Journal: Annals of Laboratory Medicine

Article Title: Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

doi: 10.3343/alm.2014.34.2.118

Figure Lengend Snippet: Alignment of the nucleotide sequences of the codon-optimized truncated ORF2 gene and the original truncated ORF2 gene by CLC sequence viewer 6.5.3 (CLC bio, Aarhus, Denmark). Abbreviaition: ORF, open reading frame.

Article Snippet: The pBluescript II SK (+) vector carrying the optimized ORF2.1 gene (pBluescript II SK-ORF2.1) was digested by NdeI and XhoI restriction enzymes (New England BioLab, Ipswich, MA, USA).

Techniques: Sequencing

Journal: Annals of Laboratory Medicine

Article Title: Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

doi: 10.3343/alm.2014.34.2.118

Figure Lengend Snippet: PCR amplification of ORF2.1 (amino acids 112-660) and ORF2.2 (amino acids 112-608) with T7 promoter and T7 terminator primers. Lanes 1 and 7, 1-kb DNA ladder (Vivantis, Selangor Darul Ehsan, Malaysia); Lanes 2 and 3, amplified ORF2.1 gene; Lanes 4-6, amplified ORF2.2 gene. Abbreviation: ORF, open reading frame.

Article Snippet: The pBluescript II SK (+) vector carrying the optimized ORF2.1 gene (pBluescript II SK-ORF2.1) was digested by NdeI and XhoI restriction enzymes (New England BioLab, Ipswich, MA, USA).

Techniques: Amplification

Journal: Annals of Laboratory Medicine

Article Title: Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

doi: 10.3343/alm.2014.34.2.118

Figure Lengend Snippet: Proteins expressed using pET30a-ORF2.1 recombinant expression vector and pET30a-ORF2.2 recombinant expression vector in E. coli BL21 (DE3) competent cells. The expressed and purified proteins were analyzed by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R250. The ORF2.1 and ORF2.2 proteins bands are visualized at approximately 60 and 55 kDa, respectively. Lanes 1-4, purified eluates of ORF2.2 protein from the Ni column; Lane 5, washing step; Lane 6, flow-through; Lane M, pre-stained protein ladder (Fermentas, Vilnius, Lithuania); Lane 7-12, purified eluates of ORF2.1 protein from the Ni column; Lane 13, washing step; Lane 14, flow-through. Abbreviations: ORF, open reading frame; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Article Snippet: The pBluescript II SK (+) vector carrying the optimized ORF2.1 gene (pBluescript II SK-ORF2.1) was digested by NdeI and XhoI restriction enzymes (New England BioLab, Ipswich, MA, USA).

Techniques: Recombinant, Expressing, Plasmid Preparation, Purification, SDS Page, Staining, Polyacrylamide Gel Electrophoresis

Journal: Annals of Laboratory Medicine

Article Title: Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

doi: 10.3343/alm.2014.34.2.118

Figure Lengend Snippet: Western blot analysis of the expressed and purified ORF2.1 and ORF2.2 protein in E. coli . (A) The ORF2.1 protein band is visualized at approximately 60 kDa. Lane 1, induction for 2 hr; Lane 2, induction for 4 hr; Lane 3, uninduced control; Lane 4, BL21 control; Lane M, pre-stained protein ladder (Cinagen, Tehran, Iran). (B) The ORF2 protein band is visualized at approximately 55 kDa. Lane M, pre-stained protein ladder (Cinagen, Tehran, Iran); Lane 1, uninduced control; Lane 2, induction for 4 hr; Lane 3, flow-through; Lane 4, wash; Lane 5-7, the first, the second, and the third eluates. Abbreviation: ORF, open reading frame.

Article Snippet: The pBluescript II SK (+) vector carrying the optimized ORF2.1 gene (pBluescript II SK-ORF2.1) was digested by NdeI and XhoI restriction enzymes (New England BioLab, Ipswich, MA, USA).

Techniques: Western Blot, Purification, Staining

Journal: Annals of Laboratory Medicine

Article Title: Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

doi: 10.3343/alm.2014.34.2.118

Figure Lengend Snippet: Comparison of the immunoreactivities of the 2 in-house ELISAs and DIA.PRO HEV IgG ELISA kit. Data are expressed as OD values of the positive and negative serum samples. The cut-off value is indicated for each assay by a horizontal line (0.35 for the in-house ELISA based on ORF2.1, 0.4 for the in-house ELISA based on ORF2.2, and 0.37 for DIA.PRO HEV IgG ELISA kit). Abbreviations: ORF, open reading frame; OD, optical density.

Article Snippet: The pBluescript II SK (+) vector carrying the optimized ORF2.1 gene (pBluescript II SK-ORF2.1) was digested by NdeI and XhoI restriction enzymes (New England BioLab, Ipswich, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay