Establishment of an ELISA Method for Quantitative Detection of PAT/pat in GM Crops

Round 1

Reviewer 1 Report

Please see attached file for comments

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments :

 

1. Not clear what other methods are currently available (Introduction)

 

Response: Thanks for your valuable comments.

The directional detection methods, polymerase chain reaction (event-specific and gene-specific PCR) and ELISA, detect exogenous gene and protein in GM crops specifically and accurately. However, the sensitivity of the commercial ELISA kits, AP014 (Lot:030240) from Envirologix, and AA2241(Lot: 20042201) from Youlong Biotech., are not enough for PAT/pat detection of GM maize C0010.3.1. So the development of a highly sensitive ELISA of PAT/pat is very necessary.

 

2. Line 53. Fermentas is part of ThermoFisher Scientific. Incorrect reference?

 

Response: Thanks for your valuable comments.

Here, Fermentas should be ThermoFisher Scientific and Uelandy Inc., and was corrected in the revised version as follows.

 

... The protein markers (26610 and P8028L) were purchased from ThermoFisher Scientific (Waltham, USA) and Uelandy Inc. (Suzhou, China). ...

 

3. Line 63. pET28a plasmid has T7 promoter and thus requires T7 RNA polymerase which is not available in E.coli BL21, it is available in BL21(DE3). Perhaps there is an error in the text.

 

Response: Thanks for your valuable comments.

BL21(DE3) was used to overexpress proteins. All BL21 were corrected as BL21(DE3) in the revised version.

 

4. Line 110. This is a standard protocol. Does everything needs to be hare?

 

Response: Thanks for your valuable comments.

The standard protocol section was removed in the revised version.

 

5. Line 155. E. coli BL21(DE3) ?Sample volumes used to produce Figure 1 should be stated. What is the expected MW of His-PAT/pat, calculated from the sequence? is this a concentrated sample or after Sephadex75?

 

Response: Thanks for your valuable comments.

Yes, It should be E. coli BL21(DE3).

Sample volumes were supplemented, and the MW of His-PAT/pat was corrected in the revised version as follows.

Yes, the sample was concentrated after Superdex75 in Figure 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 1,5ml Ni-resin, washed with 5ml washing buffer 5 times and eluted with 2.5ml 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. ...

 

Figure 1. Purification of the overexpressed PAT/pat protein. Sup., Supernatant of Cell lysate. F.T., Flowthrough after Ni-resin binding.

 

6. Line 162. Traditional antibody preparation

 

Response: Thanks for your valuable comments.

It should be traditional monoclonal antibody preparation, and corrected in the revised version as follows.

 

... traditional monoclonal antibody ...

 

7. Line 176. What was the method used to do this? What was the sequencing method used to obtain these sequences?(Table 2)

 

Response: Thanks for your valuable comments.

The method was supplied in Materials and Methods (2.7 antibody characterization) in the revised version as follows.

 

... 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.

 

8. Where do these sequences come from? （Figure 3）

 

Response: Thanks for your valuable comments.

The sequence source was supplemented in the revised version as follows.

 

... 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.

 

9. This figure and all figure panes should be fully explained. protein markers look different from stated in methods. (Figure 4)

 

Response: Thanks for your valuable comments.

The information was supplemented in the revised version as follows.

 

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.).

 

 

10. Line 202. perhaps this should be a relative quantitative method.

                                                    

Response: Thanks for your valuable comments.

Yes, It's a relative quantitative method.

 

11. Decimal point values should be standard(Table 4)

 

Response: Thanks for your valuable comments.

Decimal point values were adjusted consistent in the revised version as follows.

 

Table 4. Descent rate (%) in the 7-day thermal stabilization experiment at 37℃ of the developed PAT/pat ELISA kit

His-PAT/pat (ng/ml)	OD450 (0 day)	OD450 (7 day)	Descent rate  (% 7 day)
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%

 

 

12. Line 229. Clear explanation of both kits is needed

 

Response: Thanks for your valuable comments.

The information of both commercial ELISA kits was supplemented in the revised version as follows.

 

... then diluted and detected by the ELISA method established in the study, commercial PAT/pat ELISA kits of YouLong Biotech. AA2241(Lot: 20042201) and Envirologix Co. AP014 (Lot:030240). ...

 

13. Some guiding text to explain the data is the table is needed (Table 6)

 

Response: Thanks for your valuable comments.

Some explain was added as follows.

