Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays.
To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.
Takahashi
M
M
Allelix Diagnostics, Inc., Mississauga, Ontario, Canada.
Fuller
S A
SA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D001375:Azaguanine / Q000494:pharmacology; D002459:Cell Fusion; D006063:Chorionic Gonadotropin / Q000032:analysis / Q000276:immunology; D018997:Chorionic Gonadotropin, beta Subunit, Human; D002851:Chromatography, High Pressure Liquid; D004351:Drug Resistance; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D010042:Ouabain / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D014510:Urease / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D007074:Immunoglobulin G; D010446:Peptide Fragments; D010042:Ouabain; D014510:Urease; D001375:Azaguanine