Sandwich ELISA was applied for the expression verification of the spike protein. Rabbit anti-SARS-CoV-2 S protein polyclonal antibody was used to coat a 96-well plate. Then, the produced Model-S was placed with different diluted ratios after washing and blocking the plates. After that, the Rabbit anti-M13 pAb-HRP was added and aimed to form a sandwich structure. Followed by the reaction with the TMB substrate, OD values at 450 nm were collected and analyzed to decide whether the Model-S could express the spike protein of SARS-CoV-2.
In order to estimate the infection ability of the virus-like model, the plaque assay was used to determine the titer of the produced Model-S since it is the gold standard for titer measurement. The displayed phages Model-S from different dilution ratios were employed to infect E. coli TG1. After that, LB solid culture media with kanamycin was applied to provide a space for cultivation. Moreover, the mixtures were cultivated at 37 °C overnight. The plates were collected, and all the plaques were counted to obtain the titer of Model-S the following day.
The molecular interaction instrument ForteBio Octet K2 (SARTORIUS, Göttingen, Germany) was used to assess the combination between the produced Model-S and the corresponding anti-SARS-CoV-2 spike protein antibody (Rabbit pAb). The interaction between these biomolecules could be monitored and collected through the shift of the probe surface reflection interference spectrum following the thin film interferometry technology. The process, including association, dissociation, and regeneration, was performed repeatedly according to the preprogramming. After the measurement, the association constant (ka) and dissociation constant (kd) were obtained. Moreover, the affinity constant could be calculated.
After production and verification, the two virus-like models: Model-N [45 (link)] and Model-S, were prepared. Accompanied by these two models (as biothreats), M13 phage, bovine serum albumin (BSA), ovalbumin (OVA), commercial N protein, commercial S1 protein, commercial S2 protein, and E. coli TG1 were used as control samples for further study. All selected samples are related to the actual SARS-CoV-2 virus. The produced Model-S and Model-N are the substitutions of SARS-CoV-2 in this research, which could be regarded as the combination of M13 phage (main body) and N/S protein (p3 site). The M13 phage used in the phage display to synthesize the models is a kind of virus that belongs to the virus family, as SARS-CoV-2 does. BSA and OVA are common proteins for scientific research. In addition, the N protein, S1 protein, and S2 protein are the structural proteins of SARS-CoV-2. Furthermore, as common pathogen types, both bacteria and viruses can cause the outbreak of large-scale infectious diseases. Thus, E. coli TG1 was also included for identification research since one of the hosts of the M13 phage is E. coli TG1, and it was also used to produce the models. Therefore, the nine selected samples are more or less related to the SARS-CoV-2 virus. The higher the relation between the sample and the SARS-CoV-2 virus (or its substitution), the more difficult it might be to distinguish. In general, the nine selected samples, including viruses, proteins, and bacteria, might be representative of the research. The recognition of the Model-S and Model-N among those related samples could provide a possibility for the nondestructive detection and identification of targeted biothreats. Therefore, the nine samples were prepared for spectroscopy analysis.
In order to estimate the infection ability of the virus-like model, the plaque assay was used to determine the titer of the produced Model-S since it is the gold standard for titer measurement. The displayed phages Model-S from different dilution ratios were employed to infect E. coli TG1. After that, LB solid culture media with kanamycin was applied to provide a space for cultivation. Moreover, the mixtures were cultivated at 37 °C overnight. The plates were collected, and all the plaques were counted to obtain the titer of Model-S the following day.
The molecular interaction instrument ForteBio Octet K2 (SARTORIUS, Göttingen, Germany) was used to assess the combination between the produced Model-S and the corresponding anti-SARS-CoV-2 spike protein antibody (Rabbit pAb). The interaction between these biomolecules could be monitored and collected through the shift of the probe surface reflection interference spectrum following the thin film interferometry technology. The process, including association, dissociation, and regeneration, was performed repeatedly according to the preprogramming. After the measurement, the association constant (ka) and dissociation constant (kd) were obtained. Moreover, the affinity constant could be calculated.
After production and verification, the two virus-like models: Model-N [45 (link)] and Model-S, were prepared. Accompanied by these two models (as biothreats), M13 phage, bovine serum albumin (BSA), ovalbumin (OVA), commercial N protein, commercial S1 protein, commercial S2 protein, and E. coli TG1 were used as control samples for further study. All selected samples are related to the actual SARS-CoV-2 virus. The produced Model-S and Model-N are the substitutions of SARS-CoV-2 in this research, which could be regarded as the combination of M13 phage (main body) and N/S protein (p3 site). The M13 phage used in the phage display to synthesize the models is a kind of virus that belongs to the virus family, as SARS-CoV-2 does. BSA and OVA are common proteins for scientific research. In addition, the N protein, S1 protein, and S2 protein are the structural proteins of SARS-CoV-2. Furthermore, as common pathogen types, both bacteria and viruses can cause the outbreak of large-scale infectious diseases. Thus, E. coli TG1 was also included for identification research since one of the hosts of the M13 phage is E. coli TG1, and it was also used to produce the models. Therefore, the nine selected samples are more or less related to the SARS-CoV-2 virus. The higher the relation between the sample and the SARS-CoV-2 virus (or its substitution), the more difficult it might be to distinguish. In general, the nine selected samples, including viruses, proteins, and bacteria, might be representative of the research. The recognition of the Model-S and Model-N among those related samples could provide a possibility for the nondestructive detection and identification of targeted biothreats. Therefore, the nine samples were prepared for spectroscopy analysis.
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