Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin

Journal Pre-proof Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin

Ning Lu, Li Ling, Tian Guan, Lanteng Wang, Dian Wang, Jiahui Zhou, Ting Ruan, Xing Shen, Xiangmei Li, Yuanming Sun, Hongtao Lei PII:

S0041-0101(19)30759-7

DOI:

https://doi.org/10.1016/j.toxicon.2019.12.003

Reference:

TOXCON 6251

To appear in:

Toxicon

Received Date:

14 September 2019

Accepted Date:

05 December 2019

Please cite this article as: Ning Lu, Li Ling, Tian Guan, Lanteng Wang, Dian Wang, Jiahui Zhou, Ting Ruan, Xing Shen, Xiangmei Li, Yuanming Sun, Hongtao Lei, Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin, Toxicon (2019), https://doi.org/10.1016/j.toxicon.2019.12.003

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof

Broad-specificity ELISA with a heterogeneous strategy for sensitive detection of microcystins and nodularin

Ning Lu1, Li Ling1, Tian Guan1, Lanteng Wang1, Dian Wang1, Jiahui Zhou1, Ting Ruan1, Xing Shen1, Xiangmei Li1, Yuanming Sun1, Hongtao Lei1*

1Key

Laboratory of Food Quality and Safety of Guangdong Province, College of

Food Science, South China Agricultural University, Guangzhou 510642, China

 Authors

to whom any correspondence should be addressed.

H. T. Lei, E-mail: [email protected]; Tel: +8620-8528 3925. Fax: +8620-8528 0270. `` 1

Journal Pre-proof ABSTRACT A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay (ciELISA) method was developed for the simultaneous detection of nine microcystins (MCs) and nodularin (NOD) using MC-LR-keyhole limpet hemocyanin (KLH) for New Zealand white rabbit immunization to produce antibodies. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared and heterogeneous coating strategy was found to significantly improve the sensitivity of detection, as evident from the appropriate structure. Comparison of the half-maximum inhibitory concentration (IC50) with MC-LR and MC-LR-BSA coating techniques (0.29 ng/mL) revealed the superior performance of 0.054 ng/mL for NOD-BSA coating. NOD-BSA was selected as the coating antigen, because it showed ultrahigh sensitivity for the detection of MC-LR with a limit of detection (LOD) of 0.0016 ng/mL, which was below the maximum residue level (MRL) of 1 ng/mL. In addition, high reproducibility, good stability, and acceptable spiked sample detection, as validated by liquid chromatography tandem mass spectrometry (LC-MS/MS), indicated the possible application of this method for the analysis of MCs and NOD in water sample. KEYWORDS: Microcystin-LR, ELISA, Heterogeneous coating, Broad-specificity

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Journal Pre-proof 1. Introduction Microcystins (MCs) produced by freshwater cyanobacteria are a class of more than 90 structurally similar potent hepatotoxins. The substitution of different amino acids at positions 2 and 4 leads to the formation of different MC structures (Figure 1A)[1-3]. Humans may be exposed to high levels of MCs through drinking water or ingestion of contaminated food, leading to severe liver damage or even death[4-6]. The rapid development in industrialization and population explosion have caused serious problems of algal blooms and increased the exposure of humans to high levels of MCs[7-9]. Nodularin (NOD), another hepatotoxic, is a cyclic pentapeptide compound (Figure 1B). NOD and MCs have a common side chain ADDA, which is considered as the poisoning group[10]. Microcystin-LR (MC-LR) is the most toxic member of the MC variants[11-13]. The regulatory limit of MC-LR has been set up at 1 µg/L in drinking water by the World Health Organization (WHO)[14]. Therefore, the development of rapid and reliable methods with ultrahigh sensitivity for the detection of MCs and NOD is desirable.

Fig. 1. Structures of microcystins (A) and nodularin (B).

