Novel laboratory technique for immunofluorescent detection of intraocular ranibizumab in a rat model

We propose a novel method for detecting ranibizumab molecules by immunohistochemistry in an animal model. Four brown Norway rats weighing 200-300g each (Envigo RMS, Indianapolis, IN), were intravitreally injected with 3μl ranibizumab. Eyes were processed with different reagent combinations. One eye was intravitreally injected with aflibercept as a positive control to demonstrate the function of antibodies; two eyes were intravitreally injected with ranibizumab; and one eye was not injected with an anti-vascular endothelial growth factor (anti-VEGF) agent as a negative control. Immunofluorescence signal intensity was measured under three antigen retrieval methods (pH 4.0, 6.0, 9.0) at one antibody concentration. Marker expression was high in the different combinations examined under all conditions, particularly pH 6.0, for all antibodies tested: mouse monoclonal anti-alpha-smooth muscle actin (α-SMA), goat polyclonal anti-human immunoglobulin G (IgG)-Fab, goat polyclonal anti-IgG (Fab specific), and rabbit polyclonal anti-human IgG (H/L). The validity of the methodology was verified against a control group. Parallel ligations were seen between anti-Fab antibodies and anti-IgG antibodies against αSMA. The change in sequence of incubation prevented the cross reactivity. The combination of two different antibodies may strengthen results of ranibizumab detection in brown Norway rats following intravitreal injection. Anti-Fab and anti-IgG (H/L) antibodies yielded a high immunofluorescent signal for ranibizumab identification. This method, including the sequence incubation change, made it possible to accurately identify ranibizumab molecules alone by using the specific anti-Fab together with anti-IgG antibodies. This technique may enhance further research involving the demonstration of ranibizumab in an animal model.