One-step purification of chicken growth hormone from a crude pituitary extract by use of a monoclonal immunoadsorbent.
The immunization of mice with an affinity-purified glycoprotein preparation from chicken pituitary tissue yielded several monoclonal antibodies towards the recently described glycosylated variant of chicken GH. As all these antibodies recognize the classical (non-glycosylated) GH molecule equally well, they provide a suitable tool for the development of both a specific immunoadsorbent and an assay method. This paper deals with the surprising purification power of the immunoadsorbent that was produced with one of the monoclonal antibodies. The resulting preparation was more than 99% pure as assessed by reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, so that no further purification steps were needed before the determination of the amino acid sequence of the material. The efficiency of the purification protocol as determined by a homologous, monoclonal antibody-based radioimmunoassay was virtually absolute. Moreover, the affinity-purified GH preparation was a mixture representing the multiple molecular forms of pituitary chicken GH, including both oligomeres and glycosylated GH. The purified preparations were finally used to demonstrate the hepatic 5'-monodeiodinase-stimulating activity of GH in the chicken embryo (results not shown), in order to prove that the biological activity of the molecule had not been damaged by elution from the immunoadsorbent.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D013006:Growth Hormone / Q000302:isolation & purification*; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010902:Pituitary Gland / Q000032:analysis*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D013006:Growth Hormone