Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody

S. Dokoh, Mark R Haussler, J. W. Pike

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

A radioligand immunoassay for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] receptors was developed utilizing a specific, high-affinity (K(d) = 1.8 x 10-11 M) monoclonal antibody (9A7γ) obtained from suspension cultures of rat spleen x mouse myeloma hybrid SP2/0-9A7. A standard curve was established, based on the competition between 1,25(OH)2D3-receptor (18 fmol/tube) and increasing concentrations of radioinert 1,25(OH)2D3-receptor (0-240 fmol/tube) for the binding site on 9A7γ. Samples, prepared in identical buffer, contained 0-100 fmol of receptor/tube. After an equilibrium incubation of 1,25(OH)2D3-receptor with either standard or sample (16h at 4°C), antibody-bound receptor was immunoprecipitated with rabbit anti-(rat immunoglobulin) prelinked to Staphylococcus aureus and quantified. The assay is statistically sensitive to 2fmol of receptor/tube, with intra- and inter-assay variations of 7 and 12% respectively. Occupied, unoccupied and denatured receptor were observed to compete equally in the assay. This quantitative technique has been successfully applied to the characterization of receptors after fractionation by sedimentation analysis and DNA-cellulose chromatography. Finally, the measurement of total receptor by this assay, in conjunction with 1,25(OH)2D3 binding assays, has revealed that rachitic, normal and 1,25(OH)2D3-injected chicks have respectively 13, 20, and 56% of receptor in the occupied form. From these results we consider that this radioligand immunoassay will be a useful tool in further research on quantifying 1,25(OH)2D3 receptors in tissue and cell extracts.

Original languageEnglish (US)
Pages (from-to)129-136
Number of pages8
JournalBiochemical Journal
Volume221
Issue number1
StatePublished - 1984

Fingerprint

Calcitriol Receptors
Immunoassay
Assays
Monoclonal Antibodies
Rickets
Tissue Extracts
Calcitriol
Cell Extracts
Staphylococcus aureus
Chromatography
Immunoglobulins
Suspensions
Buffers
Spleen
Binding Sites
Rabbits
Antibodies
Rats
Research
Fractionation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody. / Dokoh, S.; Haussler, Mark R; Pike, J. W.

In: Biochemical Journal, Vol. 221, No. 1, 1984, p. 129-136.

Research output: Contribution to journalArticle

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abstract = "A radioligand immunoassay for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] receptors was developed utilizing a specific, high-affinity (K(d) = 1.8 x 10-11 M) monoclonal antibody (9A7γ) obtained from suspension cultures of rat spleen x mouse myeloma hybrid SP2/0-9A7. A standard curve was established, based on the competition between 1,25(OH)2D3-receptor (18 fmol/tube) and increasing concentrations of radioinert 1,25(OH)2D3-receptor (0-240 fmol/tube) for the binding site on 9A7γ. Samples, prepared in identical buffer, contained 0-100 fmol of receptor/tube. After an equilibrium incubation of 1,25(OH)2D3-receptor with either standard or sample (16h at 4°C), antibody-bound receptor was immunoprecipitated with rabbit anti-(rat immunoglobulin) prelinked to Staphylococcus aureus and quantified. The assay is statistically sensitive to 2fmol of receptor/tube, with intra- and inter-assay variations of 7 and 12{\%} respectively. Occupied, unoccupied and denatured receptor were observed to compete equally in the assay. This quantitative technique has been successfully applied to the characterization of receptors after fractionation by sedimentation analysis and DNA-cellulose chromatography. Finally, the measurement of total receptor by this assay, in conjunction with 1,25(OH)2D3 binding assays, has revealed that rachitic, normal and 1,25(OH)2D3-injected chicks have respectively 13, 20, and 56{\%} of receptor in the occupied form. From these results we consider that this radioligand immunoassay will be a useful tool in further research on quantifying 1,25(OH)2D3 receptors in tissue and cell extracts.",
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