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Journal Pre-proof The various methods used for the analysis of MC-LR include high-performance liquid chromatography (HPLC)[15-17], liquid chromatography-mass spectrometry (LC-MS)[18,19],

enzyme-linked

immunosorbent

assay

(ELISA)[20,21],

immuno-chromatographic assay, protein phosphatase inhibition assay (PPIA), and immunosensor[22-24]. HPLC and LC-MS are reliable and widely accepted owing to their high accuracy and precision. However, these expensive instrumentation methods are time consuming and demand extensive sample preparation procedures by trained operators[25-27]. PPIA and immuno-chromatographic assay are also commonly used for the detection of MC-LR in environmental and biological samples. However, these methods show poor repeatability and high inaccuracy[28-31]. Immunosensors are rapid and sensitive tools but require chemical instruments and may not be used for field testing[32-35]. ELISA is the method of choice, considering its salient features such as rapid procedure, high sensitivity, and simplicity. Amplification of detectable signals and high sensitivity often depends on high-affinity antibodies and appropriate labels[36-37]. Due to its simplicity, rapid procedure, and accuracy, ELISA usually used to match results for other immunoassay methods. For example, Zhongbin Luo obtained good well-matched results by using AFP ELISA kit as the reference from NIR light-based PEC immunoassay were acquired for the analysis of human serum specimens[38-39]. Here, a highly sensitive and broadly specific ELISA method was developed to detect MCs and NOD. MC-LR was coupled with carrier proteins (keyhole limpet hemocyanin [KLH]), and antibody was prepared by immunizing New Zealand white

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Journal Pre-proof rabbits with the immunogen MC-LR-KLH. The MC-LR-bovine serum albumin (BSA) and NOD-BSA coating antigens were compared, and NOD-BSA was selected as the coating antigen. We used competitive indirect immunoassay for the construction of this ultrahigh sensitivity and broad-specificity ELISA to detect MCs and NOD in water samples and aquatic products. 2. Materials and methods 2.1. Materials and apparatus MC-LR, MC-YR, MC-RR, MC-WR, MC-LA, MC-LF, MC-LW, MC-LY and NOD were purchased from Veterinary Medicine Supervisory Institute of China (Beijing,

China).

Bovine

serum

albumin

(BSA),

ovalbumin

(OVA),

1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS), complete and incomplete Freund’s adjuvant were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals and organic solvents, which were analytical grade or better, were obtained from a local chemical supplier (Yunhui Trade Co., Ltd., Guangzhou, China). New Zealand White Rabbit, 12 weeks old, were raised at the Laboratory Animal Center of South China Agricultural University (Guangzhou, China). Tween 20 was purchased from Acros Organics. Phosphate buffer solutions (PBS) of various pH was adjusted by mixing 1/15M stock solutions of KH2PO4 and Na2HPO4 at different ratios, while the washing buffer in the immunoassay was PBST by adding 0.5% tween-20 in 0.01M PBS 7.4. Ultraviolet−visible (UV−vis) spectroscopy was detected on a UV-4000 spectrophotometer (Hitachi). ELISA plates were washed in a Wellwash MK2

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Journal Pre-proof microplate washer (Thermo Scientific). ELISA absorbance values were measured at a wavelength of 450 nm with a Multiskan MK3 microplate reader (Thermo Scientific). 2.2. Preparation of immunogens and coating antigens NHS (1 mg), EDC (1 mg) and MC-LR (1 mg) were dissolved in 0.5 mL of DMF. NHS (1 mg), EDC (1 mg) and NOD (1 mg) were dissolved in 0.5 mL of DMF. After the reaction mixture was stirred for 12 h at 4 ℃, KLH (4 mg) and BSA (10 mg) were dissolved in 1 mL of 0.01M carbonate buffer. MC-LR activation solution (0.5 mL) and NOD activation solution (0.5 mL) was added dropwise to the protein solution (KLH or BSA). The mixture was stirred for 3 h. Finally, the reaction product was dialyzed against 0.9% (m/v) NaCl solution for 3 days at 4 ℃ and stored at -20 ℃ until use[40-41]. The immunogen produced by this method was designated as MC-LR-KLH. The coating antigen MC-LR-BSA, NOD-BSA was prepared via the same procedure and characterized by UV-vis spectra. 2.3. Production of polyclonal antibodies Three New Zealand White Rabbit aged 12 weeks were immunized subcutaneously with 325 μg of MC-LR-KLH in a mixture of 325 μL of phosphate-buffered saline (PBS) and 325 μL of Freund’s complete adjuvant. At intervals of 3 week after the initial injection, booster injections were given with the same amount of immunogen emulsified with incomplete Freund’s adjuvant. Subsequent immunizations were performed by mixed emulsification with Freund's incomplete adjuvant and immunization every three weeks. A small amount of rabbit blood can be taken for testing one week after the third immunization to observe the immune effect. The

