo and Soberon, 2008). Among these Cry toxins, Cry1Fa is constructive to protect crops from the Lepidoptera insects by killing their larvae, which is especially sensitive to Osrinia mubilais Hubner and O. furnacalis Güenee (Tian et al., 2013). However, A main concern with the widespread of Bt crops is their potential to adverse
* Corresponding author. Institute of Life Sciences, Southeast University, Sipailou No.2, Nanjing 210096, PR China. E-mail address: [email protected] (Y. Wan). http://dx.doi.org/10.1016/j.toxicon.2014.10.024 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
affects on food safety, human health and non-target organisms including biological control organisms (Romeis et al., 2006). It is important to examine the different potential impacts of Bt crops. The concerns currently being asked of GM crops frequently demand a sensitive and accurate detection method (Dale et al., 2002). In the past decades, numerous detections have been developed for Bt toxins. DNA/proteins of transgenic crops could be conducted by polymerase chain reaction (PCR) and real-time PCR (Asensio et al., 2008). However, they can't determine the expression level of Cry toxins and usually require sophisticated instruments (Randhawa and Singh, 2012). With the development of bioengineering and biotechnology, antibodies against Cry toxins such as single-chain antibody (ScFv), have been developed and used in ELISA and biosensors (Zhang et al., 2012). However, the technique of ScFv proved to be troublesome because of the low yield and instability of these antibodies. Camelids, such as camels, llamas and alpacas, could produce heavy-chain only antibodies, refers to the variable domain of the heavy chain (VHH) or nanobody (Nb), with a molecular mass of approximately 15 kD, which is one kind of the
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smallest known antibodies (Muyldermans, 2013). The decreased size, improved yield and excellent stability of the camelids heavychain fragments form the basis of a new generation of antibodies for the diagnostic application. With these unique features and promising values, many studies have been devoted to their development of the immunoassays (He et al., 2014; Li et al., 2014; Ma et al., 2014). It is known that biotin and streptavidin (SA) show great binding ability (Ka ~ 1013 1015 M1), one SA could specifically bind four molecules of biotin (Culouscou et al., 1993). The SA-biotin set is one of the most widely used conjugation pairs in biotechnological applications and immunoassay (Sun et al., 2014). For immunoassays, these methods often encompass an immune capture of analytes on a solid phase followed by contacting with a labeled, second antibody and sensitive detection of the label. Taking advantage of Biotin-Steptavidin system, we exploited a novel sandwich immunoassay to detect Cry1Fa toxin based on biotechnology of Nb. Sandwich ELISA is the commonly used method for biomarkers detection. The advantage of Sandwich ELISA is that the samples do not need to be purified before analysis, especially for the samples that almost impossible to be purified, such as sera samples. In addition, the assay can be more sensitive than direct or indirect ELISA. Herein, we have obtained Cry1Fa-specific VHHs or nanobodies (Nbs) with high affinity and thermal stability for the first time. The purified anti-Cry1Fa Nbs have been modified with biotin and Horseradish Peroxidase (HRP), respectively. Therefore, a novel sandwich immune ELISA based on biotin-SA interaction was established and it showed good precise of Cry1Fa toxin detection. The immunoassay displayed a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. The immunoassay indicates VHHs derived from camel immunization are promising for sensitive and accurate detection of Cry1Fa toxins. 2. Materials and methods 2.1. Camel immunization To obtain Cry1Fa-specific binding heavy chain-only antibodies (VHHs), a healthy bactrian camel was injected with high purity Cry1Fa (1 mg) mixed with Freund's complete adjuvant (Sigma) for the first time, and Freund's incomplete adjuvant (Sigma) for the following 5 times (Veloso et al., 2014). This camel was provided by “Joint Center for Nanobody Research & Development between SEU and Egens Bio”. After 6 times immunization, 100 mL of blood from the Cry1Fa-immunized camel was collected for experimental use. The camel was still alive and kept on the farm, all camel experiments were performed according to guidelines approved by Southeast University. The peripheral blood lymphocytes (PBLs) were isolated (GE healthcare) and total RNA was purified for library generation. 