RESUMO
The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax = 100 RU response level (KD(R100)).
Assuntos
Técnicas Biossensoriais , Cinética , Ligantes , Ressonância de Plasmônio de Superfície/métodosRESUMO
A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs.
Assuntos
Eritrócitos/citologia , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/imunologia , Antígenos/imunologia , Eritrócitos/imunologia , HumanosRESUMO
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated monoclonal antibodies from three convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concentration as low as 0.007 micrograms per milliliter. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addition to providing guidance for vaccine design, the antibodies described here are promising candidates for COVID-19 treatment and prevention.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Afinidade de Anticorpos , Antígenos Virais/imunologia , Subpopulações de Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19 , Linhagem Celular Tumoral , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Epitopos/imunologia , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores de Coronavírus , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Herpes simplex virus (HSV)-specific T cells are essential to control and resolve genital herpes (GH). To investigate the potential involvement of gamma delta T cells in GH, T cells were recovered and expanded, by mitogenic stimulation, to T cell lines from the genital lesions of 17 patients with GH and 5 control subjects who had other diseases. Relatively high numbers of gamma delta T cells--predominantly, V gamma 9V delta 2 T cells--were detected only in the T cell lines of the patients with GH. Intralesional V gamma 9V delta 2 T cell clones did not recognize HSV-infected cells, but they showed reactivity to isopentenyl pyrophosphate and Daudi cells. The T cell clones secreted interferon- gamma, tumor necrosis factor- alpha, interleukin (IL)-8, macrophage inflammatory protein-1 alpha, and RANTES (regulated on activation, normally T cell expressed or secreted), but they secreted no or limited IL-4. The results of the present study suggest the infiltration and putative involvement of isopentenyl pyrophosphate-reactive V gamma 9V delta 2 T helper 1-like cells in individuals with GH.