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Clin Biochem. 2007 Jan;40(1-2):98-103. Epub 2006 Oct 19.

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

Author information

  • 1Physicians Committee for Responsible Medicine, 5100 Wisconsin Ave. NW, Suite 400, Washington, DC 20016, USA. shaheven@gmail.com

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.

PMID:
17123500
[PubMed - indexed for MEDLINE]
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