1. Introduction
Phosphinothricin acetyltransferase (PAT, Uniprot ID: Q57146) converts phosphinothricin (PPT) into a nonherbicidal acetylated form by transferring the acetyl group from acetyl-CoA to the free amino group of PPT [1,2]. PAT/bar and PAT/pat have been widely used to confer herbicide resistance in a variety of GM crops [3].
With the first transgenic corn produced in 1990 and commercialized in 1995, transgenic crops are developing rapidly [4]. Consequently, an increasing number and variety of GM crops are used for the production of food and feed, and the unintended environmental and food safety issues of GM crops have received more and more attention. The efficient and consolidated analysis of the exogenous components in GM crops is becoming more and more urgent and important. Numerous methods are available to detect foreign genes [5,6], and typically, the identification of each transformation event and quantitative detection is carried out by event-specific and real-time polymerase chain reaction (PCR) [7,8] due to its time-saving and low cost. However, quantitative detection of foreign gene coding products is equally important because it is these products that ultimately confer resistance. ELISA, which employs stable reagents and inexpensive equipment, is a sensitive and convenient method for detecting foreign proteins in GM crops and products [9,10,11,12,13]. Many existing ELISA methods and products for PAT detection are only suitable for PAT/bar. Therefore, developing a highly sensitive ELISA for PAT/pat is very necessary.
In this study, overexpressed His-PAT/pat was used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). The selected mAbs were characterized and used to develop a sandwich ELISA for the detection of PAT/pat concentrations in GM crops and products. The quantitative detection ELISA method established is conducive to promoting the development of PAT/pat containing GM crops, seed production and preservation of these GM crops, and the stable promotion of these GM crops industrialization and the sustainable application of the related GM crops.
2. Materials and Methods
2.1. Reagents, Strains, and Animals
The 100 bp Plus DNA Ladder (BM311-01), FastPfu Fly DNA Polymerase (AP231-01), and Protease Inhibitor Cocktail (DI101-01) were obtained from TransGen Biotech Co. (Beijing, China). The protein markers (26610 and P8028L) were purchased from ThermoFisher Scientific (Waltham, MA, USA) and Uelandy Inc. (Suzhou, China). PAT/bar (AA1010), CP4 EPSPS (5-enolpyruvlshimimate-3-phosphate synthase, AA0810), G2 EPSPS (AA120), and G10 EPSPS (AA110) proteins were purchased from YouLong Biotech Co. (Shanghai, China). E. coli Trans10 and BL21(DE3) chemically competent cells were purchased from TransGen Biotech. (Beijing, China). Murine myeloma cells SP2/0 were obtained from Genecreate Biological Engineering Co. (Wuhan, China). BABL/c mice were obtained from SBF company (Beijing, China). Nitrocellulose (NC) membranes were purchased from Bio-RAD.
2.2. His-PAT/Pat Protein Expression
The pat gene was cloned from the genomic DNA of GM maize C0010.3.1 and subcloned into the pET-28a. The pET28a-pat reconstituted plasmid was transformed into E. coli BL21(DE3) for His-PAT/pat protein overexpression. The His-PAT/pat protein was pulled down with Ni-Resin (GE) and further purified by a Superdex75 column (GE) with 25 mM Tris-HCl, pH 7.6, 250 mM NaCl, and 5% glycerol, and dialyzed with PBS before immunization.
2.3. Mice Immunization Protocols
The immunization protocol followed conventional subcutaneous (s.c.) injection with slight modifications [14,15]. A total of 6 male BALB/c mice, 8-weeks-old and weighing about 20 g, were immunized with His-PAT/pat dissolved in 250 μL of sterile PBS adjuvant with complete or incomplete Freund’s adjuvant at the nape of the mouse neck at weeks 1, 4, 6, and 8. The immunizing dose was fixed at 50 μg by subcutaneous multi-point injection per mouse each time. Then, 3 days prior to cell fusion, the mice were boosted with 50 μg of adjuvant-free His-PAT/pat. Blood samples were collected from the mouse tail, and the titers were determined by ELISA.
