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  • Title: Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium.
    Author: Even MS, Sandusky CB, Barnard ND, Mistry J, Sinha MK.
    Journal: Clin Biochem; 2007 Jan; 40(1-2):98-103. PubMed ID: 17123500.
    Abstract:
    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.
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