Flow cytometric assays for monitoring production of recombinant HIV-1 gp160 in insect cells infected with a baculovirus expression vector.
A baculovirus expression system for the HIV-1 envelope glycoprotein gp160 has been used as a model for development of flow cytometric assays for monitoring production of cell-associated recombinant antigen. Using monoclonal antibodies to the transmembrane (gp41) or envelope (gp120) portion of gp160, gp120, but not gp41, could be reproducibly detected on the surface of insect cells 48 h after infection with the recombinant baculovirus. In contrast, fixation and permeabilization of infected cells prior to staining, to allow access of monoclonal reagents to the intracellular compartment, markedly improved the sensitivity of detection, with reactivity to both monoclonal antibodies observed at 24 h post-infection. Specificity of the intracellular immunofluorescence was verified by demonstrating that the appropriate native or recombinant HIV-1 protein blocked reactivity of monoclonal antibody with infected cells. In addition, it was observed that production of gp160 following baculovirus infection was associated with a marked increase in the 90 degrees light scatter of insect cells, as determined by flow cytometry, and that this correlated with the kinetics of cell-associated gp160 production as determined by immunofluorescence. These procedures should be of great utility for routine monitoring of recombinant proteins produced in insect cells in response to infection with recombinant baculovirus.
Farmer
J L
JL
Abbott Laboratories, Diagnostics Division, Abbott Park, IL 60064.
Hampton
R G
RG
Boots
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015686:Gene Products, env / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D005822:Genetic Vectors; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000378:metabolism*; D007304:Insect Viruses / Q000235:genetics; D007313:Insecta; D007700:Kinetics; D010539:Permeability; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D015686:Gene Products, env; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D011994:Recombinant Proteins