Enzyme-Linked Immunosorbent Assay Detection of Pyrrolizidine Alkaloids: Immunogens Based on Quaternary Pyrrolizidinium Salts

Polyclonal antibody-based enzyme-linked immunosorbent assays (ELISAs)
were developed for the
detection of retrorsine (1, 351 g/mol), monocrotaline
(2, 325 g/mol), and retronecine (3, 155 g/mol)
in
the parts per billion (ppb) range. A set of three bifunctional
linking arms (6−8) was synthesized.
By
N-alkylation of pyrrolizidine alkaloids (PAs) retrorsine,
monocrotaline, and retronecine acetonide (9),
six haptens (6.1, 6.2, 7.1,
7.2, 7.9, and 8.9) were synthesized
and used to generate rabbit antisera.
The resulting anti-retrorsine antiserum gave a 50% inhibition
(I50) value of 0.9 ± 0.2 ppb for
retrorsine
with detection limits of 0.5−10 ppb. The same ELISA system also
detected isatidine (4, retrorsine
N-oxide) dihydrate (403 g/mol) with an
I50 of 1 ppb and senecionine (5, 352
g/mol) with an I50 of 100
ppb. A second monocrotaline-based ELISA detected monocrotaline
with an I50 of 36 ± 9 ppb 2
with
detection limits of 5−500 ppb and shows no cross-reactivity with
1 or 5; this ELISA demonstrates the
potential for the substrate-specific detection method. A third
retronecine-based ELISA detects 3 with
an I50 of 3000 ± 600 ppb (3 ± 0.6 ppm) and
detection limits of 600−10000 ppb. None of these
ELISAs
cross-react with the structurally similar swainsonine (10)
or lupinine (11) alkaloids. PAs were
detected
in extracts of Senecio vulgaris and Crotalaria
retusa, but not in Lupinus spp., as a demonstration
of
the ELISA's usefulness.