 |
Engineered Antibodies to Biomarkers of Laser-induced Retinal Damage
The misuse of directed energy, whether intentional or accidental, constitutes a threat to aircraft pilots. Considering the reduction in cost of high power lasers and their wide spread use, it is important to develop a simple, effective assay for detecting retinal damage to pilots as a way of assessing their ability to operate an aircraft. Currently there are no rapid methods to detect subclinical laser ablation to the retina other than visual checks by an ophthalmologist. To develop sensitive immunoassays to retinal injury biomarkers present in serum, saliva, or tear duct fluids, we are currently exploring a two-step process.
In the first step, we have generated monoclonal antibodies to short peptides of four retinal proteins that have been identified in serum by LC/MS after laser-induced retinal damage in rabbits in the laboratory of our collaborator. A phage-display library, displaying single-chain Fragments of variable regions (scFv), was used for affinity selection against synthetic peptides corresponding to the identified biomarkers. Fluorescence polarization or photonic crystal biosensing was then used to determine the dissociation constants of the antibody clones. The best were used in western blots of rabbit and mouse retinal lysates to demonstrate recognition of the endogenous protein. These antibodies are then enhanced through mutagenic PCR, chain shuffling, or converted to a novel scFv-Fc divalent scaffold. Surface plasmon resonance is then used to obtain more precise affinity measurements. We have previously generated scFvs to short peptide segments of membrane proteins, and found them to bind to the native antigen.
The second step will involve the formatting of an immunoassay where engineered scFv or scFv-Fc antibodies are incorporated into a biosensor platform.
| 
|
Lab Researcher:Michael Kierny
|
Light-sensitive Neuromodulators
In age-related macular degeneration and certain other retinal diseases, the degeneration process primarily targets the rod and cone photoreceptors, but is thought in many cases to preserve the viability of inner retinal neurons. This raises the possibility that a molecular device, consisting of a light-sensitive receptor ligand (γ-amino butyric acid, GABA) anchored to the ectodomain of GABAA receptors, might serve to restore visual function of the cells possessing these receptors. To generate the anchoring component of such device, we developed affinity reagents based on single-chain Fragments of immunoglobulin variable regions (scFvs). We screened two phage libraries, displaying human scFvs by affinity selection with synthetic peptides corresponding to extracellular regions of the rat α1and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Measurements by Fluorescence Polarization and with the SRU BIND™ System revealed all four scFvs to have low micromolar affinities. These scFvs were capable of detecting the fully folded GABAA receptors expressed in Xenopus laevis oocytes while preserving channel activity. Moreover, the anti-α1 subunit specific scFv, A10 was also capable of detecting endogenously expressed GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to conjugate the light-sensitive receptor ligand to an anchor, we have fused the G8 scFv to the CH2-CH3 hinge domains of human IgG, thus creating an scFv-Fc bivalent fusion scaffold. Preliminary binding studies with the fusion protein indicate a 45-fold increase in affinity of the scFv-Fc protein, with no loss in specificity. Our long-term goal is to conjugate the ligand to these fusion proteins for regulating channel activity in retinal neurons in a light-dependent manner.
| 
|
Lab Researcher:Sujatha Koduvayur
|
|
|
|
|