2.2. Generation of phagemid library The variable heavy repertoires of the heavy chain-only antibodies were amplified by 2-steps nested PCR. In the first PCR, the VH regions were amplified using the cDNA synthesized from mRNA as the template with the primers CALL001 and CALL002 (Conrath et al., 2001). The first PCR fragments have evident bands around 700 bp. From these PCR products, the VHH genes were re-amplified with the VHH-Back and VHH-For primers (Hmila et al., 2008), which contain Pst I and Not I (NEB) restriction enzymes sites, respectively. The final PCR fragments around 400 bp were gelpurified, in company with the phage-display phagemid vector
187
pMECS (Vincke et al., 2012), digested with Pst I and Not I, and gelpurified again, then ligated at 16 C with T4 DNA ligase (NEB). The recombinant plasmids were electro-transformed in electrocompetent E. coli TG1 cells, then plated onto 2 yeast extract and tryptone growth medium (2 YT) supplemented with glucose (1% w/v) and ampicillin (100 mg/mL), cultured at 37 C overnight. The size of the library was measured by calculating the number of colonies after gradient dilutions. Generally, 24 colonies were chose to detect the correct insertion rate by PCR. Subsequently, the correct inserted colonies would be preserved in 2 YT with 1% glucose and 50% glycerol, and finally stored at 80 C. 2.3. Phage-displayed bio-panning The library of phage-displayed VHHs was constructed after infecting the bacterial cells with the VCSM13 helper phage (Pardon et al., 2014). The Cry1Fa antigen diluted at 100 mg/mL in 100 mM NaHCO3 was coated on one well of a sterile 96-well plate (Nunc Maxisorp) at 4 C overnight. Next, the well was washed with PBST (PBS with 0.05% Tween-20), then 0.1% casein was added to block residual protein binding sites. Next the antigen well and control well without Cry1Fa were incubated 100 mL phage particles in PBS at room temperature for 1 h. The phage particles eluted from immobilized antigen after each round of bio-panning are subsequently transferred to infect E. coli TG1 cells to further amplify phagemids for the next round of bio-panning (Pardon et al., 2014). Through three consecutive rounds of bio-panning, Cry1Fa specific VHHs expressed on phages' coat proteins were enriched enough for the following screening. 2.4. Periplasmic extract ELISA screening From the bio-panning plates, 95 individual colonies were randomly patched, cultured in Terrific Broth (TB) medium, and the Cry1Fa-specific Nbs were overexpressed with 1 mM isopropyl b-D1-thiogalactopyranoside (IPTG) overnight at 28 C. Cells were collected and the TES buffer (0.5 M sucrose, 0.2 M TriseHCl, pH8.0 and 0.0005 M EDTA) was used to burst the cells and release proteins. A 96-well plate was coated with 2 mg/mL Cry1Fa in 100 mM NaHCO3 (100 mL/well) at 4 C in advance. After washing with PBST, 1% BSA in PBS (200 mL/well) was used to block non-specific binding sites at 25 C for 2 h. Bacterial extracts including soluble Nbs were transferred into coated wells (100 mL/well) and incubated for 1 h. Afterwards, the mouse anti-HA tag antibody (Covance) and antimouse IgG-alkaline phosphatase (Sigma) were added one after another for 1 h, respectively. Finally, the plate was read using an ELISA reader (Bio-rad imark™) at OD405 nm. 2.5. Generation and purification of VHHs Individual Cry1Fa-targeting VHHs were selected on positive clones in PE-ELISA followed by sequence analysis. Nbs were classified into different families derived from great different amino acids sequences especially in CDR3 hypervariable areas. The plasmids of the different families were extracted and electrotransformed into electrocompetent E. coli WK6 cells, which could overexpress Nbs by culturing in TB medium and inducing with 1 mM IPTG. Cells were subjected to the osmosis of TES, and burst to release periplasmic Nbs. Cry1Fa Nbs were produced as C-terminal hexahistidine-tag (His6) proteins, purified via affinity binding with NI-NTA Superflow Sepharose columns (Qiagen). After washing with PBS, the binders were eluted with 500 mM imidazole. Ultimately, the Cry1Fa specific Nbs were ultra-filtrated by Ultra-filtration column (Millipore) to remove imidazole and the final Nbs were concentrated in PBS (pH7.4).
