ANALYSIS OF NEUROTROPHIN RECEPTOR INTERACTIONS WITH A GD-FLAG-MODIFIED QUANTITATIVE KINASE RECEPTOR ACTIVATION (GD.KIRA) ENZYME-LINKED-IMMUNOSORBENT-ASSAY/

A rapid, sensitive, and high capacity assay has been developed to quan tify ligand-induced receptor tyrosine kinase activation in terms of re ceptor phosphorylation. The assay, termed a ''kinase receptor activati on'' or KIRA-ELISA, utilizes two separate microtiter plates, one for c ell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of ne urotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag de rived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptor s were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with go-specific mAb. The degree of receptor autophospho rylation was quantified by anti-phosphotyrosine ELISA. Reproducible st andard curves were generated with an EC50 of approximately 16 ng/ml NG F for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trk B KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA a ssay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. Whe n the gD.trkA KIRA assay was used to test several NGF variants, as wel l as NGF stability samples, the capacity of the assay to quantify liga nd bioactivity compared well with the more widely used radioreceptor b inding and PC 12 cell survival assays. The gD.trk KIRA assays show gre at potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses. (C) 1997 Academic Press.