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 quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a "kinase receptor activation" or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC50 of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.
Sadick
M D
MD
Department of BioAnalytical Technology, Genentech, Inc., South San Francisco, California 94080, USA. sadick.michael@gene.com
Galloway
A
A
Shelton
D
D
Hale
V
V
Weck
S
S
Anicetti
V
V
Wong
W L
WL
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008024:Ligands; D009414:Nerve Growth Factors / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity; D018139:Simplexvirus; D014759:Viral Envelope Proteins / Q000235:genetics
D008024:Ligands; D009414:Nerve Growth Factors; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D020794:Receptor Protein-Tyrosine Kinases