2.4. Antibody Preparation
The mouse spleen samples were used to prepare a single-cell suspension. The cell suspensions were mixed at a ratio of 10:1 with murine myeloma cells SP2/0 and fused using the method described by Galfre G et al. [16]. The fusion cell suspension was distributed into 6-well plates and incubated in 5% CO2 at 37 °C. After one week, many white dots formed. These white dots were transferred to 96-well plates to screen hybridoma cell lines secreting the anti-PAT/pat antibody. Next, the absorbance of the culture supernatant of anti-PAT/pat antibody-secreting hybridoma cell lines was evaluated at 450 nm (A450). The positive clones with culture supernatant (A450 > 0.5) were selected for subcloning (negative control A450 < 0.2, positive control A450 > 1.0) and subjected to limited dilution and subcloning until the monoclonal cell lines that stably secreted anti-PAT/pat antibodies were finally selected for expanded culture. Finally, the anti-PAT/pat antibody-producing hybridoma cell lines were injected into the abdomens of 10-week-old BALB/c mice [17,18,19,20], and the ascitic fluid was collected 7 days later. The anti-PAT/pat mAbs were purified from the ascitic using protein A-columns (GE, Boston, MA, USA) [21].
2.5. Antibody Characterization
PAT/pat mAb titers were determined by indirect ELISA. A total of 100 μL of 1 μg/mL His-PAT/pat was added into each well of the plate and incubated at 4 °C overnight. The solution in the wells was discarded the next day. Then, following the indirect ELISA method, the reaction was terminated by the addition of 50 μL of 1 M H2SO4, and the absorbance at 450 nm was measured. The antibody titer was defined as the highest antibody dilution that gave an absorbance greater than 2.1-fold of the absorbance of the negative control [22]. Subclass estimates of the PAT/pat mAbs were performed by direct ELISA using a commercially available kit from Sigma (Shanghai, China) [23,24]. The total RNA of hybridoma cells was extracted and reverse transcribed. The heavy and light chains of antibodies were amplified by using designed mouse antibody degenerate primers, and the products amplified were subcloned in plasmid Puc57 and sequenced.
2.6. Total Protein Extraction
The leaves of GM maize C0010.3.1 and corresponding negative control maize NH106 (Dabeinong Co., Beijing, China) were quickly ground in liquid N2, resuspended in lysis buffer (100 mM Tris, pH 7.5, 300 mM NaCl, 5 mM EDTA, 5% glycerol, 0.1% SDS, protease cocktail and 1 mM PMSF) at 4 °C for 15 min, and then centrifuged at 15,000 rpm and 4 °C for 30 min. The supernatants were used for Western blotting analysis or ELISA detection.
2.7. Protein Sample Analysis
The His-PAT/pat protein, GM maize C0010.3.1 and negative control NH106 samples, and other herbicide resistance proteins PAT/bar, G2 EPSPS, and G10 EPSPS were electrophoresed in 4–12% NuPAGE gels (Life Technologies Co., Carlsbad, CA, USA), stained with Coomassie Blue, or transferred to NC membranes, then blocked with 5% skim milk and incubated with His-PAT/pat mAbs [15].
2.8. ELISA Method Development
A total of 100 μL of 2 μg/mL capture antibody 1F5-2F2 was incubated in the wells of the plate overnight at 4 °C. After being washed 3 times, each well was blocked with 200 μL of 5% skim milk. After washing 3 times, 100 μL of His-PAT/pat protein serially diluted was added to each well. The plate was incubated at 37 °C for 2 h. After discarding the solution in the wells, 1 μg/mL of biotin-labeled anti-His-PAT/pat mAb 1B6-2D3 was added and incubated for 1 h at 37 °C. The plates were washed 3 times and incubated with 100 μL avidin-HRP at 37 °C for 30 min. The wells were then washed 5 times, and 90 μL of TMB solution was added. The reaction was terminated by the addition of 50 μL of 1 M H2SO4, and the absorbance at 450 nm was measured.
2.9. Validation of ELISA Method
The limit of detection (LOD) was estimated as the average concentration of 16 replicates of the 0 standard plus twice the standard deviation. The inter-assay coefficient of variation (CV%) was estimated as the percentage of the standard deviation/the average A450 of 8 replicates of the 50 ng/mL, 12.5 ng/mL, and 3.125 ng/mL PAT/pat, respectively. The intra-assay coefficient of variation (CV%) was estimated as the percentage of the standard deviation/the average A450 of 24 replicates (8 per batch) of the 50 ng/mL, 12.5 ng/mL, and 3.125 ng/mL PAT/pat, respectively.
2.10. Detection of PAT/pat Containing Sample by Sandwich ELISA Established
A total of 100 microlitres of His-PAT/pat protein, leaf extracts of GM maize C0010.3.1, and negative control NH106 were added to a 1F5-2F2 coated well. Other operation steps were the same as the ELISA development. PAT/pat concentration detection with other commercial ELISA kits was performed according to the instructions
2.11. Statistical Analysis
Data are expressed as the average value (AV) and standard deviation (SD). The limit of detection (LOD) was calculated using the standard formula, with a slight modification [25,26,27].