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Journal Pre-proof polyclonal antibody can be collected for ten days after the fourth immunization and stored at -20 ℃ until use. The animal experiments was carried out in accordance with the U.K. Animals (Scientific Procedures) Act. 2.4. Enzyme-linked immunosorbent assay The ELISA was developed for MCs and NOD. The 96-well plates were coated with coating antigens (100 μL/well) in carbonate buffer at 37 °C overnight. The concentration of the coating antigens MC-LR-BSA was 50 ng/mL, the concentration of the coating antigens NOD-BSA was 6.25 ng/mL. Next, the well was washed twice with PBST (300 μL/well) (phosphate-buffered saline solution containing 0.1% Tween-20) and blocked with 5% skimmed milk (120 μL/well) in PBST at 37 °C for 3 h, and the 96-well plates were dried at 37 °C for 1 h. The wells were then incubated with diluted MC-LR standard solution (50 μL/well) and diluted antibody (50 μL/well) in PBST. After incubation in a 37 °C water bath for 40 min, the wells were washed five times with 300 μL of PBST. Then 100 μL/well HRP-IgG (diluted 1:5000 in PBST) was added and the wells were incubated at 37 °C for 30 min. After five times washes with 300 μL of PBST, 100 μL of TMB solution (400 μL of 0.6% TMB−dimethyl sulfoxide and 100 μL of 1% H2O2 diluted with 25 mL of citrate−acetate buffer, pH 5.5) was added to the wells and they were incubated at 37 ℃for 10 min. Finally, the reaction was stopped by addition of 50 μL of 2 M H2SO4. Absorbance of the reaction solution at 450 nm (A450) was recorded. 2.5. Statistical analysis and curve fitting A series of different concentrations of MC-LR standard solution were mixed with

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Journal Pre-proof the antibody and tested under the optimal test conditions. The standard curves were obtained by plotting absorbance against analyte concentration. A four-parameter equation was used to plot the sigmoidal curve by use of Origin 8.5 software (Origin Lab Corp., Northampton, MA): y = (A - D) / [1 + (x/C) B] + D A and D correspond to maximal and minimal absorbance, respectively. B is the slope of the curve and C is the MC-LR concentration that inhibited 50% of antibody binding. 2.6. Cross-reactivity The cross-reactivity was determined by use of MCs and NOD under optimized ELISA conditions. Cross-reactivities (CR) were calculated according to CR = IC50(MC-LR)/ IC50(structurally related compounds) × 100% where IC50 refers to the concentration that inhibited 50% of antibody binding. 2.7. Sample pretreatment The water sample containing MCs was centrifuged and passed through a 0.22 μm filter membrane. It can be directly used for ELISA detection. The C18 SPE column was activated with 10 ml of methanol and 10 ml of water. Add the sample at a flow rate of 1-2s. Rinse with 10ml of 20% methanol at a flow rate of 1-2s. Eluted with 10 ml of methanol containing 0.1% formic acid with a flow rate of 1-2 s. The eluate was blown dry with nitrogen and reconstituted with 1 ml of methanol. Machine inspection after through film. The matrix interference could be eliminate by add seven times 0.1 mol/L of

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Journal Pre-proof phosphate buffer solution to the water sample to be tested[42]. 3. Results and discussion 3.1. Preparation of artificial antigen UV-vis spectra of artificial antigens, BSA, KLH, and MC-LR are shown in Figure 2. BSA and KLH showed absorption peaks at 280 nm that were absent for MC-LR and NOD. The coupling of MC-LR and NOD to the carrier protein resulted in a significant shift in the characteristic peaks of the artificial antigen. MC-LR-KLH, MC-LR-BSA, and NOD-BSA showed absorption peaks at 280 nm wavelength that significantly changed as compared with the peaks for MC-LR, NOD, and carrier protein. Thus, the carrier protein was successfully coupled to MC-LR and NOD[43].

