Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (5): 1071-1080.doi: 10.3864/j.issn.0578-1752.2020.05.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Development of High Sensitive Zeranol Monoclonal Antibody Based on the Cross Reactivity of Structural Analogs

HU XiaoFei1,LI QingMei1,YAO JingJing1,HU SiYu2,SUN YaNing1,XING YunRui1,DENG RuiGuang1,ZHANG GaiPing1,3()   

  1. 1 Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002
    2 College of Life Sciences, Henan Agricultural University, Zhengzhou 450002
    3 Henan Agricultural University, Zhengzhou 450002
  • Received:2019-01-08 Accepted:2019-10-08 Online:2020-03-01 Published:2020-03-14
  • Contact: GaiPing ZHANG E-mail:zhanggaip@126.com

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Abstract:

【Objective】The aim of this study was to obtain highly sensitive monoclonal antibody of Zearalanol (ZAL). 【Method】Zearalanone (ZAN), as the structural analogs of ZAL, was employed to synthesize the artificial complete antigen. ZAN structure was modified by oximation reaction to obtain ZNA-O. ZAN-O was coupled with bovine serum albumin and ovalbumin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) respectively to prepare ZAN-O-BSA and ZAN-O-OVA. ZAN-O-BSA was used to immunize the mice with the immune dose of 50 μg protein/mouse. The mouse with higher serum titer and sensitivity was selected for cell fusion. During the process of cell fusion to screen the positive hybridoma cells, ZAL was used as a blocker instead of ZAN to screen the hybridoma cell lines that could secrete monoclonal antibody against ZAL. ZAL monoclonal antibody was prepared in batches by ascites induction in vivo, and the immunological properties of the monoclonal antibodies were identified. 【Result】By cell fusion and screening for positive hybridoma cell lines, one hybridoma cell line named 12B10A7 that could secrete monoclonal antibody against ZAL was obtained. The sensitivity (half inhibitory concentration, IC50) of the prepared monoclonal antibody was 577 pg?mL -1, the affinity constant Ka was 6.21×10 7 L?mol -1, the cross-reactivity rate with structural analogues such as β-zearalanol, α-zearalenol, β-zearalenol, zearalanone and zearalenone were 43.06%, 15.51%, 15.22%, 77.65% and 9.79%, respectively, and the cross reaction rates with other toxins and carrier proteins were all less than 0.06%. 【Conclusion】In conclusion, based on the cross reaction characteristics of antibody, in this study, we prepared the artificial antigen of ZAN, and got the monoclonal antibody against ZAL, which had high affinity, sensitivity and specificity.

Key words: Zearalanol, Zearalanone, structural analogs, cross reaction, monoclonal antibody

Fig. 1

Roadmap of ZAN structural renovation by oximation"

Table 1

The ZEN McAb’s titer detected by microplates coated with the prepared ZAN-O-BSA or ZAN-O-OVA"

ZEN mAb稀释倍数
Dilution ratio of ZEN mAb		 1×104 2×104 4×104 8×104 16×104 32×104 64×104 空白
Blank
ZAN-O-BSA >3.5 3.044 1.847 1.028 0.579 0.343 0.243 0.064
ZAN-O-OVA 1.182 0.869 0.653 0.387 0.289 0.196 0.163 0.042

Table 2

The serum titer of mice immunized with ZAN-O-BSA"

小鼠编号
Number of mice		 血清稀释倍数 Dilution ratio of serum
200 400 800 1600 3200 6400 12800 空白Blank
1 3.471 2.438 1.462 0.718 0.418 0.329 0.240 0.105
2 2.656 1.898 1.047 0.611 0.345 0.244 0.205 0.083
3 >3.50 2.937 2.084 1.120 0.656 0.384 0.309 0.107
4 2.179 2.012 1.828 1.432 1.043 0.822 0.670 0.089
5 1.879 1.855 1.392 0.938 0.692 0.539 0.373 0.105
6 2.977 2.728 2.122 1.560 1.049 0.692 0.502 0.089

Table 3

The serum sensitivity of mice immunized with ZAN-O-BSA"

小鼠编号
Number of mice		 ZAL浓度 ZAL concentration (ng·mL-1) 阳性
Positive		 空白
Blank
500 250 125 62.5 31.25 15.62
1 0.681 0.836 1.099 1.148 1.286 1.802 1.910 0.104
2 0.736 0.844 0.915 0.986 1.085 1.128 1.550 0.109
3 1.078 1.043 1.101 1.176 0.993 1.168 1.544 0.092
4 0.569 0.608 0.731 0.820 0.987 1.069 1.591 0.107
5 0.865 0.879 0.927 0.972 0.994 1.077 1.346 0.103
6 0.288 0.402 0.510 0.621 0.653 0.741 1.276 0.106

Fig. 2

The serum inhibition curve of NO.6 immunized mouse"

Table 4

The titer of ZAL McAb"

ZAL单抗稀释倍数
Dilution ratio of ZAL mAb		 4×103 8×103 1.6×104 3.2×104 6.4×104 1.28×105 2.56×105 空白
Blank
OD450值 OD450 value >3.50 2.235 1.224 0.739 0.547 0.294 0.238 0.094

Fig. 3

The serum inhibition curve of ZAL McAb"

Table 5

The specificity of ZAL McAb"

Fig. 4

The affinity curve of ZAL McAb"

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