Aqueous Humor VEGF-A Levels Detected with Millipore and R&D Multiplexing Immunoassays after Ranibizumab Treatment

Abstract

Purpose : To detect VEGF-A levels in aqueous humor (AH) of eyes with choroidal neovascularization (CNV) before and after intravitreal injection of Ranibizumab (IVL) using Millipore and R&D multiplexing immunoassays

Methods : Treatment-naïve patients with CNV, treated with IVL between March 2017 and August 2023 at St. Michael’s Hospital (Toronto, Ontario) were recruited.The eyes with history of trauma were excluded. 60-80μl of AH was extracted via paracentesis from the inferotemporal limbus of study eyes pre-IVL, at baseline and at follow-up visits. Each sample was divided into two portions, one for VEGF-A detection using the R&D assay and the other using the Millipore assay. VEGF-A levels were assessed with the Magpix device incorporating Luminex’s xMap multiplexing immunoassay technology. The Wilcoxon paired test was employed to compare VEGF-A levels between assays at pre and post IVL administration. A p-value <0.05 was considered statistically significant.

Results : 16 participants (17 eyes) were enrolled in this study, 12 were female (75%) and the average age of the cohort was 75.5 years (SD 9.6). The number of samples collected from each eye ranged from 3 to 17. At the pre-IVL baseline visit, median VEGF-A levels were measured at 134.56 pg/ml (IQR 83.56 to 161.24) by the Millipore assay and 225.39 pg/ml (IQR 109.30 to 842.19) by the R&D assay (p>0.05). At the one-month visit following the first IVL administration, the Millipore assay detected a median 718.90 pg/ml (IQR 335.49 to 1454.94) of VEGF-A, which was significantly increased when compared to the baseline (p<0.001). By contrast, the R&D assay detected a median 4.44 pg/ml (IQR 1.48 to 13.42) of VEGF-A at one month post IVL, which was significantly decreased when compared to the baseline (p<0.001).

Conclusions : Baseline measurements revealed comparable VEGF-A detection between the Millipore and R&D assays, indicating a similar sensitivity for free VEGF-A. Following IVL administration, the Millipore detected substantially higher VEGF-A levels compared to baseline, whereas R&D detected significantly lower VEGF-A levels compared to baseline. This discrepancy likely arises from Millipore’s ability to detect VEGF-A in both its free and drug-bound states, whereas R&D is specific to free VEGF-A. Implementing both assays concurrently may provide valuable insight on the VEGF-A behavior and distribution post Ranibizumab treatment for CNV.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.