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2.6. Cross-reactivity assay The specificity of Cry1Fa Nbs was characterized via indirect ELISA. As the Cry1Fa antigen contains a His-tag, 100 mL different toxins Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1Fa and His-tag (2 mg/mL) in 100 mM NaHCO3 were coated on 96-well plate (Nunc Maxisorp), and a blank control was set. After washing and blocking, 100 mL purified Cry1Fa Nbs at the concentration of 2 mg/mL were incubated to detect the specificity. All analysis was repeated 3 times, the signal was read at OD405 nm. 2.7. Thermostability analysis Nb 55, Nb 76 and Nb 84 were diluted to 1 mg/mL in PBS, and placed at 37 C and 70 C for 0, 0.5, 1, 2 and 3 h, respectively. Afterwards, the thermal-treated and untreated Nbs were added into antigen-coated wells for incubating. The rest was performed as indirect ELISA described above. 2.8. Conjugated Nbs with HRP To determine whether the screened Nbs can recognize different antigen epitopes, we used Horseradish Peroxidase (HRP) (Sigmaaldrich) to conjugate the purified Nbs firstly. 1 mg HRP was dissolved in 200 mL ddH2O, and 100 mL freshly prepared 0.1 mol/L NaIO4 was mixed in, standing for 30 min at 4 C, then 200 mL 2.5% glycol was added, and the mixture was placed at room temperature. After 1 h, 1 mg of Nb in PBS was added in and 1 M CB solution (carbonate and bicarbonate solution, pH 9.5) to adjust pH to 9.0, and then was incubated at 4 C overnight. The next day, 20 mL of NaBH4 (5 mg/mL) was added into the mixed solution, placed at 4 C for 3 h. After the above steps are completed, the mixture was transferred to an ultrafiltration tube to replace the buffer with PBS. 2.9. Epitope mapping ELISA plate (Nunc Maxisorp) was coated with 10 mg/mL Nbs (Nb 55, Nb76, Nb84) at 4 C overnight, these three Nbs were coated for three rows and three columns, each Nb was in 3 antigen wells and 3 control wells. After washing and blocking, Cry1Fa (2 mg/mL) in PBS and PBS without Cry1Fa was added to the antigen wells and the control wells, respectively. After 1 h, three different HRP-Nbs (2 mg/ mL) were added to each Nbs coated wells. After extensive washing, the peroxidase reaction was developed by adding 100 mL of tetramethylbenzidine (TMB) substrate solution and incubated for 5 min at room temperature. The enzyme reaction was stopped by the addition of 2 M H2SO4, and the absorbance was read at 450 nm. 2.10. Biotinylation of Nb The VHH genes were subcloned into the vector pBAD17 after digested by Nco I and BstE II restricted enzymes, then cotransformed with pBirA plasmid into E. coli WK6 cells. The cells were cultured in TB medium and induced with 1 mM IPTG to express proteins followed by adding 50 mM D-biotin (Bio Basic Inc). Biotinylated Nbs catalyzed by BirA ligase were purified by Streptavidin Mutein Matrix (Roche) with 6 mM D-biotin elution buffer. The residual D-biotin was removed out by ultra-filtrating with 0.01 M PBS for several times. 2.11. Establishment the immunoassay of the Nbs based on biotin-SA interaction SA were directional coated with high density and even arranged on wells of BeaverNano™ Streptavidin Matrix Coated 96-Well Plate,
the coated density is up to 9.3 1013 molecules/cm2. 100 mL of biotinylated Nb 84 (BiNb84, 1 mg/mL) in PBST were coated on a SA coated plate (BeaverNano™) at room temperature for 1 h. After washing and blocking with 5% BSA, the wells were incubated with Cry1Fa with serial dilutions ranged from 1 to 3000 ng/mL for 2 h. Finally, 100 mL of HRP-Nb76 (1 mg/mL) in 5% BSA was added for antigen binding. The rest of the experiment was performed as described above in epitope mapping. 2.12. Assessed by spiked crop samples Cry1Fa toxins in non-transgenic corn and soybean samples were determined by VHH-based ELISA. One gram of the dried and homogenized corn or soybean powder samples were spiked with Cry1Fa toxin at seven concentration levels (0.05, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.00 mg/kg) and then 10 mL of the protein extraction solution (0.1 M PBS containing 0.1% BSA and 0.05% Tween-20) was added. After incubating with gentle shaking at room temperature for 2 h, the suspensions were centrifuged at 10,000 g for 10 min. Then the supernatant was used for sample analysis directly by performing ELISA that described above, each spiked sample was analyzed with three replicates. 