Bioluminescence emission of selected aequorin variants in an <i>in vivo</i> mouse model.

<p>Mice received a 5 μL intrastromal or antechamber injection in the right eye with variant aequorin and the left eye with HEPES Buffer. (A,B) Aequorin injected intrastromally at a concentration of 2.7 x 10⁻⁵ M, 30 s exposure. (C) Aequorin with L-4-methoxyphenylalanine at positions 82 and 86. (D) HEPES Buffer only in both eyes. (E) Aequorin injected in the antechamber at a concentration of 3.2 x 10⁻⁵ M, 60 s exposure. (F) Aequorin with L-4-iodophenylalanine at positions 82 and 86. Images generated with Matlab R2014b and LivingImage 4.4. Reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158579#pone.0158579.ref047" target="_blank">47</a>] under a CC BY license, with permission from <i>Open Access Dissertations</i>, original copyright 2015.</p

Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon‑γ

Bioorthogonal conjugation eliminates the shortcomings of classical conjugation methods. The conjugation of antibodies to reporter proteins, such as bioluminescent protein, can be controlled with orthogonal conjugation methods. Here we report a bioluminescent immunoassay for the sensitive detection of interferon-γ (IFN-γ) that utilizes orthogonal conjugation of bioluminescent protein, <i>Gaussia</i> luciferase to anti-IFN-γ antibody. The IFN-γ is produced by the immune system and the detection of the IFN-γ is pivotal for the detection of persistent viral and bacterial infections. A bioorthogonal conjugation approach is used to conjugate an anti-IFN-γ antibody with a GLuc mutant containing the N-terminal tyrosine using formylbenzene diazonium hexafluorophosphate reagent (FBDP) in hydrophilic mild pH environment yielding high conjugation efficiency (60%). This reagent is shown to be specific for tyrosine (Tyr) residues. Therefore, conjugation through Tyr was orthogonal and not detrimental to the bioluminescence activity of GLuc. The immunoassay described in this paper is a sandwich type assay and involves a capture and a detection antibody. The assay was validated for its robustness, precision, accuracy, limit of detection, and recovery

Emission characteristics of selected aequorin variants.

<p>Emission characteristics of selected aequorin variants.</p

Structures of wild-type aequorin and the non-natural amino acid analogs employed for site-selective incorporation into the protein.

<p>(A) Crystal structure of aequorin with the location of the Tyr82-His16-Trp86. The substituted tyrosine is at position 82 and the substituted tryptophan is at position 86. (B) Chemical structure of non-natural amino acids: L-4-aminophenylalanine, (C) L-4-bromophenylalanine, (D) L-4-iodophenylalanine, (E) L-4 methoxyphenylalanine (PDB ID: 1EJ3). Reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158579#pone.0158579.ref047" target="_blank">47</a>]under a CC BY license, with permission from <i>Open Access Dissertations</i>, original copyright 2015.</p

Increased recruitment of systemically infused Ac-G5-sE-sel nanocarrier-coated BMC into grafted corneas enhances neovascularization.

<p><i>Top</i>: increased recruitment of Ac-G5-sE-sel coated EGFP⁺ BMC to the grafted cornea compared to cells coated with control nanocarrier. <b>A & B.</b> Representative images. The enlarged box is an area along the surgical incision that shows the enhanced recruited cells coated with Ac-G5-sE-sel. <b>C.</b> Quantitative intensity of the recruited EGFP⁺ BMC (mean ± SD). <i>Bottom</i>: Enhanced corneal neovascularization. Blood vessel density was measured by DiI perfusion and confocal microscopy. <b>D, E, F.</b> Representative images of neovascularization 2-days post injection of BMC either uncoated or coated with Ac-G5-sE-sel nanocarrier, respectively (OD (oculus dexter (the right eye))-sutured cornea; OS (oculus sinister (the left eye))-non-sutured eye). <b>G.</b> Fluorescent intensity of new-vessels in two groups mean ± SD, n = 5 mice/group.</p

Increased expression of E-selectin ligand, CD44, in luminal EC of skin wounds compared to normal skin.

<p>A. Co-expression (yellow) of CD44 (green) and KDR (red) in vessels was detected by immunostaining. Representative images were showed (n = 6 mice/group). The lower panels are enlarged highlighted areas in the upper panels. B. Quantification of CD44 expression in vessels. Data are presented as mean ± SD of KDR (red fluorescence intensity), CD44 (green fluorescence intensity) and ratio of CD44: KDR signals from 5 random selected sections of low power field (LPF, X 10).</p