Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (7): 1473-1481.doi: 10.3864/j.issn.0578-1752.2020.07.015

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Improving the Sensitivity of ELISA by Large-Capacity Reaction System of Aflatoxigenic Fungi-Biomarker in Agro-products

Xiao WEI1,2,4,Qi ZHANG1,2,3,Wen ZHANG1,3,5,Hui LI1,2,4,PeiWu LI1,2,3,4,5   

  1. 1. Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062
    2. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062
    3. Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062
    4. Laboratory of Risk Assessment for Oilseeds Products (Wuhan) , Ministry of Agriculture, Wuhan 430062
    5. Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062
  • Received:2019-08-09 Accepted:2019-11-05 Online:2020-04-01 Published:2020-04-14

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Abstract: 【Objective】In order to prevent and reduce the contamination by Aspergillus species from the source, a highly sensitive large-capacity reaction system DAS-ELISA was established, based on the biomarker PO8 protein of aflatoxigenic fungi. This study aimed to provide key technical support for pollution source monitoring. 【Method】 In this study, the dry mycelium was used as a reference for the biomarker PO8 protein, the mycelia lysate made by high pressure homogenization was used as envelope antigen, the purified PO8-VHH was used as capture antibody, and rabbit polyclonal antibody against Aspergillus was used as detection antibody. The antigen and antibody were added to the 96-well microtiter plate at 200 μL/well, and the sandwich ELISA for the large-capacity reaction system was carried out. Based on the principle that the positive hole OD450nm≥1.0, the positive hole OD450nm/negative hole OD450nm was higher to determine the optimal experimental conditions and to establish a standard curve. The performance of the established sandwich ELISA method was evaluated by spike-and-recovery test, repeatability test and specific test. 【Result】 Assays were performed in the PO8-VHH (3 μg?mL -1) coated ELISA format, in which the detection antibody was 2.5 μg?mL -1 diluted. The optimized physicochemical factors in the performance were obtained: the antibody coating condition was 4℃ overnight, the blocking reagent was 3% BSA, the blocking condition was 37℃ 2 h, and the polyclonal antibody working time was 50 min. The standard curve was established under the optimal conditions, the minimum detectable limit was 0.1 μg?mL -1. This method was specific, with no cross reaction with HBHA, FO, FV, and AO. Meanwhile, the inter-assay repetition rate was 1.5%-5.8% and intra-assay repetition rate was 0.4%-3.2%, both lower than 10%, indicating it was good repeatability. Non-aflatoxigenic fungi had lower values, close to negative value. 【Conclusion】 The large-capacity reaction system sandwich ELISA method established in this study could quickly and accurately detect aflatoxigenic fungi, which laid a foundation for further control of aflatoxin contamination from the source.

Key words: agro-products, Aspergillus flavus, nanobody, ELISA, biomarker

Fig. 1

SDS-PAGE electrophoresis analysis of purified Nanobodies"

Fig. 2

Sensitivity of PO8-VHH determined with different concentrations of A. flavus"

Table 1

The optimum antibody coating concentration and the best dilution of the polyclonal antibody"

包被抗体稀释度
Antibody coating dilution (μg∙mL-1)		 多抗稀释度 Polyclonal antibody concentration (μg∙mL-1)
4 3 2.5 2 1.5 1 0.5
0.5 1.4758 0.8651 0.5182 0.3603 0.3344 0.227 0.2148
1 1.5511 0.8667 0.5633 0.4631 0.3676 0.3353 0.2937
2 1.5714 0.9132 0.6063 0.4855 0.3974 0.3598 0.2949
3 1.8485 1.3707 1.0641 0.7815 0.5402 0.3655 0.3570

Fig. 3

The specific value in different coating time Different lowercase letters in the figure indicate significant differences among different treatments (P<0.05). The same as below"

Fig. 4

The specific value in different blocking solution"

Fig. 5

The specific value in different blocking time"

Fig. 6

The specific value in different polyclonal antibody working time"

Fig. 7

Standard curve of sandwich ELISA method for large- capacity reaction system"

Table 2

Results of specificity test"

项目
Project		 检测样品 Test sample
阳性
Positive		 阴性
Negative		 HBHA FO FV AO
OD450nm 1.043 0.179 0.172 0.221 0.176 0.169

Table 3

ELISA for detecting Aspergillus-contaminated peanut samples"

组别
Sample number		 OD450nm值
OD450nm value		 AFB1的含量
Concentration of AFB1 (μg∙kg-1)
1 0.201 0.000
2 0.202 0.000
3 0.209 0.000
4 0.199 0.000
5 0.193 0.000
6 0.389 1237.053
7 0.417 2294.187
8 0.388 3135.416
9 0.470 4212.220
10 0.466 5434.766

Table 3

Inter-assay repeatability"

组别
Sample number		 平均值
Average value		 标准差
Standard deviation		 变异系数
Variation coefficient (%)
阳性1 Positive1 1.975 0.033 1.6
阳性2 Positive2 1.043 0.047 4.5
阳性3 Positive3 0.581 0.015 2.6
阳性4 Positive4 0.523 0.008 1.5
阴性5 Negative5 0.188 0.011 5.8

Table 4

Intra-assay repeatability"

组别
Sample number		 平均值
Average value		 标准差
Standard deviation		 变异系数
Variation coefficient (%)
阳性1 Positive1 1.725 0.006 0.4
阳性2 Positive2 1.069 0.012 1.2
阳性3 Positive3 0.585 0.019 3.2
阳性4 Positive4 0.532 0.014 2.6
阴性5 Negative5 0.184 0.004 2.0

Table 5

The detection results of analog samples"

样品
Sample		 添加量
Dosage (μg∙mL-1)		 检出浓度
Detection concentration (μg∙mL-1)		 回收率
Rate of recovery
(%)

花生
Peanut		 100 107.32±3.20 107.32
10 8.19±1.08 81.90
1 0.97±0.12 97.00

玉米
Corn		 100 105.55±3.80 105.55
10 9.6±0.98 96.00
1 1.09±0.13 109.00
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