Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies.
A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was 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, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator 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). Mean 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 human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. 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.
Kjems
L L
LL
Steno Diabetes Center, Gentofte, Denmark.
Røder
M E
ME
Dinesen
B
B
Hartling
S G
SG
Jørgensen
P N
PN
Binder
C
C
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D002096:C-Peptide / Q000097:blood; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D011384:Proinsulin / Q000097:blood*; D012016:Reference Values
D000911:Antibodies, Monoclonal; D002096:C-Peptide; D011384:Proinsulin