The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation

Figures

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  Figure 1. Humoral immune response in chicken. (A) The EV71 virus particles and BSA were immobilized on ELISA wells. Purified IgY from pre-immunization and each immunization were used to monitor the humoral immune response in chicken from the pre-immunized (None) to the fourth week after the 7th immunization (7th(4)). (B) Purified IgY antibodies from pre-immunization and the 7th immunization were in a series of two-fold dilutions (500× to 256,000×) to test the binding abilities to EV71 virus particles and BSA, respectively. ELISA experiments were performed in a duplicated manner and statistically analyzed as mean ± standard deviation (SD).

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  Figure 2. Determination of the phage titers and their specific binding activity after each round of bio-panning. (A) Two antibody libraries displaying scFv antibodies were constructed with short and long linkers and used for the bio-panning procedure. Recombinant phages were eluted to determine the colony-forming units (CFU) after each round of bio-panning. (B) Each amplified phage containing 1011 to 1012 plaque-forming units (PFU) was used as primary binders against EV71-infected lysates or BSA on phage-based ELISA. Purified IgY from the 7th immunization was used as a positive control. The labels Lib (original library phages) to 5th represent the phages isolated before and after 1 to 5 rounds of bio-panning. The experiments were performed in a duplicated manner and statistically analyzed as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 3. Expression and characterization of scFv antibodies. After bio-panning, 13 randomly selected clones from (A) short linker or (B) long linker library were induced by IPTG for scFv antibody expression. These expressed scFv antibodies were further confirmed using anti-HA antibody on Western blots. The expression of anti-Coxsackievirus 16 (CA16) S1 scFv antibody was included as a positive control (lane P). (C) After sequence analysis and classification, the binding activity of 5 represented clones, EV71S1, EV71L1, EV71L3, EL71L4 and EV71L9 scFv antibodies was tested against the lysate of EV71 infected cells at 1:10 dilution in ELISA. The IgY antibodies from 7th immunized chicken was used as a positive control. ELISA experiments were run in a duplicated manner and statistically analyzed as mean ± SD.

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  Figure 4. Purification of EV71S1 scFv antibody. After IPTG induction, the His-fused scFv was purified using Ni2+ sepharose and analyzed on SDS-PAGE stained with Coomassie blue dye. The purity of recombinant scFv antibody is genuinely high as demonstrated in lane 3. The scFv antibody was not entirely removed from the Ni2+ sepharose after elution (lane 4). Lane 1: flow through. Lane 2: washing. Lane 3: elution. Lane 4: sepharose.

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  Figure 5. Cross-reactive analysis of IgY and EV71S1 antibodies against Enteroviruses 71. The EV71-infected, CA16-infected, non-infected cell lysates and BSA were coated on ELISA wells, respectively. The purified (A) IgY and (B) EV71S1 scFv antibodies were used for probing the viral proteins in lysates. *: p < 0.05. **: p < 0.01. (C) After SDS-PAGE separation, the PVDF membranes immobilized with EV71-infected (lane 1), CA16-infected (lane 2), and non-infected cell lysates (lane 3) were detected by purified (D) IgY and (E) EV71S1 scFv antibodies. ELISA experiments were run in a duplicated manner and statistically analyzed as mean ± SD.

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  Figure 6. Analysis of IgY and scFv antibodies neutralizing activity. The EV71-infected RD cells were incubated with various concentrations of antibodies. After incubation, the density of the well at 570 nm was measured and the 50% inhibitory concentrations (IC50) were calculated according to the formula described in the material and method. All experiments were carried out in a triplicated manner.