3. Results
3.1. His-PAT/pat Protein Purification
The pat gene was cloned from the genomic DNA of GM maize C0010.3.1 and heterogonous expressed in BL21(DE3). His-PAT/pat was crudely separated by His-affinity purification; about 25 mL cell lysate supernatant was incubated with 1.5 mL Ni-resin, washed with 5 mL washing buffer 5 times, and eluted with 2.5 mL elution buffer twice. Then, further fractionated with a gel filtration chromatography Superdex75 column. The molecular weight of His-PAT/pat was approximately 22 kDa (Figure 1), consistent with expectations.
3.2. PAT/pat mAb Preparation
PAT/pat mAbs were produced using the traditional monoclonal antibody produce method, four times immunization, boosting, cell fusion, subcloning, and screening. In the first subcloning, approximately 300 hybridomas were screened out. Among them, hybridomas with 450 nm absorbance values > 2.0 were selected for further subcloning (Figure 2a). After 2 rounds of subcloning and screening, 11 PAT/pat antibody-secreting hybridoma clones were screened out. Five of them, 1B6-2D3, 1F5-2F2, 2F1-1B5, 2G8-2D2, and 3E5-2F11, were amplified and injected into the BALB/c mice for ascitic fluid preparation. These anti-PAT/pat mAbs were purified from the ascitic fluid with a protein A-column (Figure 2b). They specifically identify PAT/pat positive maize leaf samples by Western blotting screening and recognize different antigen sites between each other (data not shown). The purified mAbs 1F5-2F2 and 1B6-2D3 performed the best detection range and sample measurement results in ELISA paired pre-experiments and were selected to establish the ELISA method. The anti-PAT/pat mAbs 1F5-2F2 and 1B6-2D3 were IgG1 subtypes. Their titers were 1:1,024,000 and 1:896,000, respectively (Table 1). The variable region sequence of the anti-PAT/pat mAbs was detected and is shown in Table 2.
3.3. PAT/pat mAb Cross-Reaction Detection
Several other herbicide resistance proteins, including PAT/bar (Uniprot ID: P16426, 84.8% identity of PAT/pat, Figure 3), CP4 EPSPS, G2 EPSPS, and G10 EPSPS, widely used in GM crops, were applied to analyze the cross-reactivity of the 1F5-2F2 and 1B6-2D3 mAbs. The two mAbs specifically recognized the His-PAT/pat protein and the PAT/pat in C0010.3.1 maize leaf but not the other herbicide resistance proteins (Figure 4).
3.4. Establishment of the Sandwich ELISA
To establish the quantitative sandwich ELISA method, 1F5-2F2 was employed as the capture antibody, and the biotin-labeled 1B6-2D3 was applied as the detection antibody. An initial 200 ng/mL PAT/pat of standard dilutions served to develop a standard curve (Figure 5a). The linear working range of the assay was defined as the part of the curve with the equation y = 0.0376x + 0.1595 and a coefficient of R2 > 0.9999. The linear range ranges from 1.5625 ng/mL to 12.5 ng/mL (Figure 5b), with an LOD of 0.085 ng/mL (Table 3). The descent rates of the developed PAT/pat ELISA assay kit were less than 25% in the 7-day thermal stabilization experiment at 37 °C (Table 4). The variability of the established PAT/pat ELISA method was detected using the coefficient of variation (CV). The inter- and intra-assay CV of this ELISA were 2.270% and 4.572%, respectively (Table 5). These results indicate that the ELISA established for PAT/pat detection is a specific, sensitive, and stable assay.
3.5. Detection of PAT/pat in GM Maize Leaves
Protein samples were extracted from the leaves of the GM maize C0010.3.1 and non-GM maize NH101, then diluted and detected by the ELISA method established in the study, commercial PAT/pat ELISA kits of YouLong Biotech. (Shanghai, China) AA2241 (Lot: 20042201) and Envirologix Co. (Portland, ME, USA) AP014 (Lot:030240). The PAT/pat contents detected by different ELISA methods are shown in Table 6. The PAT/pat contents are 11.35 ± 0.12 μg/g detected by the ELISA established in the study. The ELISA method established in this study is the most suitable detection method for PAT/pat in GM crops.
