2/18/2023 0 Comments Ivcd cell culture![]() Utilizing a stabilized version of the full-length SARS-CoV-2 spike protein, a very robust and accurate serological enzyme-linked immunosorbent assay (ELISA) for antibodies in patient sera has recently been developed (Amanat et al., 2020) and approved for use by the US FDA. The spike glycoprotein that protrudes from the surface of the virus is highly immunogenic with the receptor-binding domain (RBD) being the target of many neutralizing antibodies (Yuan et al., 2020). Immune response represents the first line of defense against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that has caused the coronavirus disease 2019 (COVID-19) pandemic. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32☌ to permit extended duration cultures. We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity.
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