Absorbance/(AU)

1.5

BSA KLH MC-LR MC-LR-BSA MC-LR-KLH NOD NOD-BSA

1.0

0.5

0.0 200

300

400

Wavelength (nm)

Fig. 2. UV-vis spectra of artificial antigens, BSA, KLH, and MC-LR

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Journal Pre-proof 3.2. Standard curve of MC-LR Proper antigen coating is essential to ensure high sensitivity in ELISA. Here, NOD-BSA and MC-LR-BSA were used as the coating antigens to establish a standard curve for MC-LR. After using MC-LR-BSA as the coating antigen, the IC50 of MC-LR was 0.29 ng/mL. The IC50 of MC-LR was 0.054 ng/mL with the use of NOD-BSA as the coating antigen (Figure 3). Thus, the sensitivity improved by nearly six times. NOD is similar to MC-LR, it shows ADDA and four-arginine structure, which is an important site for antibody binding. The difference between NOD and MC-LR structures contributed to the weaker competitiveness of NOD for antibody binding than MC-LR, hence, the sensitivity of ELISA significantly improved.

1.0

MC-LR-BSA NOD-BSA

0.8

B/B0

0.6

0.4

0.2

IC50=0.29 ng/mL IC50=0.054 ng/mL

0.0 1E-4

1E-3

0.01

0.1

1

10

100

1000

10000

Concentration of MC-LR (ng/mL)

Fig. 3. ELISA standard curve for MC-LR (MC-LR-BSA and NOD-BSA were the coating antigens)

10 ``

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Therefore, NOD-BSA was selected to complete the establishment of the standard curve. The working range was 0.0059-0.49 ng/mL. The assay revealed an IC50 value of 0.054 ng/mL and the limit of detection (LOD) of 0.0016 ng/mL for MC-LR, indicating its suitability to meet the requirements for MC-LR quantitation considering the WHO-recommended standard limit of 1.0 μg/L for MC-LR in drinking water. 3.3. Immunoassay specificity As shown in Table 1, upon MC-LR-BSA coating, the antibody failed to recognize MC-LF but could detect other seven MCs and NOD. The cross-reactivity of the antibody to the MCs with arginine at position four (MC-LR, MC-RR, MC-YR, MC-WR and NOD) was all greater than 72%, and the cross-reactivity to the non-arginine-substituted structures at the fourth position (MC-LY, MC-LA, MC-LW, MC-LF) showed less than 23%. The sensitivity of identifying structures substituted with arginine at position four was significantly higher than that for the detection of other structures, probably because the antibody recognition site included ADDA and the four-arginine structure. The change in the four-arginine structure resulted in the change in the steric hindrance of antibody recognition site, thereby leading to a decrease in antibody recognition ability.

Molecules

IC50 (ng/mL)

CR (%)

LOD (ng/mL)

MC-LR

0.29

100.0

0.0340

MC-RR

0.21

109.6

0.0140

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0.24

95.0

0.0150

MC-YR

0.30

75.5

0.0180

NOD

0.32

72.5

0.0080

MC-LY

1.01

22.6

0.0200

MC-LA

1.71

13.4

0.0530

MC-LW

1.97

11.6

0.0730

MC-LF

-

-

-

Table 1 Cross-reactivity of MC-LR-related molecules (MC-LR-BSA as the coating antigen)

After applying NOD-BSA as the coating antigen (Table 2), the antibody could recognize all the common eight MCs and NOD structures. As the antibody recognition site included ADDA and the four-arginine structure, the sensitivity of identification of structures substituted with arginine at 4th position (MC-LR, MC-RR, MC-YR, MC-WR, and NOD) was better than that for detection of other structures. As shown in Figure 4A, the left-side structure of the MCs having the arginine structure at the fourth position could be superposed, and the change in the right structure had a negligible steric hindrance effect on the ADDA and four-arginine structure on the left. Hence, these structures could show sensitive binding to the antibody.

Molecules

IC50 (ng/mL)

CR (%)

LOD (ng/mL)

MC-LR

0.05

100.0

0.0016

MC-WR

0.02

226.0

0.0013

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Journal Pre-proof MC-RR

0.11

48.1

0.0065

MC-YR

0.23

23.6

0.0018

NOD

0.29

18.4

0.0510

MC-LW

0.39

14.0

0.0020

MC-LA

0.65

8.3

0.0015

MC-LY

0.70

7.7

0.0140

MC-LF

1.31

4.1

0.0130

Table 2 Cross-reactivity of MC-LR-related molecules (NOD-BSA as the coating antigen)

Fig. 4. Minimum energy configuration of MCs (A. MC-LR, MC-YR, MC-WR, MC-RR; B. MC-LF, MC-LW, MC-LA, MC-LY; and C. MCs)

