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 is being presented by the authors at the Fifth
World Congress on Alternatives and Animal Use in the Life Sciences,
which is taking place in August 2005 in Berlin.
Media
Center | Health | Research
| About PCRM | Catalog
| Join Us | Search
| Site Index | Home
The site does
not provide medical or legal advice. This Web site is for information purposes
only.
Full Disclaimer | Privacy Policy
|
Animal
Experimentation Issues 