3. Results 3.1. VHH library construction To generate VHHs specific binding Cry1Fa, we immunized a healthy Bactrian camel with highly pure and immunogenic Cry1Fa (Fig. 2A). The VHH genes were amplified from the lymphocyte cDNA, and the first PCR fragments have evident bands around 700 bp (Fig. 1B). The 700 bp fragments were gel purified and used as the template in a nested PCR to re-amplify the VHH-only fragments (Fig. 1B). Transforming the ligation of the VHH genes and the pMECS vector into E. coli TG1 cells, and the cells were plated on medium to generate the VHH library. Library titer and correct insertion rate can indicate the quality of the VHH library. The titer of the Cry1Fa-specific VHHs library is 2 109 cfu/mL, which was enough to meet high specificity and sequence diversity. Twentyfour individual colonies were randomly selected and the result of PCR revealed a correct insertion rate of 100% (Fig. 1C). In brief, we have successfully constructed a high quality immunized VHH library belonged to Cry1Fa. 3.2. Phage-displayed bio-panning and PE ELISA screening Phage-displayed Cry1Fa-specific VHHs were enriched after a consecutive of bio-panning. After each round of bio-panning, phage particles were eluted from the well of Cry1Fa, and co-infected TG1 cells together with about 2 1011 helper phages. The infected cells were linear coated on the ampicillin resistant plate. We counted the clones and reckoned the relative enrichment times. The bar graph in Fig. 2B revealed the enrichment folds increased to 20-fold after 3 rounds of panning, while it is possible to select Nbs with diverse sequences. 95 individual colonies were randomly patched to perform PE-ELISA assay. Ten positive clones of anti-Cry1Fa VHH were sequenced (Fig. 2C) and they were classified into 3 families according to the distinct amino acid sequences in CDR3 region. Nb 55, Nb 76 and Nb 84 were chosen as the represents of 3 classes of Cry1Fa Nbs. As shown in Fig. 3, amino acids with blue color stand for the same amino acids and the yellow (in web version) represents the different ones. The amino acid sequences of these three individual Nbs showed great difference in CDR3, they only share a few same amino acids with each other, and the CDR3 size of Nb 55 was longer than that of Nb 76 and Nb 84.
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Fig. 1. Construction of phage-displayed library. (A) SDS-PAGE of the immunogen Cry1Fa toxin reveals molecular mass around 65 kD and high purity. (B) Scheme of VHH genes generation. (C) The first PCR fragments had evident bands around 700 bp (left) and the VHH genes were re-amplified by second PCR (right). (D) The correct insertion rate detected by PCR of 24 individual clones was nearly 100%.
Fig. 2. Cry1Fa-specific Nbs selection. (A) Through three consecutive cycles of bio-panning, the enrichment factor reached to 20. (B) Ten positive clones selected from PE-ELISA.
Fig. 3. Alignment of amino acid sequence of Cry1Fa specific binding Nbs. Nbs were classified into three families Nb55, Nb76 and Nb84 based on great diversity in CDR3. In CDR areas, blue stands for the same amino acids and yellow represents the different ones. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.3. Expression and purification of Cry1Fa specific VHHs According to the structure of the recombined vector, Nbs contain an HA-tag and a His6-tag at C-terminal, thus they could be purified through NI-NTA affinity chromatography. After ultrafiltration, the bands of Nbs were revealed by 15% SDS-PAGE around 16 kD, and the purity was greater than 90% (Fig. 4A), though they appeared not
coincide with the mass calculated. The yields of soluble Nbs were all above 10 mg/L, and other characters are showed in Table 1. 3.4. Cross-reactivity assay To ensure the purified Nbs were Cry1Fa specific, equal quantity of different toxins and His-tag was coated for specificity validation.
