High performance liquid chromatography of the 1, 25-dihydroxyvitamin D3 receptor: Its application toward resolution of receptor-monoclonal antibody complexes

J. Wesley Pike, Michael A. Kelly, Mark R Haussler

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4 Citations (Scopus)

Abstract

The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.

Original languageEnglish (US)
Pages (from-to)902-907
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume109
Issue number3
DOIs
StatePublished - Dec 15 1982

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Calcitriol Receptors
High performance liquid chromatography
High Pressure Liquid Chromatography
Monoclonal Antibodies
Hormones
Calcitriol
Radioactivity
Macromolecules
Methionine
Pressure
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "High performance liquid chromatography of the 1, 25-dihydroxyvitamin D3 receptor: Its application toward resolution of receptor-monoclonal antibody complexes",
abstract = "The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91{\%}) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.",
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N2 - The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.

AB - The present report describes the application of high performance liquid chromatography (HPLC) towards the analysis of chick intestinal 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) receptors and their interaction with anti-receptor monoclonal antibodies. Hormone-labeled vitamin D3 receptors were chromatographed under high pressure on an Altec Spherogel TSK 3000 sw column. Both radioactivity and DEAE-filtration measurements of the eluted fractions indicated a high yield (91%) of 1, 25(OH)2D3 receptor. Specific binding was both displaceable with 100-fold excess radioinert hormone and dissociable with 2 mM p-chloromercuribenzene sulfonate. In contrast, when 1, 25(OH)2D3 receptors were preincubated with anti-receptor monoclonal antibodies, the receptor's elution time was significantly accelerated to that expected for a larger macromolecule. This position corresponded to that observed for either pure or internally [35S]methionine-labeled monoclonal antibody. These results suggest that HPLC analysis and resolution of both receptor and receptor-antibody complex may be of future practical value.

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