Radioimmunoassay with Heterologous Antibody(Hetero-Antibody RIA): Utilization of Highly Cross-Reactive Antibody Present in Polyclonal Antiserum.

Iwasawa, Atsushi; Hayashi, Hiroaki; Wakabayashi, Keiji

doi:10.1507/endocrj1954.38.673

Cited by 3 publications

(1 citation statement)

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“…We first performed small scale purification from 10 canine pituitaries (0.5 g) to obtain cLH and cTSH for hetero-antibody RIA [21] provided by the National Hormone and Pituitary Program, consisting of NIDDK rat LH I-9, NIDDK rat LH RP-3, and anti rat S-11 for LH and NIDDK rat TSH I-9, NIDDK rat TSH RP-2, and NIDDK anti rat TSH S-5 for TSH. In the second large scale purification, cLH and cTSH activities were monitored by hetero-antibody RIA with cLH and cTSH, which were obtained from the first purification for radioiodination and standardization, and antisera to rat hormones.…”

Section: Radioimmunoassay (Ria) Of Clh and Ctshmentioning

confidence: 99%

Isolation and Partial Characterization of LH, FSH and TSH from Canine Pituitary Gland.

1997

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Abstract. A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography.In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDa, respectively.The purified hormones showed two bands corresponding to a (20 kDa) and f3 subunits (cLHJ3:16 kDa, cFSHJJ: 22 kDa, cTSHJJ:16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Crosscontamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

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Section: Radioimmunoassay (Ria) Of Clh and Ctshmentioning

confidence: 99%