Quantitative immunoassay of recombinant murine neuroleukin.
A eukaryotic, transient expression system was used to produce recombinant neuroleukin, a growth factor for neurons. Neuroleukin in media conditioned by transfected monkey COS-1 cells was purified to homogeneity in one step by high-performance cation-exchange chromatography. Purified neuroleukin was used to establish a quantitative two-site enzyme-linked immunosorbent assay and the accuracy of the assay was confirmed by analytical high-performance liquid chromatography. The amount of recombinant neuroleukin secreted by the transfected COS-1 cells and the content of endogenous neuroleukin in various murine cell lines was determined. Neuroleukin levels were nearly undetectable in Balb/3T3 embryo cells, intermediate in several leukocytic cell lines and highest in mouse LBRM-33 T lymphoma cells. Maximal survival of sensory neurons was obtained with approximately 10(-9) M recombinant neuroleukin although tissue derived neuroleukin appeared to be significantly more active. Dialysis of the transfected COS-1 cell conditioned medium resulted in increased neuroleukin bioactivity, while binding to the cation-exchange column reduced bioactivity. The expression and purification of the recombinant protein and the detection of natural sources expressing high levels of neuroleukin will greatly facilitate studies of its biological effects.
Spear
G T
GT
Department of Pharmacological and Physiological Sciences, University of Chicago, IL 60637.
Caldwell
J
J
Lee
M R
MR
Gurney
M E
ME
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005956:Glucose-6-Phosphate Isomerase; D006133:Growth Substances / Q000032:analysis* / Q000494:pharmacology; D008222:Lymphokines / Q000032:analysis* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000032:analysis*
D000596:Amino Acids; D003470:Culture Media; D006133:Growth Substances; D008222:Lymphokines; D011994:Recombinant Proteins; D005956:Glucose-6-Phosphate Isomerase