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Fig. 4. Specificity and thermostability of purified Nbs. (A) Purified soluble Nbs were analyzed by SDS-PAGE nearly 15 kD. (B) Cross-reactivity analysis. Analysis based on ELISA revealed Nb55, Nb76 and Nb84 were specific against Cry1Fa, no other cross-reactivity could be detected. (C) Thermostability assay. Nbs were treated at 37 C and 70 C for 0.5, 1, 2 and 3 h, then the activities tested by ELISA were approximately 100% compared to the untreated Nbs.
ELISA experiment demonstrated great specificity of three Nbs (Nb 55, Nb 76 and Nb 84) to Cry1Fa, no cross-reactivity could be detected with other toxins as well as His-tag (Fig. 4B).
Nb84 can bind to different epitopes on the Cry1Fa, as well as Nb76 and Nb84, but Nb55 and Nb76 appeared to recognize the same epitope. According to results of epitope mapping and specificity, we chose Nb76 and Nb84 to establish the immunoassay.
3.5. Thermostability analysis 3.7. Biotinylation of Nbs The endurance of Nbs to temperature is a key point to their functions. Nb 55, Nb 76 and Nb 84 were diluted to 1 mg/mL in PBS, and placed at 37 C and 70 C for 0, 0.5, 1, 2 and 3 h. Their reactivity with Cry1Fa was analyzed by ELISA. Compared to untreated Nbs, data showed that Nb 55, Nb 76 and Nb 84 could keep nearly 100% of activity after incubated at 37 C and 70 C for different times (Fig. 4C). 3.6. Epitope mapping A coupled enzymatic ELISA requires a first Nb to capture antigen and a second Nb to target its antigen conjugated of HRP for detection. Firstly, we coated enough first Nb (10 mg/mL) on the ELISA plate to capture the Cry1Fa antigen as much as possible. Then the second Nb was added to combine antigen and detection. If the two Nbs recognize two different epitopes, the second Nb could successfully bind to the Cry1Fa on anther epitope, and then the coupled HRP would catalyze the color reaction. When they combine the same epitope of the antigen, no signal or very small signal would be detected. Our results here show that Nb55 and Table 1 Properties of Cryl Fa toxin-specific nanobodies. Nbs
Molecular mass (kD)a
Isoelectric pointa,b
Yield (mg/L)
Nb55 Nb76 Nb84
16.39 15.68 15.63
5.59 6.38 6.02
12.5 11.0 10.5
For establishing a simple, rapid and sensitive immune detection using of Biotin-SA interaction, a biotin domain was added to the Ctermini of Nb. We took the advantage of plasmid pBAD17 that it includes a biotin acceptor domain cloned downstream of the Nb84 sequences. The recombinant plasmid was co-transformed into WK6 cells with the pBirA plasmid encoding biotin protein ligase that catalyzes the ligation between biotin and biotin acceptor domain on pBAD17. By which, the Nb84 realized biotinylation, named BiNb84. The BiNb84 was purified with Streptavidin Mutein Matrix for further study. 3.8. Immunoassay of the Nbs based on biotin-SA interaction On the basis of conventional ELISA, a novel detection based on double Nbs sandwich was established by advantage of the biotinSA interaction, which can directionally detect and amplify signal highly. 0.1 mg/well BiNb84 was coated, antigens were incubated with serial dilutions and 0.1 mg/well HRP-Nb76 were used to develop signals. The results displayed that the signal was stable when the concentrate of antigen was up to 2000 ng/mL (Fig. 5B), the linear range of the direct detection was 1e100 ng/mL with a acceptable correlation coefficient of 0.9898, and the limit of detection was 0.88 ng/mL for Cry1Fa (Fig. 5C). 3.9. Assessed by spriked crop samples
a
Molecular mass includes HA and His6tags. Theoretical isoelectric point and Molecular mass were calculated by the ExPASy ProtParam Tool. b
The recoveries of Cry1Fa from the spiked non-transgenic corn and soybean samples are showed in Table 2. The recoveries ranged
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Fig. 5. Detection of Cry1Fa by sandwich immunoassay. (A) Schematic program of sandwich immunoassay based on biotin-SA interaction. (B) The results displayed that the signal was stable when the concentrate of antigen was up to 2000 ng/mL. (C) Assay standard curve of Cry1Fa toxin. The linear range of detection was 1e100 ng/mL. The linear equation was calculated as Y ¼ 0.0072 X þ 0.1056 with an acceptable correlation coefficient of 0.9898 (R2).
