Ocular toxicity of intravitreal clarithromycin.

OBJECTIVE: To investigate the ocular toxicity and clearance of intravitreal clarithromycin lactobionate (Klaricid) and to determine the highest nontoxic dose. MATERIALS AND METHODS: To evaluate toxicity, 24 New Zealand white rabbits were divided into six groups (four rabbits each). Rabbits were examined preoperatively and electroretinography (ERG) was performed. The left eyes of the animals served as controls and received intravitreal injection of 0.1 mL sterile water. Klaricid (0.1 mL) was injected into the midvitreous cavity of the right eyes at concentrations of 25 microg, 250 microg, 500 microg, 1.0 mg, 2.0 mg, and 4.0 mg/0.1 mL. The animals were followed up to 15 days postinjection by clinical examination and ERG. The animals were killed and the eyes were enucleated and processed for light microscopy. Ten New Zealand rabbits were used for the vitreous clearance study as drug test rabbits and two additional rabbits were used to generate control retina and vitreous. The highest nontoxic dose (1 mg) was injected into the vitreous and the concentration of clarithromycin in the vitreous was determined using high-performance liquid chromatography at various time intervals after injection. RESULTS: Cataract occurred after intravitreal doses of 2.0 and 4.0 mg. Electroretinography showed decreasing b-wave amplitude with both dark- and light-adapted stimulus in the 4.0-mg group; it was normal in other groups. Histopathologic sections showed localized retinal necrosis and disorganization with the 2.0 and 4.0 mg dosage. No histologic changes were found in the other groups. The half-life of intravitreal clarithromycin was found to be 2 hours. No metabolites of clarithromycin were observed in the vitreous samples. CONCLUSION: Intravitreal clarithromycin lactobionate is nontoxic to rabbit eyes up to a dose of 1.0 mg. Because of its broad-spectrum antibiotic effect and appropriate half-life in the vitreous, it may be a good choice for intravitreal treatment of susceptible organisms.

Surfaces modified with PEO by the Williamson reaction and their affinity for proteins.

Poly(ethylene oxide) (PEO) was immobilized by the Williamson ether synthesis onto halogenated surfaces such as poly(vinylidene chloride), surface-brominated polypropylene, and surface-brominated poly(ethylene terephthalate). PEO (Mw 20,000) was converted to its sodium salt and reacted for various times with the halogen-containing surfaces at 95 degrees C in the melt, or as a solution in 2-methoxyethyl ether. X-ray photon spectroscopy and water contact-angle measurements confirmed the surface-immobilization of PEO. The adsorption from phosphate buffer of human serum albumin, immunoglobulin G (IgG) and fibrinogen, and murine IgG was reduced after modification of the surfaces. In addition, IgG adsorption from pooled human sera was diminished. The difference in protein adsorption between test samples and controls exhibited a strong dependence on input protein concentration.

Specific activity of polypyrrole nanoparticulate immunoreagents: comparison of surface chemistry and immobilization options.

Polypyrrole-based colloids with differing surface chemistries were compared with respect to the specific activity of immobilized antibody. Monoclonal antibody to the alpha subunit of human chorionic gonadotropin (hCG) was modified by incorporation of cystamine into the Fc-carbohydrate, followed by reduction with dithiothreitol resulting in the generation of 4.5 free thiols per IgG. The reduced IgG was added to clean, unmodified and surface-modified polypyrrole colloids. Functionalized colloids included carboxylate-modified polypyrrole, poly[pyrrole-co-1-(2-carboxyethyl) pyrrole]-silica composite, and amine forms of the carboxylated colloids. The amine-functionalized colloids were subsequently treated with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to provide thiol-reactive maleimide surface groups. Following the conjugation of IgG to the colloids, bound and soluble antibody activity was quantitated using a sequentially competitive immunoassay for hCG, based on an automated commercial hCG kit. The results indicated that all forms of polypyrrole retained the equivalence of between 12 and 33 micrograms of IgG activity/mg of colloidal solids, relative to the unmodified soluble IgG.

Absorption-enhanced solid-phase immunoassay method via water-swellable poly(acrylamide) microparticles.

Water-swellable hydrogel microspheres based on cross-linked polyacrylamide were used as solid supports in immunoassay formats. Capture antibody was covalently bound at the surfaces and the pore size of particles was such that these antibodies were excluded from the interiors. Upon contact with a solution containing immunological reactants, water molecules quickly penetrated the microspheres, causing them to swell, thereby concentrating analyte at the surface. Using swellable particles, the antigen capture rate during the first 5 min of incubation with antigen was two times that of antibody-coated 0.79 cm polystyrene beads. In addition, antibodies attached to lightly cross-linked swellable microparticles proved resistant to inactivation by ultrasonic energy, which can be used to accelerate immunological interactions.

Immunoassay employing surface-enhanced Raman spectroscopy.

Surface-enhanced Raman scattering (SERS) was used to measure binding between biomolecules with mutual affinity, including antigen-antibody interactions. The conjugation of nitro groups onto bovine serum albumin enhanced their specific SERS activity 10(4)-fold. A dye, 2-[4'-hydroxyphenylazo]benzoic acid (HABA), with a major absorption at the Raman excitation frequency, demonstrated surface-enhanced resonance Raman scattering (SERRS) when captured from solution by avidin-coated silver films. Individual peak intensities showed a logarithmic relationship to the HABA concentration in solution over the range 10(-8) to 10(-5) M. Another resonance dye, p-dimethylaminoazobenzene (DAB) was covalently attached to an antibody directed against human thyroid stimulating hormone (TSH), without loss of antibody activity. The resultant conjugate was used in a sandwich immunoassay for TSH antigen: silver surfaces coated with anti-TSH antibody captured TSH antigen which in turn captured the DAB-anti-TSH antibody conjugate. A linear relationship was observed between the intensity of the resultant SERRS signals and the TSH antigen concentration over a range of from 4 to 60 microIU/ml. These results demonstrate the potential utility of the SERRS effect as a readout in a one-step, no wash immunoassay system.

The immunochemistry of sandwich-ELISAs. IV. The antigen capture capacity of antibody covalently attached to bromoacetyl surface-functionalized polystyrene.

The antigen capture capacity of antibodies covalently immobilized on injection-molded polystyrene beads was evaluated. Bromoacetyl groups on the bead surfaces rendered them reactive to protein nucleophilic groups. The bromoacetyl surface exhibited up to a ten-fold greater capacity for protein compared to unmodified polystyrene, with no detectable dissociation such as occurs with simple adsorption. Biotinylated anti-fluorescein was immobilized on this surface both through direct covalent attachment and indirectly via streptavidin, which was first covalently attached to the bead. Comparisons of the resulting biological activity, normalized to the amount of anti-fluorescein on the bead, were made between the attachment methods and simple passive adsorption. The presence of the streptavidin spacer on the bromoacetyl surfaces improved the antigen capture capacity of antifluorescein, for fluoresyl-albumin by 45% compared to direct covalent linkage of the antibody to modified polystyrene and by 160% relative to antibody adsorbed on unmodified polystyrene.

2,3-Diaminophenazine is the product from the horseradish peroxidase-catalyzed oxidation of o-phenylenediamine.

NMR and mass spectroscopic evidence has been obtained which indicates that the product of the oxidation of o-phenylenediamine by hydrogen peroxide, uncatalyzed or catalyzed by horseradish peroxidase, is 2,3-diaminophenazine. These results settle disparate literature descriptions. The process is most likely free radical in nature starting with the abstraction of a labile amino hydrogen atom.