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Rapid screening of monoclonal antibodies against porcine circovirus type 2 using colloidal gold-based paper test

100%

Jin Q. Y., Feng L. L., Wang Y. B., Li P., Yang J. F., Teng M., Chai S. J., Xing G. X., Zhang G. P.

Polish Journal of Veterinary Sciences

2022

tom 25

nr 1

A proof of concept for using paper test as a suitable method in the production of monoclonal antibodies (MAbs) is reported. The paper test which detects antibodies against porcine circovirus type 2 (PCV2) using colloidal gold-labelled capsid protein as the antigen probe was applied exclusively in the screening of anti-PCV2 MAbs. It allowed the detection of 118 single cell clones within 30 min using naked eyes. MAbs with specific binding to authentic epitopes on the virus were selected using a blocking strategy in which the antibody was pre-incubated with PCV2 viral sample before applying to the test paper. Five hybridomas secreting MAbs against the capsid protein were obtained, with only three of them capable of binding to PCV2. The results were validated and confirmed using enzyme-linked immunosorbent assay and immunofluorescence assay. The paper test is simple, rapid, and independent on professional technicians and proves to be an excellent approach for the screening of MAbs against specific targets.

Expression of water-soluble nucleocapsid protein of SARS-CoV-2 and analysis of its immunogenicity

81%

Wang Y. B., Wang S. W., Jin Q. Y., Chen L. P., Zhang F. Q., Shi J. J., Yin Y., Fan Z. X., Liu X. Y., Wang L. P., Li P.

Polish Journal of Veterinary Sciences

2023

tom 26

nr 4

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major public health concern. Nucleocapsid (N) protein is the most abundant structural protein on SARS-CoV-2 virions and induces the production of antibodies at the early stage of infection. Large-scale preparation of N protein is essential for the development of immunoassays to detect antibodies to SARS-CoV-2 and the control of virus transmission. In this study, expression of water-soluble N protein was achieved through inducing protein expression at 25°C with 0.5 mM IPTG for 12 h. Western blot and ELISA showed that recombinant N protein could be recognized by sera collected from subjects immunized with Sinovac inactivated SARS-CoV-2 vaccine. Four monoclonal antibodies namely 2B1B1, 4D3A3, 5G1F8, and 7C6F5 were produced using hybridoma technology. Titers of all four monoclonal antibodies in ELISA reached more than 1.28×106.0. Moreover, all monoclonal antibodies could react specifically with N protein expressed by transfection of pcDNA3.1-N into BHK-21 cells in IPMA and IFA. These results indicated that water-soluble N protein retained high immunogenicity and possessed the same epitopes as that of native N protein on virions. In addition, the preparation of water-soluble N protein and its monoclonal antibodies laid the basis for the development of immunoassays for COVID-19 detection.

Ograniczanie wyników

2 Polish Journal of Veterinary Sciences

2 Jin Q. Y.

2 Li P.

2 Wang Y. B.

1 Chai S. J.

1 Chen L. P.

1 Fan Z. X.

1 Feng L. L.

1 Liu X. Y.

1 Shi J. J.

1 Teng M.

1 Wang L. P.

1 Wang S. W.

1 Xing G. X.

1 Yang J. F.

1 Yin Y.

1 Zhang F. Q.

1 Zhang G. P.

1 2023

1 2022

Wyniki wyszukiwania

Rapid screening of monoclonal antibodies against porcine circovirus type 2 using colloidal gold-based paper test

Expression of water-soluble nucleocapsid protein of SARS-CoV-2 and analysis of its immunogenicity