Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Immune response against inactivated EVA71 and phage ELISA. (A) The serum titers for the test bleed were much higher than in the preimmune bleed. No significant difference was observed between 1st (after the fourth immunization) and 2nd (after the sixth immunization) bleed samples of different tests. NC, negative control. (B) Phage ELISA was performed to estimate the positive rate of the output phage against the target antigen. A total of 94 phage antibody clones were randomly picked from the 1st-round panning output.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Clone Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Amino acid alignment of EVA71-specific sdAbs. Amino acid sequences of different sdAbs were aligned using the CLC Sequence Viewer (Qiagen). The CDRs were highly variable between each clone and are labeled in yellow. The dots represent conserved amino acids.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Sequencing, Labeling

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Binding affinity and neutralization activity of different sdAbs for EVA71 and other viruses

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Binding Assay, Neutralization, Activity Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Analysis of binding of sdAb-hFc to EVA71 capsid proteins by indirect ELISA and immunoblotting. (A) An ELISA was performed by coating wells with recombinant viral capsid proteins or formalin-inactivated EVA71. Various concentrations of target proteins were added in duplicate and were detected with an HRP assay. The amount of bound target proteins is presented as the average ± standard deviation (SD) OD450. Anti-EVA71 VP1-specific antibody (D5 IgG) was used as a positive control and E18 IgG can recognize conformational epitopes on the surface of EVA71 virions but not individual recombinant EVA71 capsid proteins by ELISA. (B) An ELISA was performed by coating wells with recombinant sdAb-hFc, EVA71 D5, and E18 IgG. Target protein (2 μg/ml) was added in duplicate, and bound proteins were detected with an HRP assay. The amount of bound target protein is presented as the average ± SD OD450. (C) An ELISA was performed by coating wells with recombinant viral proteins. MAB979 antibodies were added in duplicate, and bound antibody was detected with an HRP assay. The amount of bound MAB979 is presented as the average ± SD OD450. Anti-EVA71 VP2-specific antibody (MAB979) was used as a positive control for VP2 adsorption. (D) Formalin-inactivated EVA71 was separated by SDS-PAGE and transferred onto a membrane. The membrane was probed with F1-hFc and MAB979 antibody.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Binding Assay, Indirect ELISA, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation, Positive Control, Adsorption, SDS Page

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Antiviral activity of the tetravalent sdAb-hFc construct F1×F1-Fc against various viruses

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Activity Assay, Construct

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Epitope mapping study by ELISA-based competition binding assay and VP3 overlapping peptides-ELISA. (A) An ELISA-based competitive binding assay was performed by coating wells with formalin-inactivated EVA71. The tested antibodies (1 μM) were competitively bound with or without F1×F1-hFc (0.25 or 0.5 μM), and bound antibodies were detected by an HRP assay. (B) E19 IgG (0.5 μM) was competitively bound with or without F1-hFc at concentrations ranging from 0.25 to 2 μM, and bound E19 IgG was detected by an HRP assay. (C) E19 IgG (0.2 μM) was competitively bound with or without F1×F1-hFc at concentrations ranging from 0.1 to 2 μM, and bound E19 IgG was detected by an HRP assay. (D) Different concentrations of antibodies were incubated with a 20 μg/ml EVA71 VP3 (58 to 74 aa) synthetic peptide-coated plate, and bound antibodies were detected by an HRP assay. Anti-EVA71 VP1 specific antibody (D5 IgG) was used as a negative control. (E) A panel of an EVA71 VP3 peptide library (47 × 15-mer peptides) was used in an overlapping peptide-ELISA for epitope mapping against F1×F1-hFc (200 nM), and bound antibody was detected by an HRP assay.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Competitive Binding Assay, Incubation, Negative Control, Peptide ELISA

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Sequence comparison of VP3 region of different genotype EVA71. VP3 sequences of different genotype EVA71 were aligned using the CLC Sequence Viewer (Qiagen). Dots indicate positions that have a single, fully conserved residue. Strain diversity is labeled in bright green. The discontinuous epitopes of EVA71 VP3 protein recognized by sdAb clone F1 are highlighted in yellow in the aligned sequences. The GenBank accession numbers are AAB39968.1 for EVA71 genotype A, ACD63040.1 for EVA71 genotype B1, AAB39969.1 for EVA71 genotype B2, AFJ40582.1 for EVA71 genotype B3, AAK13008.1 for EVA71 genotype B4, ACK37353.1 for EVA71 genotype B5, ADQ54215.1 for EVA71 genotype C1, ACH87535.1 for genotype C2, AFN20478.1 for EVA71 genotype C3, AGK07542.1 for genotype C4, AFN20481.1 for EVA71 genotype C5.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Sequencing, Labeling

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Tetravalent F1×F1-hFc is more protective than bivalent F1-hFc in vivo. (A and B) A Kaplan-Meier survival curve (A) and disease scores (B) of 3-week-old hSCARB2 transgenic mice intraperitoneally challenged with sdAbs-pretreated EVA71 (EVA71 TW/73/12) are shown. The total number (n) of mice per group from two independent experiments is shown. A log rank test was used to analyze the statistic difference of survival rate. One-way analysis of variance (ANOVA) with Kruskal-Wallis test was used to analyze the statistical differences of the individual groups with disease score. The symbols *, **, and *** are used to indicate P values of <0.05, <0.01, and <0.001, respectively.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: In Vivo, Transgenic Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Single treatment using tetravalent F1×F1-hFc or bivalent F1-hFc can alleviate EVA71 in vivo. (A and B) Kaplan-Meier survival curve (A) and disease scores (B) of 3-week-old hSCARB2 transgenic mice challenged with lethal-dose EVA71 (EVA71 TW/73/12) 1 day after administration of sdAb fusion proteins. (C and D) Kaplan-Meier survival curves for different doses of bivalent F1-hFc (C) and tetravalent F1×F1-hFc (D) administered to EVA71-challenged 3-week-old hSCARB2 transgenic mice. The total numbers (n) of mice per group from two independent experiments are shown. A log rank test was used to analyze the statistical differences in survival rate. One-way ANOVA with Kruskal-Wallis test was used to analyze the statistical differences of the individual groups with disease score. The symbols ** and *** are used to indicate P values of <0.01 and <0.001, respectively.

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: In Vivo, Transgenic Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

doi: 10.1128/AAC.01922-19

Figure Lengend Snippet: Survival days of F1×F1-hFc or minocycline-treated hSCARB2 transgenic mice preinfected with EVA71 mouse-adapted strain MP4. (A) Three-week-old mice preinfected with 1 × 106 PFU of EVA71 were given 200 μg/mouse tetravalent F1×F1-hFc via intraperitoneal injection at 24 h postinfection (n = 6). The symbol *** was used to indicate a P value of <0.001. (B) Three-week-old mice preinfected with 2 × 105 PFU of EVA71 were given 100 μg/mouse tetravalent F1×F1-hFc or 25 mg/kg minocycline via intraperitoneal injection at 4, 24, and 48 h postinfection (n = 7 or 8).

Article Snippet: E. coli -produced recombinant EVA71 viral capsid proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen).

Techniques: Transgenic Assay, Injection