Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium.
OBJECTIVE
Development of an ELISA for human insulin that utilizes monoclonal antibodies (mAbs) produced in serum-free medium.


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.
Even
Megha S
MS
Physicians Committee for Responsible Medicine, 5100 Wisconsin Ave. NW, Suite 400, Washington, DC 20016, USA. shaheven@gmail.com
Sandusky
Chad B
CB
Barnard
Neal D
ND
Mistry
Jehangir
J
Sinha
Madhur K
MK
D000328:Adult; D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002417:Cattle; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005215:Fasting; D005260:Female; D006801:Humans; D007328:Insulin / Q000097:blood* / Q000276:immunology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D019518:Postprandial Period; D011933:Reagent Kits, Diagnostic; D012044:Regression Analysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D007328:Insulin; D011933:Reagent Kits, Diagnostic