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GB van Setten, TD Blalock, GR Grotendorst, GS Schultz; Identification and Measurement of Connective Tissue Growth Factor In Human Anterior Chamber Fluid . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4204.
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Purpose: To determine if connective tissue growth factor (CTGF) is present in the human anterior chamber fluid (ACF), or aqueous humor, and establish its average level. Methods: After informed consent was obtained, samples of ACF were collected from 11 consecutive patients undergoing routine cataract extraction without exfoliation syndrome. Samples of ACF were collected at the beginning of the intraocular procedure with a 1 ml syringe then samples were frozen at -80° C. Levels of CTGF were measured in the ACF samples using a non-commercially available sandwich ELISA that is specific for CTGF. Briefly, 100 ul of ACF were added to 96 well plates that had been coated with an affinity purified polyclonal goat antibody generated to recombinant human CTGF. After incubation for 2 hours at room temperature, wells were washed and incubated with a biotinylated goat anti-CTGF antibody for an additional 2 hours at room temperature, washed and incubated with an alkaline phosphatase-avidin conjugate, washed and color generated for 30 minutes by addition of p-nitrophenylphosphate substrate. The limit of detection for CTGF was 0.1 ng/ml with a range from 0.5 to 100 ng/ml. There was no detectable cross-reactivity with other growth factors at 100 ng/ml. Results: Serial dilutions of pooled ACF generated a curve that was parallel to the recombinant CTGF standard indicating that ACF samples contained immunoreactive CTGF. Levels of CTGF were above the detection limit of the ELISA for 9 of the 11 ACF samples (80 %). The average concentration of CTGF in the anterior chamber fluid was 1.2 ng/ml with a SD ± 0.3 ng/ml. Conclusion: These data indicate that CTGF is present in a high percentage of human anterior chamber fluid samples, and the mean concentration of CTGF (1.2 ng/ml, 32 pM) is in a physiologically relevant range since CTGF at 0.2 ng/ml stimulated significant increase in collagen synthesis (FASEB J 13:1774), and CTGF at 1 ng/ml stimulated proliferation of NRK fibroblasts (J Invest Dermatol 107:404). The source of CTGF in ACF and its roles in normal or pathological functioning of cells in the anterior segment are not known. However, because CTGF is a very potent mitogen and fibrotic factor that mediates TGF-b's induction of collagen synthesis in cultured fibroblasts, it is possible that CTGF in the ACF may play important roles in normal or pathological functioning of anterior segement cells.
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