ELISA for Hepatitis C Virus Antigens

Microtiter plate wells (Corning) were coated with 1 μg GNA-lectin (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) overnight at 4°C and then blocked for 1 h with 4% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS containing 0.2% Tween 20 (PBST). After washing of the wells with PBST, WT or ΔHVR1 gpE1/gpE2 antigens (100 ng/well) were added for 1 h. gpE2-specific MAbs (H77.16, AP33, HC33.1, HC33.4, HC84.26, and AR3b) (16 (link), 52 (link), 53 (link), 55 (link), 56 (link)), gpE1/gpE2-specific MAbs (AR4a and AR5a) (54 (link)), or a control MAb (B6) (16 (link)) was added for 1 h (50 μl/well) and detected by an anti-human or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000; Jackson Immuno Research, West Grove, PA, USA) and KPL peroxidase substrate (SeraCare Life Sciences, Milford, MA). The absorbance (450 to 570 nm) was read by using an Enspire plate reader (Perkin-Elmer, Waltham, MA, USA).

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Law J.L., Logan M., Wong J., Kundu J., Hockman D., Landi A., Chen C., Crawford K., Wininger M., Johnson J., Mesa Prince C., Dudek E., Mehta N., Tyrrell D.L, & Houghton M. (2018). Role of the E2 Hypervariable Region (HVR1) in the Immunogenicity of a Recombinant Hepatitis C Virus Vaccine. Journal of Virology, 92(11), e02141-17.

Publication 2018

GNA-lectin bovine serum albumin (BSA) KPL peroxidase substrate Enspire plate reader

Anti antibody Antigens Bovine serum albumin Horseradish peroxidase Human Lectin Mouse Peroxidase Phosphate Saline Tween 20

Corresponding Organization :

Other organizations : University of Alberta

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Variable analysis

independent variables

Presence or absence of ΔHVR1 gpE1/gpE2 antigens

dependent variables

Binding of gpE2-specific MAbs (H77.16, AP33, HC33.1, HC33.4, HC84.26, and AR3b) and gpE1/gpE2-specific MAbs (AR4a and AR5a) to WT or ΔHVR1 gpE1/gpE2 antigens
Absorbance (450 to 570 nm) measured using an Enspire plate reader

control variables

Coating of microtiter plate wells with 1 μg GNA-lectin in phosphate-buffered saline (PBS) overnight at 4°C
Blocking of wells with 4% bovine serum albumin (BSA) in PBS containing 0.2% Tween 20 (PBST) for 1 h
Addition of 100 ng/well of WT or ΔHVR1 gpE1/gpE2 antigens for 1 h
Addition of monoclonal antibodies (MAbs) (50 μl/well) for 1 h
Detection of bound MAbs using an anti-human or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000) and KPL peroxidase substrate

positive controls

GpE2-specific MAbs (H77.16, AP33, HC33.1, HC33.4, HC84.26, and AR3b)
GpE1/gpE2-specific MAbs (AR4a and AR5a)

negative control

Control MAb (B6)

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