Intravitreal infliximab clearance in a rabbit model: different sampling methods and assay techniques.
OBJECTIVE
To investigate the clearance of intravitreal infliximab with the use of different sampling techniques and immunoassay methods in rabbits.


METHODS
Infliximab (1.6 mg) was intravitreally injected into both eyes of 47 rabbits. Two approaches were used to collect the vitreous: the classic method and a microsampling technique. Whereas the classic method consists of collection of the whole vitreous after enucleation, the microsampling technique consisted of the aspiration of small (10-15 microL) samples with a 200-microL syringe. Samples were taken from 30 minutes to 40 days using both methods and were then compared. Infliximab concentration was estimated with competitive ELISA, dot blot analysis, and Western blot analysis.


RESULTS
The vitreous half-life of infliximab was estimated to be 6.5 +/- 0.6 days. The data indicated monoexponential decay reaching its conclusion after approximately 40 days. This decay was preceded by 4-day-long diffusion in the vitreous. Microsampling proved to be effective in the vitreous collection, giving statistically comparable signals (+/- 4%, P = 0.68) with respect to the classic procedure. ELISA proved to be the best analytical technique--especially if coupled with microsamplings--because of its lower detection limit, precision, and reduced amount of sample needed. No differences were observed between half-life values obtained by ELISA and dot blot analysis (P = 0.081) and Western blot analysis (P = 0.614).


CONCLUSIONS
The findings of this study added to the knowledge of infliximab clearance in the vitreous and confirmed the validity of a microsampling technique that was compared with the classic one. ELISA was found to be the best analytical technique when using microsampling.
Giansanti
Fabrizio
F
Department of Oto-Neuro-Ophthalmological Surgical Sciences, Eye Clinic, University of Florence, Florence, Italy. fabriziogiansanti@interfree.it
Ramazzotti
Matteo
M
Giuntoli
Matteo
M
Virgili
Gianni
G
Vannozzi
Lorenzo
L
Degl'Innocenti
Donatella
D
Menchini
Ugo
U
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000493:pharmacokinetics*; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006207:Half-Life; D000069285:Infliximab; D007267:Injections; D023421:Models, Animal; D011817:Rabbits; D013048:Specimen Handling / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000493:pharmacokinetics; D014822:Vitreous Body / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D014409:Tumor Necrosis Factor-alpha; D000069285:Infliximab