As shown in Figure 4B, the substitution of the amino acid at 4th position with other amino acids (MC-LA, MC-LF, MC-LW, and MC-LY) resulted in an increase in the distance between the ADDA structure and the amino acid at the 4th position, leading

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Journal Pre-proof to significant steric effects. This seems to be the reason underlying the low cross-reaction observed with the 4th position of non-arginine structure. However, ADDA is an important structure for antibody binding; hence, the 4th position of non-arginine MCs could still be identified. The antibody could recognize all the common eight MCs and NOD structures. The recognition sensitivity of all structures was better than that of MC-LR-BSA as the coating antigen, owing to the heterogeneous coating strategy employed herein. 3.4. Recovery To verify the practical applicability of this immunoassay for the detection of the MC-LR in real samples, we performed an addition and recovery experiment using the west lake water of South China Agricultural University. To investigate the recovery rate, we added a series of concentrations of MC-LR, MC-RR, and NOD into west lake water (0.5, 1, and 2 ng/mL) to prepare different levels MC-LR solutions. The recovery ranged from 81.2% to 106.5%, and the CV ranged from 2.8% to 10.8% (Table 3). The value of R2 between ELISA and LC-MS/MS ranged from 98.8% to 99.8%. The acceptable recovery and R2 value indicated the great potential of this assay for the detection of MC-LR in water samples.

ELISA Sample

Add

Mean

LC-MS/MS

Recovery

CV

concentration

MC-LR

Mean

Recovery CV

R2

concentration

(ng/mL)

(ng/mL)

(%)

(%)

(ng/mL)

(%)

(%)

(%)

2.0

2.055±0.06

102.8

2.8

1.839±0.08

92.0

4.5

99.8

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Journal Pre-proof MC-LR

1.0

1.065±0.09

106.5

8.9

0.998±0.07

99.8

7.4

MC-LR

0.5

0.510±0.02

102

3.7

0.527±0.02

105

4.7

MC-RR

2.0

1.740±0.08

87

4.5

1.698±0.12

84.9

7.2

MC-RR

1.0

1.035±0.05

103.5

4.8

1.100±0.04

110

3.4

MC-RR

0.5

0.495±0.03

99.8

6.4

0.538±0.04

108

7.1

NOD

2.0

2.005±0.12

100.3

6.0

1.837±0.06

91.9

3.1

NOD

1.0

0.950±0.10

95.0

10.8

0.924±0.08

92.4

8.6

NOD

0.5

0.406±0.03

81.2

8.4

0.459±0.04

91.8

8.5

99.3

98.8

Table 3 Results of MC-LR field detection by ELISA and LC-MS/MS (n=3)

4. Conclusions In this work, an antibody that could recognize all the common eight MCs and NOD structures was prepared. The recognition site was ADDA, and the four-arginine structure also played an important role in the binding process. The sensitivity for the detection of structures substituted with arginine at 4th position (MC-LR, MC-RR, MC-YR, MC-WR, and NOD) was better than that for the detection of other structures. A highly sensitive and broadly specific ELISA method for the rapid detection of eight common MCs and NOD was thus developed with NOD-BSA coating. This method shows ultrahigh sensitivity for the detection of MC-LR with an LOD of 0.0016 ng/mL, which was below the MRL of 1 ng/mL. The IC50 of MC-LR was 0.054 ng/mL. The acceptable recovery shows that this immunoassay could be used for the detection of eight MCs and NOD. Acknowledgments This work was supported by the National Natural Science Foundation of China 15 ``

Journal Pre-proof (31871883, 31701703, 31601555, 71633002), Science and Technology Planning Project of Guangdong Province and Guangzhou (2017B020207010，201803020026) Notes and references There are no conflicts of interest to declare. Appendix A. Supporting information There is no supporting information.

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Author Contributions Section NL and HL conceived and designed the study. NL, LL, TG, LW, DW, JZ, and TR performed the experiments. NL and HL wrote the paper. NL, XS, XL, YS, and HL reviewed and edited the manuscript. All authors read and approved the manuscript.

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Declaration of interests ☑The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights 1. An antibody recognizing 9 microcystins and nodularin was obtained for the first time. 2. A heterogeneous coating strategy resulted to a high sensitivity. 3. A highly sensitive and broadly specific competitive indirect enzyme-linked immunosorbent assay method was developed for the simultaneous detection of 9 microcystins and nodularin.