4. Discussion
In recent years, GM crop cultivation has developed rapidly and led to increased crop yield, reduced labor costs, decreased pesticide use, and so on. At the same time, the unintended food and environmental safety issues caused by GM crops have also been widely studied [6,28,29,30,31]. Detection of exogenous ingredients in GM crops, their products, and even their growing environments is becoming more and more urgent and important. The common methods are event-specific PCR and ELISA, which are based on the foreign genes and their encoding proteins, respectively. Proteins not only play important roles in the biological functions of genes but also act as allergens or toxins. Therefore, establishing an ELISA method and quantifying the detection of exogenous proteins in GM crops is crucial. Several ELISA methods for monitoring insecticidal crystal proteins (ICPs) have been established [15,32,33,34]. Some commercial ELISA kits for the detection of exogenous protein in transgenic crops have been successfully developed by Agdia Inc. (Elkhart, IN, USA) and Envirologix Co. (Portland, ME, USA), and YouLong Biotech. (Shanghai, China). However, a kind of ELISA kit only targets a specific transgenic protein. The detection sensitivity of the same protein ELISA kits by different manufacturers is different for the same protein antigen.
The content of PAT/pat protein in transgenic crops is very low and difficult to enrich and purify. The N-terminus of the PAT/pat is fused with six His residues to facilitate enrichment and purification. In addition, with a few steps in the purification process and mild conditions, the purified PAT/pat protein maintains native conformation and immunogenicity. Monoclonal antibodies are antibodies with high specificity and uniformity against a single epitope, have the advantages of high sensitivity and specificity, and can be repeatable and scalable produced, which overcomes some disadvantages of polyclonal antibodies. Therefore, a sandwich mAb-applied ELISA method was established to detect PAT/pat in GM materials.
The PAT/pat ELISA method established is sensitive, with a LOD of 0.085 ng/mL, which is more sensitive than the previously published method such as the Cry1Ie ELISA method with a LOD of 0.27-0.51 ng/mL [33], the Cry1 ELISA with a LOD of 15 ng/mL [17], the Cry1F method with a LOD of 0.88 ng/mL [13], and the Cry1Ab ELISA method with a LOD of 0.008 μg/mL [35]. The PAT/pat ELISA method established is stable, with CV less than 5.0%, which is similar to the ELISA method established before, such as the Cry1Ab ELISA method with CV less than 5.0% of [35], the Cry1C [36], Cry1F [13], and Cry1B [37] ELISA method with CV less than 6.0%. These results indicate that the PAT/pat ELISA method established in this study has potential for commercial usage.
The results showed that the established ELISA method was without any cross-reactivity with other herbicide resistance proteins, including CP4 EPSPS, G2 EPSPS, and G10 EPSPS, especially PAT/bar showing the high homology with PAT/pat. This may be because the two proteins have different structured epitopes, even though the amino acid sequence is highly homologous. The content of PAT/pat in the leaves of the GM maize C0010.3.1 detected by different ELISA methods are significantly different (Table 4). This may be due to the different sensitivity of ELISA methods developed for the same protein by different manufacturers. It fully demonstrates that the established ELISA method is a necessary, specific, sensitive, and stable method for analyzing PAT/pat in different GM materials. This is conducive to promoting the development of the GM corn industry.
5. Conclusions
Two specific anti-PAT/pat mAbs, 1F5-2F2 and 1B6-2D3, were screened out and purified. A sandwich ELISA method was established to analyze PAT/pat with a linear detection range from 1.5625 ng/mL to 12.5 ng/mL, a LOD of 0.085 ng/mL, and a CV less than 5.0%. The PAT/pat ELISA method performed well in the thermal stability experiment and was successfully applied for quantitative analysis of PAT/pat protein in GM crops samples. The establishment of this ELISA method is important to promoting the development of PAT/pat GM crops.
Conceptualization, fund acquisition, and original draft writing, W.L.; Methodology, investigation, and data curation, L.M., X.L. and C.L.; Manuscript editing, W.J. All authors have read and agreed to the published version of the manuscript.
The study was conducted in accordance with approved institutional animal care and use committee (IACUC) protocols (#08-133) of the Institute of Zoology, Chinese Academy of Sciences.
Not applicable.
Not applicable.
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. Purification of the overexpressed PAT/pat protein. Sup., Supernatant of Cell lysate. F.T., Flowthrough after Ni-resin binding.
Enlarge this image.Figure 2. Preparation of the anti-PAT/pat mAbs. (a) Hybridoma screening. (b) the purified PAT/pat mAbs 1F5-2F2 and 1B6-2D3.
Enlarge this image.Figure 3. Alignment of PAT/pat (comes from Streptomyces viridochromogenes, Uniprot ID: Q57146) and PAT/bar (comes from Streptomyces hygroscopicus, Uniprot ID: P16426). Sequence identity is indicated by shading.
