Preparation of monoclonal antibodies against Pinellia ternata lectin protein and establishment of double-antibody sandwich ELISA.
10.19540/j.cnki.cjcmm.20210414.302
- Author:
Yu-Wei XIE
1
;
Hong-Li YU
2
;
Hao WU
2
;
Xing-Bao TAO
1
;
He-Peng WANG
1
;
Yan-Qiu CHENG
1
;
Cai-Xia WANG
1
;
Ping ZENG
1
;
Bing-Bing LIU
1
;
Ping ZHANG
3
;
Xiao-Bing CUI
1
Author Information
1. School of Pharmacy, Nanjing University of Chinese Medicine Nanjing 210023, China.
2. School of Pharmacy, Nanjing University of Chinese Medicine Nanjing 210023, China Jiangsu Key Laboratory of Chinese Medicine Processing Nanjing 210023, China Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing Nanjing 210023, China.
3. National Institutes for Food and Drug Control Beijing 100050, China.
- Publication Type:Journal Article
- Keywords:
Pinelliae Rhizoma;
double-antibody sandwich enzyme linked immunosorbent assay(ELISA);
lectin protein;
monoclonal antibodies
- MeSH:
Pinellia;
Drugs, Chinese Herbal;
Antibodies, Monoclonal;
Enzyme-Linked Immunosorbent Assay;
Horseradish Peroxidase
- From:
China Journal of Chinese Materia Medica
2022;47(22):6076-6081
- CountryChina
- Language:Chinese
-
Abstract:
To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 μg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.