 

Table 6. PAT/pat content in the maize leaves

Protein	Maize Sample	Content (μg/g)	AV±SD (μg/g)	Content (μg/g)	AV±SD (μg/g)	Content (ng/g)	AV±SD (ng/g)
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, R2=0.9943 (BRI, CAAS)		y=0.0372x-0.0163, R2=0.9998 YouLong Biotech. AA2241(Lot: 20042201)		y=1. 312x+0.04, R2=0.9964 Envirologix AP014 (Lot:030240)

-, no PAT/pat expression detected

*, content of PAT/pat per gram of leaves

AV, the average value

SD, the standard deviation

 

14. Line 240. Reference is missing

 

Response: Thanks for your valuable comments.

The references were added as follows.

 

... In recent years, GM crop cultivation has developed rapidly and led to increased crop yield, reduced labour 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, 28-31]. ...

 

15. Line 252. not a discussion

 

Response: Thanks for your valuable comments.

Part of the content was deleted in the revised version.

 

16. Line 279. Sandwich ELISA

 

Response: Thanks for your valuable comments.

ELISA was replaced with Sandwich ELISA in the revised version.

 

 

17. Line 306. Who are Authors and what pages are relevant?

 

Response: Thanks for your valuable comments.

The reference is a broad review of available information on genetically-engineered (GE) crops conducted by an ad hoc committee as follows.

 

 

18. Line 348. Pages? Line 352. Pages?

 

Response: Thanks for your valuable comments.

The information of the references were supplemented in the revised version.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The subject of the current manuscript is surely worthy of investigation.  However, there are some points in the manuscript that I would like you to reconsider. I evaluate this manuscript as a major revision, as several points need to be carefully revised before the next resubmission as follows:

1)      Old references have been cited, especially in the introduction section. Please update.

2)      Previous methods should be presented in more detail in the introduction. The hypothesis of the study should be clarified at the end of the Introduction section.

3)      The source of the tested chemicals (product, purity, company, city, country) should be clearly stated (e.g. Line 53).

4)      Line 69:  “A detailed immunization protocol is presented in the supplementary methods” please add the details.

5)      Line 72: add the source

6)      Line 86: How many mice were used? And add their average weight or weight range.

7)      Line 87: at 1st week…..at 4th, 6th and 8th week. Week of what? Please specify.

8)      Add the catalog number of the used kits throughout the materials and method section.

9)      The statistical methods section is missing. please add.

10)  In all Tables and Figs.: describe all abbreviations used in the tables' footnotes and figure legends.

11)  The discussion is short and truly needs to be deep with the findings of the earlier studies related to the topic. All estimated parameters should be discussed in more detail with more depth.

 

12)  In the references list: the reference styles are not the same. Please revise according to the authors' instructions for the journal.

Author Response

Response to Reviewer 2 Comments

 

Comments and Suggestions for Authors

The subject of the current manuscript is surely worthy of investigation.  However, there are some points in the manuscript that I would like you to reconsider. I evaluate this manuscript as a major revision, as several points need to be carefully revised before the next resubmission as follows:

Response: We thank you for your careful review of our manuscript, and are pleased to know you are very positive on our manuscript, and put forward many constructive suggestions.

1. Old references have been cited, especially in the introduction section. Please update.

Response: Thanks for your valuable comments.

The references in the introduction section were supplemented in the revised version.

2. Previous methods should be presented in more detail in the introduction. The hypothesis of the study should be clarified at the end of the Introduction section.

Response: Thanks for your valuable comments.

The introduction section was revised as follows.

 

... Most existing ELISA methods and products of PAT detection are just suitable for PAT/bar. The sensitivity of the commercial PAT/pat ELISA kits, AP014 (Lot:030240) from Envirologix, and AA2241(Lot: 20042201) from Youlong Biotech., are not enough for PAT/pat detection of GM maize C0010.3.1. So the development of a highly sensitive ELISA of PAT/pat is very necessary. ...

 

3. The source of the tested chemicals (product, purity, company, city, country) should be clearly stated (e.g. Line 53).

Response: Thanks for your valuable comments.

The source of the tested chemicals were supplemented in the revised version as follows.

 

... 100bp 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 (P8028L and GS4769) were purchased from Uelandy Inc. (Suzhou, China) and BioLAB Co. (Beijing, 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). Escherichia coli Trans10 and BL21 (DE3) chemically competent cells were obtained from TransGen Biotech (Beijing, China). murine myeloma cells SP2/0 were obtained from Genecreate Biological Engineering Co. (Wuhan, China). 8-week-old BABL/c male mice were obtained from SBF Co. (Beijing, China). Nitrocellulose (NC) membranes were purchased from Bio-RAD (Hercules, USA). ...

4. Line 69:  “A detailed immunization protocol is presented in the supplementary methods” please add the details.

Response: Thanks for your valuable comments.

The detailed protocol was added in the revised version as follows.

