Development of a Novel Diagnostic ELISA for Human Insulin Using Serum-Free Cell Culture
* Megha S. Even (1), Chad B. Sandusky (1), Neal D. Barnard (1), Jehangir Mistry (2), Madhur K. Sinha (2)
(1) PCRM, Research Dept, Washington DC, USA; (2) Linco Research, Inc., St. Charles, MO, USA
Background: Concerns for animal welfare led us to develop a custom ELISA, for the measurement of human insulin, that replaced both the use of the ascites method and the use of fetal calf serum, while providing sufficient precision and reliability for clinical and research applications.
Methods: Insulin monoclonal antibodies were grown in vitro (derived from hybridoma cultures) in RPMI 1640/DMEM (1:1 v/v) supplemented to 4 mmol/L L-glutamine, 4% Maxi-MAb Mark II Supplement, and 2% Complex Lipid Solution. After weaning of the cells, fetal calf serum was not used for antibody production. A two-step ELISA was developed using recombinant human insulin (standard), charcoal-treated human serum (matrix), 50 mmol/L PBS containing human serum (assay buffer), and HRP-TMB detection system.
Results: The assay characteristics include sensitivity of 1.56 uU/mL, dynamic range of 1.56 to 200 uU/mL, no cross-reactivity with human C-peptide or proinsulin, intra- and inter-assay CVs of <10%, recovery of exogenously added insulin to plasma samples of 102.2-105.7%, and linearity of dilution (1/2, 1/4, and 1/8) of insulin spiked plasma samples as 93-110% of undiluted plasma samples. Circulating insulin levels in ten healthy volunteers were measured using both conventional ELISA methods and our new ELISA with absolute values similar between the two assays.
Conclusions: A highly specific and sensitive insulin ELISA was developed without using the ascites method or fetal calf serum for monoclonal antibody production. These methods could serve as a guide for reducing animal use for antibodies produced for other types of immunoassays and diagnostic tests.
This abstract was presented by the authors at the Fifth World Congress on Alternatives and Animal Use in the Life Sciences, which took place in August 2005 in Berlin.