Epitope mapping of serum of mice infected with <i>A. fumigatus</i> on Crf1 and Crf2.

<p>A Epitope mapping membrane (15mer oligopeptide, 3 aminoacid overlap) stained with mouse serum (1∶400). The bound mouse antibodies were detected with goat anti-mouse IgG Fc specific AP conjugated (1∶2000). The stained Crf2 specific epitope was marked with a square. B Aligment of Crf1, Crf2 and Asp f9. The recognized epitopes of Crf1 and Crf2 are marked in red (the shorter polypeptide Asp f9 is shown for comparison, Asp f16 is not shown due to a frameshift resulting in a divergent amino acid sequence compared to Crf1, Crf2 and Asp f9).</p

Immunoblot of the Hyperphage packaged macaque library.

<p>1×10⁹ or 5×10⁹ scFv phage particles or 1×10⁹ Hyperphage particles per lane were separated on a reducing 10% SDS-PAGE, followed by Western blot and detection of wildtype pIII or scFv::pIII fusion using mouse mAb anti-pIII (1∶2000) and goat anti-mouse HRP (1∶5,000).</p

Properties of the Crf2 specific scFv antibody clones.

<p>Abbreviations: n.d. - not determined,</p>1<p>Epitopes were concluded from scFv binding to denatured Crf2 in Western immunoblots (linear epitope) or exclusive binding to native antigen on A. fumigatus in immunofluorescence microscopy (conformational epitope).</p>2<p>Serum half life of the scFvs were determined by incubation in PBS supplemented with human serum at 37°C.</p>3<p>Antigen detection limit of the scFv clones was measured in ELISA. Streptavidin was used for capturing biotinylated Crf2 without denaturation. Detection was performed with the scFv clones.</p

Isolation and analysis of <i>crf2</i>.

<p>A Isolation of <i>crf2</i>. PCR products derived from cDNA using primer sets for the amplification of <i>asp f16</i> (resulting in the amplification of <i>crf2</i>) or <i>asp f9</i> were separated by 1% agarose gel electrophoresis. B Alignments of the nucleotide sequences of <i>crf1</i> gene (NCBI: NC_007194.1, CADRE: AFUA_6G08510), mRNA of <i>crf1</i> (NCBI: AY169706), mRNA of <i>crf2</i>, mRNA of <i>asp f9</i> (NCBI: AJ223327) and mRNA of <i>asp f16</i> (NCBI: AF062651). The primer sequences for the amplification of <i>asp f9</i> and <i>asp f16</i> are underlined. The <i>asp f16</i> reverse primer1 binds directly downstream of the <i>crf1</i> gene stop codon. If the <i>crf1</i> gene sequence is identical with all mRNA sequences the sequences are red. The <i>crf1</i> gene sequence is in black and the mRNA sequences are in blue, if differences between the <i>crf1</i> gene sequence and any mRNA sequence. The leader sequences are marked in green.</p

Classification of the selected anti-Crf2 scFv gene fragments with human gene segments according to VBASE2.

<p>Abbreviations: HV: V (variable) gene segments of the heavy chain; D: D (diversity) gene segment; HJ: J (joining) gene segment of the heavy chain; LV: V gene segment of the light chain; LJ: J gene segment of the light chain; HAL4/7: human, naive antibody gene library; immune library: immune library derived from Crf2 immunized macaque. The germinality index describes the similarity of the anti-Crf2 scFvs to the most similiar human germline genes identified by VBASE2 (<a href="http://www.vbase2.org" target="_blank">www.vbase2.org</a>) indicated by percental identity of the VH or VL framework region.</p

Fluorescence microscopy using anti-Crf2 scFv-Fc fusion proteins.

<p>A Binding of anti-Crf2 scFv-Fc fusion proteins to <i>A. fumigatus</i> (left panel brightfield, right panel FITC). B Binding of MS112-IIB1 to different <i>Aspergillus</i> strains (<i>A. fumigatus</i>, <i>A. nidulans</i>, <i>A. terreus</i>, <i>A. flavus</i>, <i>A. clavatus</i>) and <i>Candida albicans</i>. The staining was performed using 2 µg/mL scFv-Fc fusion protein and goat anti-human IgG (Fc specific) conjugated with Alexa 488 (1∶500).</p

Detection of recombinant biotinylated Crf2 in human serum using anti-Crf2 scFvs.

<p>400 ng anti-Crf2 scFvs were coated for capturing. Human serum was spiked with a dilution series of recombinant Crf2. The bound Crf2 was detected with 100 µL streptavidin HRP conjugate (1 µg/mL).</p

Production and purification of Crf2.

<p>IMAC and IEC purified Crf2 was separated on a reducing 12% SDS-PAGE. A Coomassie staining. B the recombinant Crf was detected with mouse anti-his tag (1∶10,000) and goat anti-mouse IgG (Fc specific) AP conjugate (1∶10,000).</p

Analysis of the anti-Crf2 scFv stability by titration ELISA.

<p>Antigen: 400 ng biotinylated Crf2 coated on streptavidin microtitre plates. ScFvs were incubated in PBS (A), human serum (B) and inactivated human serum (C) for 0 to 27 days at 37°C and used for Crf2 detection. The scFvs were detected with mouse anti-myc (1∶1,000) and goat anti-mouse IgG HRP conjugated (1∶10,000).</p