 

... Six male BALB/c mice 8-week-old and weighing about 20 g were immunized with His-PAT/pat dissolved in 250 μ l of sterile PBS adjuvant with complete Freund's adjuvant or incomplete Freund's adjuvant at the nape of the 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. Three days prior to cell fusion, the mice were boosted with 50 μg of adjuvant-free immunogen. Blood samples were collected from the mouse tail, and the titers were determined by ELISA. ...

 

5. Line 72: add the source

Response: Thanks for your valuable comments.

The source was added in the revised version as follows.

... The leaves of GM maize C0010.3.1 and corresponding negative control maize NH106 (Dabeinong Co. Beijing, China) were quickly ground in liquid N2,

6. Line 86: How many mice were used? And add their average weight or weight range.

Response: Thanks for your valuable comments.

The information of mice was added in the revised version as follows.

 

... Six male BALB/c mice 8-week-old and weighing about 20 g were immunized...

 

7. Line 87: at 1st week…..at 4th, 6th and 8th week. Week of what? Please specify.

 

Response: Thanks for your valuable comments.

The information was added in the revised version as follows.

 

... Six male BALB/c mice 8-week-old and weighing about 20 g were immunized with His-PAT/pat dissolved in 250 μ l of sterile PBS adjuvant with complete Freund's adjuvant or incomplete Freund's adjuvant at the nape of the 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. Three days prior to cell fusion, the mice were boosted with 50 μg of adjuvant-free immunogen. Blood samples were collected from the mouse tail, and the titers were determined by ELISA. ...

 

8. Add the catalog number of the used kits throughout the materials and method section.

Response: Thanks for your valuable comments.

The catalog number of the used kits was added in the revised version as follows.

 

... the sensitivity of the commercial ELISA kits, AP014 (Lot:030240) from Envirologix, and AA2241(Lot: 20042201) from Youlong Biotech., are not enough for PAT/pat detection of GM maize C0010.3.1. So the development of a highly sensitive ELISA of PAT/pat is very necessary. ...

 

Table 6. PAT/pat content in the maize leaves

Protein	Maize Sample	Content (μg/g)	AV±SD (μg/g)	Content (μg/g)	AV±SD (μg/g)	Content (ng/g)	AV±SD (ng/g)
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, R2=0.9943 (BRI, CAAS)		y=0.0372x-0.0163, R2=0.9998 YouLong Biotech. AA2241(Lot: 20042201)		y=1. 312x+0.04, R2=0.9964 Envirologix AP014 (Lot:030240)

-, no PAT/pat expression detected

*, content of PAT/pat per gram of leaves

AV, the average value

SD, the standard deviation

 

9. The statistical methods section is missing. please add.

Response: Thanks for your valuable comments.

The statistical methods was added in the revised version as follows.

 

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[25-27].

 

10. In all Tables and Figs.: describe all abbreviations used in the tables' footnotes and figure legends.

Response: Thanks for your valuable comments.

All abbreviations were described in the revise version as follows.

 

Figure 1. Purification of the overexpressed PAT/pat protein. Sup., Supernatant of Cell lysate. F.T., Flowthrough after Ni-resin binding.

 

Table 3. LOD of the developed PAT/pat ELISA method

Average of S0 concentration (ng/ml)	SD of S0 concentration	LOD (ng/ml)
-0.345	0.215	0.085

S0, 0 ng/ml of the standard product

SD, the standard deviation

LOD, the limit of detection

 

Table 5. Inter- and Intra-assay coefficients of variation of the established PAT/pat ELISA method

Theoretical value (ng/mL)	AV  (ng/mL)	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

 

Table 6. PAT/pat content in the maize leaves

Protein	Maize Sample	Content (μg/g)	AV±SD (μg/g)	Content (μg/g)	AV±SD (μg/g)	Content (ng/g)	AV±SD (ng/g)
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, R2=0.9943 (BRI, CAAS)		y=0.0372x-0.0163, R2=0.9998 YouLong Biotech. AA2241(Lot: 20042201)		y=1. 312x+0.04, R2=0.9964 Envirologix AP014 (Lot:030240)

-, no PAT/pat expression detected

*, content of PAT/pat per gram of leaves

AV, the average value

SD, the standard deviation

 

11. The discussion is short and truly needs to be deep with the findings of the earlier studies related to the topic. All estimated parameters should be discussed in more detail with more depth.

 Response: Thanks for your valuable comments.

The Discussion was supplemented in the revised version as follows.

 

... The PAT/pat ELISA method established is sensitive, with a LOD of 0.085 ng/mL, which is more sensitive than the previous 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 the potential commercial usage. ...

 

12. In the references list: the reference styles are not the same. Please revise according to the authors' instructions for the journal.

Response: Thanks for your valuable comments.

The reference styles were adjusted consistent in the revised version.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

In my opinion the revised version is much clear now and my previous suggestions have been addressed.

Reviewer 2 Report

The authors have satisfactorily revised the manuscript.