Quantitative determination of S-antigen in human ocular tissues, aqueous humour and serum

Abstract

Retinal S-antigen is thought to play an important role in the immunopathogenesis of uveitis. To investigate whether S-antigen is a sequestered antigen confined to the retina, a sensitive ELISA was developed to determine the levels of this protein in various human ocular tissues, aqueous humour and serum. The ELISA was performed by incubating S-antigen-containing samples with solid-phase bound immunospecific rabbit anti human C-ar.tigen F(ab')_? fragments and then incubating the bound S-antigen with mouse anti bovine S-antigen serum and the bound mouse antibodies with peroxidase-labelled rabbit anti mouse IgG; the peroxidase activity is developed with ABTS. This method was demonstrated to be highly sensitive and specific: S-antigen could be measured at concentrations of 5–10 nanograms per ml, irrespective of whether it was present in buffer, undiluted whole serum or tissue extracts. Human retinas were shown to contain approximately 1.2 mg immunoreactive S-antigen per retina. Of the other human ocular tissues, only the vitreous and choroid (including pigment epithelium) contained small amounts of S-antigen. Low levels of S-antigen could also be detected in the aqueous humour of two out of seven patients with posterior uveitis. No immu-noreactive S-antigen could be detected in the serum of either healthy individuals or patients with uveitis. Sera collected from diabetic patients 15 minutes after extensive laser photocoagulation also did aot contain immunoreactive S-antigen. Preliminary experiments with rats to study the clearance of intravenously injected S-antigen from the circulation indicated that the relatively short half-life (± 30 min) of circulating S-antigen might account for the absence of detectable S-antigen in the patient sera.