Journal: BMC Biotechnology

Article Title: Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

doi: 10.1186/1472-6750-8-85

Figure Lengend Snippet: Neutralizing activity of natural (■) and recombinant (○) purified 26A1 IgG . The 26A1 mAb was purified from serum-free supernatants by protein A chromatography. The neutralizing activity of HCMV infectivity was then tested either with the laboratory strain AD169 in HELF (panel A) or with the clinical isolate VR1814 in HUVEC (panel B). The effect of 26A1 IgG on HCMV infectivity was measured by staining the HCMV IEA by indirect immunoperoxidase. Five fields (HPF 20×) were counted per well from triplicate wells. Percentage of inhibition of viral infectivity was calculated using the following formula: [(means ± s.d. of IEA + nuclei of HCMV infected cells – means ± s.d. of IEA + nuclei of 26A1-treated HCMV infected cells/means ± s.d. of IEA + nuclei of HCMV infected cells) ×100]. Results represent the means ± s.d. of 3 independent experiments. Plaque reduction assay (panel C) shows the neutralizing activity of natural and recombinant 26A1 mAb against HCMV AD169 (1,000 pfu/rxn) in HELF. The values were plotted as a function of IgG concentration, and the concentrations of IgG required to inhibit viral infection by 50% (IC 50 ) were calculated by linear regression using GraphPad Prism (v. 4.0) software.

Article Snippet: The effect on HCMV infectivity was determined by an HCMV microneutralization assay based on the evaluation of the ability of clone supernatants to interfere on the infectivity of HCMV AD169 strain (a laboratory strain from ATCC, cod.

Techniques: Activity Assay, Recombinant, Purification, Chromatography, Infection, Staining, Inhibition, Concentration Assay, Software

Journal: PLoS ONE

Article Title: Exploitation of Herpesviral Transactivation Allows Quantitative Reporter Gene-Based Assessment of Virus Entry and Neutralization

doi: 10.1371/journal.pone.0014532

Figure Lengend Snippet: ( A ) Human MRC-5 cells, transiently transfected with the pTA-Control plasmid (left panel) or pTA-GAS (right panel), were split into aliquots and seeded. Cells were infected with 1 PFU/cell HCMV-AD169, HCMV-Towne, HCMV-TB40/E (black bars) or left uninfected (white bars). After 24 h the cells were treated for additional 5 h with 500 U/ml human IFN-γ. Cells were lysed and luciferase activity was measured. The experiment was performed in triplicate and the arithmetic mean with the standard deviation is shown. The significance was tested with the Student's t-test: * p<0.05 and ** p<0.01. ( B ) ARPE-19, CV-1, Vero and Jurkat cells were transfected with 2.5 µg pTA-Control plasmid using the Lonza nucleotransfection system. Cells were split so that parallel measurements and infections originated from the same transfection. 20 h post transfection cells were left uninfected (mock; open bars) infected with 3 PFU/cell HCMV-AD169, HSV-1 F strain (black bars) or UV-inactivated virus (hatched bar) for additional 20 h. Cells were lysed and luciferase activity was measured. ( C ) MRC-5 cells were infected with 3 PFU/cell HCMV-AD169 or UV-inactivated HCMV-AD169. After the indicated time cells were lysed and total RNA or protein was prepared. Protein lysates were subjected to native sodium-deoxycholate-PAGE for the analysis of IRF-3 dimerization or to SDS-PAGE for the analysis of pp72-IE1 and β-actin protein amount. Proteins were transferred to filters and probed with the indicated antibodies. IFN-β and GAPDH mRNA was assessed by semi-quantitative RT-PCR from total RNA as. For GAPDH two log 10 dilutions were used as template to confirm measurement in the linear amplification range.

Article Snippet: Purified stocks of HCMV strains AD169 , Towne (ATCC VR-977), the BAC-derived HB5 and the BAC-derived endotheliotropic strain TB40/E were used.

Techniques: Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, Standard Deviation, SDS Page, Quantitative RT-PCR, Amplification