Enlarge this image.Figure 4. Specificity of anti-PAT/pat mAbs. (a) SDS-PAGE stained with Coomassie Blue, protein marker (26610, ThermoFisher Scientific) and (b) Western blotting of PAT/pat in C0010.3.1, His-PAT/pat, and other herbicide resistance proteins (PAT/bar, CP4 EPSPS, G2 EPSPS, and G10 EPSPS) against 5 µg/mL of the mAbs 1F5-2F2 and 1B6-2D3, protein marker (P8028L, Uelandy Inc., Suzhou, China).
Enlarge this image.Figure 5. The sandwich ELISA method for PAT/pat detection. (a) Standard curve of the Pat/pat ELISA. (b) Linear detection range from the standard curve in (a).
PAT/pat mAbs for ELISA method establishment.
| Item | Antigen | Property | Host | Antibody Subtype | Titer | Application |
|---|---|---|---|---|---|---|
| 1F5-2F2 | PAT/pat | Monoclonal | Mouse | IgG1 | 1:1,024,000 | ELISA, WB |
| 1B6-2D3 | PAT/pat | Monoclonal | Mouse | IgG1 | 1:896,000 | ELISA, WB |
Variable region sequence of Anti-PAT/pat mAbs.
| Item | Sequence of Antibody Variable Region | |
|---|---|---|
| Light Chain | Heavy Chain | |
| 1F5-2F2 | DIVMTQSPPILSASPGEKVTMTCRASSTVSYIHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISGVEAEDAATYYCQQWSSYPPTFGAGTKLEIKR | DVKLQESGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGFNPNTGGTRYNQKFKGKATVTVDKSSSTAHMELRSLTSEDSAVYFCARGPYGNYDWFAYWGQGTLVTVSS |
| 1B6-2D3 | DVLMTQTPLSLPVSLGDQASISCRSSQNIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSYVPWTFGGGTKLEIKR | EVQLQQSGGGLVQPKGSLKLSCAASGFTFNTYAMHWVCQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVSDGYYPFAYWGQGTLVTVSA |
LOD of the developed PAT/pat ELISA method.
| Average of S0 Concentration |
SD of S0 Concentration | LOD |
|---|---|---|
| −0.345 | 0.215 | 0.085 |
S0, 0 ng/mL of the standard product; SD, the standard deviation; LOD, the limit of detection.
Descent rate (%) in the 7-day thermal stabilization experiment at 37 ℃ of the developed PAT/pat ELISA kit.
| His-PAT/pat |
OD450 |
OD450 |
Descent Rate |
|---|---|---|---|
| 100.000 | 2.821 | 2.612 | 7% |
| 50.000 | 2.632 | 2.337 | 11% |
| 25.000 | 2.121 | 1.941 | 8% |
| 12.500 | 1.384 | 1.221 | 12% |
| 6.250 | 0.673 | 0.611 | 9% |
| 3.125 | 0.432 | 0.394 | 9% |
| 1.563 | 0.244 | 0.204 | 17% |
| 0.000 | 0.037 | 0.028 | 23% |
Inter- and Intra-assay coefficients of variation of the established PAT/pat ELISA method.
| Theoretical Value (ng/mL) | AV |
SD | (CV%) | Average CV (%) |
|---|---|---|---|---|
| Intra-assay CV (n = 8) | ||||
| 50 | 49.328 | 0.031 | 1.233 | 2.270 |
| 12.5 | 11.645 | 0.026 | 2.123 | |
| 3.125 | 4.161 | 0.015 | 3.453 | |
| Inter-assay CV (n = 24) | ||||
| 50 | 42.046 | 0.074 | 3.055 | 4.572 |
| 12.5 | 10.528 | 0.056 | 4.902 | |
| 3.125 | 4.044 | 0.024 | 5.760 | |
AV, the average value; SD, the standard deviation; CV, the coefficient of variation.
PAT/pat content in the maize leaves.
| Protein | Maize Sample | Content * |
AV ± SD |
Content * |
AV ± SD |
Content * |
AV ± SD |
|---|---|---|---|---|---|---|---|
| PAT/pat | C0010.3.1 | 11.23 | 11.35 ± 0.12 | 0.598 | 0.60 ± 0.01 | 15.43 | 16.20 ± 1.07 |
| 11.47 | 0.605 | 15.74 | |||||
| 11.35 | 0.592 | 17.42 | |||||
| non-GM NH101 | - | - | - | - | - | - | |
| - | - | - | |||||
| - | - | - | |||||
| The equation of ELISA standard curve | y = 0.0934x + 0.0134, |
y = 0.0372x − 0.0163, |
y = 1.312x + 0.04, |
||||
-, no PAT/pat expression detected; *, content of PAT/pat per gram of leaves; AV, the average value; SD, the standard deviation.
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