Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium.

OBJECTIVES: Development of an ELISA for human insulin that utilizes monoclonal antibodies (mAbs) produced in serum-free medium. DESIGN AND METHODS: Insulin mAbs were produced in vitro by hybridomas in serum-free medium. A two-step ELISA was developed to replace bovine insulin (standard) and bovine serum albumin (assay buffer) with non-animal reagents. RESULTS: The sensitivity of the insulin ELISA was 0.73 uU/mL with a dynamic range of 2-200 uU/mL. No cross-reactivity with either human C-peptide or human proinsulin was observed. The intra- and inter-assay CVs were <7%. The mean recovery of insulin added to plasma samples was between 102.2% and 105.7%. The mean linearity of dilution was between 93% and 110% of undiluted plasma samples. The animal serum-free (ASF) insulin ELISA showed no marked degradation of any kit component when stored at 37 degrees C for up to 7 days. Significantly higher fasting insulin levels were observed in overweight or obese subjects (n=12) compared to lean subjects (n=10, p<0.05). Feeding markedly increased fasting insulin levels in both lean (p<0.02) and overweight or obese (p<0.005) subjects. Excellent correlation was observed between insulin levels measured by ASF insulin ELISA and another CE marked insulin ELISA (y=1.06x-0.44, r=1.00, n=44). CONCLUSIONS: This novel insulin ELISA provides precision and reliability equal to methods currently used in clinical research and serves as a guide for the development of other serum-free immunoassays.

Serum-free hybridoma culture: ethical, scientific and safety considerations.

Despite considerable progress in the development of cell culture techniques, including the development of the serum- and protein-free media that now routinely support hybridoma and mammalian cell growth, fetal bovine serum (FBS) supplemented media are still commonly used: a practice that raises ethical, scientific and safety concerns. The use of FBS in hybridoma culture media is examined here, with regards to the development and production of monoclonal antibodies (mAbs), and it is our recommendation that researchers adopt serum-free cell culture methods to reduce animal use in this area.