Relative binding responses for Z domain variants to mouse IgG₁.

<p>Solutions of Z domain variants (2 µM concentration) obtained after single amino acid substitutions at the seven targeted positions (K4, F5, Q9, Q10, F13, H18 and K35) were injected over a sensor chip surface containing immobilized mIgG₁ monoclonal antibody protein and the equilibrium responses (plateau values) were recorded and normalized to the response values obtained for the wild type Z domain (WT) A blank surface (activated/deactivated) was used as negative control and used for buffer effect subtraction. (<b>A</b>) A representative overlay sensorgram from injections of Z_WT, Z_F5R and Z_F5I variants over mIgG₁ monoclonal protein showing higher equilibrium response values for the two mutant variants than for the Z_WT domain. The response obtained from buffer injection only is also indicated (BUFFER). (<b>B</b>) Results from the analysis of the different variants. The horizontal line in each panel corresponds to the normalized response obtained for the wild type Z domain ( = 100).</p

Photo-conjugation efficiencies for the different Z domain probes to mouse IgG₁.

<p>The efficiencies of photoconjugation were analysed through scanning of the gel images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056597#pone-0056597-g005" target="_blank">Figure 5</a> using ImageJ software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056597#s4" target="_blank">Materials and methods</a> section). (<b>A</b>) Photo-conjugation efficiencies for 16 probe variants, derived from either the Z_WT-domain (white bars) or the Z_F5I-domain, containing the F5I substitution (black bars). The position at which a cysteine was introduced to site-specifically position the MBP probe is indicated for each probe. (<b>B</b>) Mean values of photo-conjugation efficiencies (%) from the use of either the Z_WT-Q32C-MBP (white bar; mean value  =  45.4±4.1%) or Z_F5I-Q32C-MBP (black bar; mean value  =  64.4±5.3%) probes to a large panel of different mIgG₁ antibodies (16 of the mAbs seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056597#pone-0056597-g005" target="_blank">Figure 5B</a>).</p

Analysis of labeling and comparison of the antigen binding potency of differently biotinylated antibody samples.

<p>Two samples of a mouse anti-human interferon-gamma mIgG1 mAb were biotinylated using either a direct or a Z_{F5I-Q32C-MBP-BIO} probe-mediated strategy and the resulting preparations compared with respect for their antigen binding potency. (<b>A</b>) Western blotting analysis of the samples and the probe, using a streptavidin horse radish peroxidase (HRP) conjugate. Lane 1: blank; lane 2: a sample of the anti-human interferon-gamma mIgG1 mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent resulting in "global" biotinylation of both heavy (<i>ca.</i> 50 kDa) and light chains (<i>ca.</i> 25 kDa); lane 3: the Z_{F5I-Q32C-MBP-BIO} probe alone, biotinylated using the same amine reactive sulfo-NHS-ester-biotin reagent; lane 4: a sample of the anti-human interferon-gamma mIgG1 mAb after photoconjugation to the Z_{F5I-Q32C-MBP-BIO} probe (ca. 8 kDa) showing a selective biotinylation of only the heavy chains (<i>ca.</i> 50 + 8 kDa). A small amount of unreacted probe is also visible. (<b>B</b>) Biosensor analysis of the relative antigen binding potency of the biotinylated antibody preparations. Using a streptavidin coated biosensor chip, the biotinylated antibody proteins were selectively immobilized onto separate sensor chip surfaces, followed by injection over both surfaces of a common 7.5 nM solution of the antigen human interferon-gamma. This allowed for direct comparison of the effect on the antigen binding potency from the different biotinylation strategies. Sample (i): the anti-human interferon-gamma mIgG₁ mAb biotinylated using a conventional amine reactive sulfo-NHS-ester-biotin reagent; sample (ii): the anti-human interferon-gamma mIgG₁ mAb biotinylated via photoconjugation using the Z_{F5I-Q32C-MBP-BIO} probe.</p

Photo-conjugation of Z domain probes to mouse IgG₁.

<p>(<b>A</b>): Analysis by SDS-PAGE of the mouse IgG₁ mAb photocoupling efficiency of nine different Z_WT- or Z_F5I-based probes, differing in the position of the maleimide benzophenone (MBP) group. As indicated, variants containing the MBP group at positions 2, 3, 4, 9, 11, 15, 24 or 32 were investigated. (<b>B</b>): Analysis by SDS-PAGE of the photocoupling efficiency of the Z_WT-Q32C-MBP and Z_F5I-Q32C-MBP probes, both containing the MBP group at position 32, to 19 different mouse IgG₁ mAbs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056597#s4" target="_blank">Material and Methods</a> for a list). The designations HC+P, HC, LC and P, refer to heavy chain+probe, heavy chain, light chain and probe, respectively. The M lanes refer to marker protein with molecular weights in kDa as indicated.</p

Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG₁

<div><p>Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in <i>E. coli</i> and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs). The best variant, denoted Z_F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z_F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z_F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z_F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.</p></div

Structure and sequence data.

<p>(<b>A</b>): Computer graphic representation of a part of the complex between a single staphylococcal protein A domain (domain B, closely related to the Z domain used in the present study) (yellow) and human IgG₁ Fc (brown) (PDB file: 1FC2.pdb). The amino acid side chains corresponding to the seven positions addressed for substitutions in the Z domain are highlighted in cyan. Highlighted in purple, red and green, respectively, are three IgG1 Fc subregions in close contact with the B domain. (<b>B</b>): Alignment of amino acid sequences of Fc regions of human IgG₁, mouse IgG₁, mouse IgG_2a and mouse IgG_2b, respectively, corresponding to the three contact areas shown in (A), using the same colour code. (<b>C</b>): Amino acid sequence of the 58-residue Z domain, with the seven positions included in the engineering boxed. Indicated with red dots are the ten positions at which unique cysteine residues were introduced for site-specific labeling with a photoactivable maleimide benzophenone (MBP) group.</p

Binding of antibodies to immobilized Z domain variants.

<p>(<b>A-D</b>): Overlay of sensorgram traces obtained after injection of four different mouse IgG₁ monoclonal antibodies (one per panel) over separate sensor chip surfaces containing similar amounts of the variants Z_F5I, Z_F5R or the wild type Z domain (Z_WT), respectively. (E): Overlay of sensorgram traces obtained after injection of a TNF-alpha receptor:human IgG1 Fc fusion protein (etanercept, Enbrel®) over separate sensor chip surfaces containing similar amounts of the variants Z_F5I, Z_F5R or the wild type Z domain (Z_WT), respectively.</p

Carbon Nanotubes Filled with Different Ferromagnetic Alloys Affect the Growth and Development of Rice Seedlings by Changing the C:N Ratio and Plant Hormones Concentrations - Fig 5

<p>Transmission electron micrographs of untreated rice roots (A), rice roots treated with MWCNTs (B), Fe-CNTs (C), FeCo-CNTs (D), EDS spectra of FeCo-CNTs in rice roots (E). CNTs are circled in images, cw: cell wall.</p

C: N ratio in rice roots and shoots after treatments with three different carbon nanotubes.

<p>C: N ratio in rice roots and shoots after treatments with three different carbon nanotubes.</p