If you don’t read until the end, here’s the key takeaway:
As researchers utilizing commercial assays, we bear a critical responsibility to ensure the validity and reproducibility of the data we generate. While companies provide us with essential tools, it is our duty to apply these tools correctly and rigorously, implementing proper controls to guarantee the integrity of our findings.
Science depends on the accuracy of the data we collect, and this principle is paramount.
This study underscores the importance of perseverance and critical thinking in research. I extend my gratitude to J. Schikora for their unwavering dedication and calm persistence throughout this project and to R. Pouw for emphasizing the importance of proper experimental controls.
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When I began my AMD research a professor approached me suggesting it was improbable that such an active and important, but tiny, tissue as the retina could significantly influence the complement system of the entire systemic blood circulation, and that systemic biomarker analyses might be insufficiently sensitive. At that time, I began developing the concept of detecting biomarkers locally within the eye. While studies were subsequently published regarding wet AMD, only a few exist for dry AMD.
Key Question 1:
How can we collect and analyze intraocular samples from AMD patients?
I still recall my telephone conversation with T. Bertelmann, who offered his support in collecting aqueous humor. Professor Bertelmann successfully engaged the exceptional clinicians at Göttingen Clinic in this project, and aqueous humor samples were collected from patients with dry AMD during cataract procedures—a process considerably more complex than obtaining blood samples.
Key Question 2:
What are the challenges in ocular biomarker analysis?
Our objective was to detect as many biomarkers as possible with high sensitivity, focusing on bead-based multiplex ELISA methodology. This presented the second challenge of the project. Companies provide researchers with products that benefit the entire scientific community; however, researchers bear responsibility for applying these products conscientiously. We utilized the cytokine multiplex assay as provided, but without a possibility to validate the detection of over 50 proteins (we simply trust the company).
Key Question 3:
How specific are commercial complement assays for detecting key complement components?
However, for the complement assay (my research focus and central interest), we conducted validation that I would like to share. In the referenced paper, Supplementary Figures 12 J, K, and N demonstrate that:
The beads in Panel 1, designated as C4b beads by the manufacturer, detected both uncleaved C4 and activated C4b in our assays, making them not suitable for assessing C4 activation, but rather for whole C4/C4b detection
The C4 beads in Panel 2, labeled as C4 beads, also detect both C4 and C4b, though when testing recombinant fragments specific to C4 isoforms A and B, C4B was preferentially detected—which I consider a valuable scientific tool, especially since we observed a significant difference in our samples for the beads in Panel 1 but not in Panel 2
Another important finding worth mentioning is that the beads in this assay detect not only Factor H (FH) but also the splice variant FHL-1, though not FH-related proteins
Regarding validation in this yet unpublished manuscript, we examined the iC3b assay of the panel. Supplementary File S9 reveals that:
The manufacturer's designated C3b/iC3b beads preferentially detect iC3b and C3c, but not C3b
The C3 beads are specific for C3 and C3c (which should not present an issue)
Supplementary Figures 9 (Schikora et al. submitted)
Best regards,
Diana
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