from 83.33% to 117.17%, with C.V less than 6.0%, suggesting excellent accuracy of for the quantitative detection based on biotin-SA ELISA of Cry1Fa toxin in agricultural samples. 4. Discussion In this report, we demonstrated the generation and characteristics of anti-Cry1Fa VHHs. Anti-Cry1Fa Nbs showed great specificity, high yield and thermal stability, which made Nbs attractive for wide utilization for detecting GM crops and environment residuals. The panning was stopped at the third round of selection, though the enrichment factor reached only 20, it is enough to screen several diversity Nbs and reduce the duplicates. After sequencing, we classified these Nbs into three families according to great diversity at the area of CDR3, and Nb55, Nb76 and Nb84 were chosen as the represents. Soluble VHHs were purified with a yield above 10 mg/L, ignoring the loss during ultrafiltration. By molecular biological technique, Nbs were coupled with biotin and HRP. However, we are not clear whether the interaction between the modified Nbs and Cry1Fa would be minute adjusted. Table 2 Recoveries of Cry1Fa toxin from spiked samples by nanobody-based ELISA. Sample
Spiked Cry1Fa ng/mL
Mean ± SD
Corn
5.00 10.00 20.00 40.00 60.00 80.00 100.00 5.00 10.00 20.00 40.00 60.00 80.00 100.00
4.50 12.83 19.78 39.29 65.40 93.74 102.56 4.17 9.27 19.13 36.49 57.14 82.37 94.64
Soybean
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.12 0.46 0.79 0.79 1.20 3.65 0.17 0.10 0.24 1.12 1.51 1.53 2.46 5.12
Recovery (%)
C.V (%)
90.04 111.49 98.89 98.23 109.00 117.17 102.56 83.33 92.69 95.65 91.23 95.23 102.96 94.64
2.77 3.57 3.99 2.00 1.84 3.90 0.17 2.40 2.62 5.85 4.15 2.68 2.98 5.41
Depend on strong affinity between SA and biotin molecule, we designed a novel sandwich immunoassay to directly detect Cry1Fa. The results showed that the linear range of the detection is 1e100 ng/mL and with a limit of detection of 0.88 ng/mL. By the use of SA-biotin set, biotinylated Nbs (BiNbs) were strong stuck on the plate, but the SA coated on plate was finite, so the detection would not be promoted by increasing addition of BiNbs. If the step can be smartly improved, this type of detection would be more sensitive. As to Cry1Fa, it is the first time to obtain VHHs through camel immunization, which was successfully applied in detection of GM crops in this study. These analyses indicate that the proposed immunoassay based on Cry1Fa-specific VHHs will be a promising tool for extensive research and practical applications. Ethical statement On behalf of, and having obtained permission from all the authors, I declare that: (a) the paper is not currently being considered for publication elsewhere; (b) all authors have been personally and actively involved in substantive work leading to the report, and will hold themselves jointly and individually responsible for its content; (c) all relevant ethical safeguards have been met in relation to animal experimentation. Acknowledgments This work was supported by Jiangsu Nanobody Engineering and Research Center of China (2014-02), Program for New Century Excellent Talents in University (NCET-20130127), Key topics for State Key Laboratory of Materials-Oriented Chemical Engineering (ZK20134), National Natural Science Foundation (grant number 31471692), the Key Technology R & D Program of Jiangsu ProvinceSocial Development (No.BE2014722), National Natural Science Foundation of China (Grant 31271365 and 31471216) and Jiangsu Province Natural Science Foundation (BK2011599). The authors would like to thank Prof. Serge Muyldermans's help for providing
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TG1 cells, WK6 cells, pMECS phagemid and pBAD plasmid. The authors declare that they have no conflict of interest.
Conflict of interest We declared that we have no conflict of interest.
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