PRELIMINARY STUDY ON THE COLLOIDAL GOLD IMMUNOCHROMATOGRAPHIC ASSAY BASED ON NANOBODY OF SOLUBLE EGG ANTIGENS FROM SCHISTOSOMA JAPONICUM
10.3969/j.issn.1005-0507.2017.04.001
- VernacularTitle:基于日本血吸虫SEA纳米抗体的胶体金免疫层析技术的初步研究
- Author:
Hao-Jie DING
1
;
Jian-Zu DING
;
Qing-Ming KONG
;
Bin ZHENG
;
Di LOU
;
Rui CHEN
;
Shao-Hong LU
Author Information
1. 浙江省医学科学院寄生虫病研究所
- Keywords:
Schistosoma japonicum;
SEA;
Nanobody;
Colloidal Gold Immunochromatographic Assay
- From:
Acta Parasitologica et Medica Entomologica Sinica
2017;24(4):205-211
- CountryChina
- Language:Chinese
-
Abstract:
A colloidal gold immunochromatographic assay for detection of Schistosoma japonicum based on nanobody was established. Two nanobodies, VHH-48 and VHH-52 against soluble egg antigens (SEA) of Schistosoma japonicum have been expressed in Bacillus subtilis and purified by Ni-NTA affinity chromatography. In order to confirm the optimal labeling pH and optimum concentration of nanobody proteins,the colloidal gold solution was prepared by trisodium citrate reduction, VHH-48 and VHH-52 labelled with colloidal gold were used as probes,and the SEA and goat anti-mouse IgG were coated onto the nitrocellulose membrane to form the test line and control line respectively. The colloidal gold immunochromatographic assay with double nanobody sandwich was established. Test strips were assembled with the optimized double-sandwich nanobody combination,sensitivity was evaluated with a serial dilution of SEA antigen and serum from patients of schistosomiasis positive. The specificity of the test strips was determined with sera from other parasites such as Paragonimus. Results showed every 1 mL colloidal gold labeled with VHH-48 and VHH-52 mixed with 16 and 12 μL 0.2 mol/L K2CO3respectively to reach the optimum pH. The colloidal gold solution was adjusted to the optimum pH to determine the optimal protein concentrantion. The optimal protein concentration of VHH48 and VHH-52 were 14 and 10 μg/mL. The best reaction model was using the VHH-52 to label with colloidal gold and coat onto the nitrocellulose membrane to form the test line. The detective sensitivity of SEA was 3.4 ng/mL and the serum positive rate of Schistosomiasis patients was 2.27%. Detection of other parasite-infected sera showed good specificity. The colloidal gold dipstick based on nanobody against SEA laid a certain foundation for the application of nanobody in the rapid diagnosis of Schistosomiasis.
