Ll. Kjems et al., HIGHLY SENSITIVE ENZYME-IMMUNOASSAY OF PROINSULIN IMMUNOREACTIVITY WITH USE OF 2 MONOCLONAL-ANTIBODIES, Clinical chemistry, 39(10), 1993, pp. 2146-2150
A highly sensitive two-site sandwich ELISA measuring total proinsulin
immunoreactive material in serum or plasma was developed. The assay wa
s based on two monoclonal antibodies, an anti-C-peptide antibody bound
to a microtest plate and a biotin-labeled anti-insulin antibody. The
detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, cor
responding to 0.25 pmol/L in human serum (diluted 1:5). The linear cal
ibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median
(range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.
3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). M
ean analytical recovery of added human proinsulin (hPI) (2, 5, and 10
pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and hum
an C-peptide did not cross-react at 5000 and 10 000 pmol/L, respective
ly. The four major proinsulin conversion intermediates reacted 65-99%:
split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des
(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were
above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.