A. Krief et al. / Tetrahedron Letters 43 (2002) 1843–1846
1845
were collected from days 56 and 60 and stored at 4°C
until used. The yolk was carefully separated from the
white. The resulting IgY antibodies were isolated from
the egg yolk, purified through an affinity column8,‡ and
tested by enzyme linked immunosorbent assay
(ELISA)9,§ by antibody binding to solid-phase bound
BSA-2 antigen (Fig. 3). The presence of immunoglobu-
lins ABY-KLH-2 specific to the hapten 2 or part of it,
in the egg yolk was confirmed by indirect ELISA§ (Fig.
4, ꢀ).
length and lower (IC50: 50 mM) when the side chain
length decreases (6b R=Et, *) or increases 6d (R=Hex,
+).
Furthermore a polar group, such as a carbomethoxy
group, on the side chain of sufficient length has little
effect on the inhibition (6e, IC50: 50 mM) and neither
6c%, the diastereoisomer of 6c bearing a hydroxy group
in endo-position, nor 7 missing the exo- or endo-methyl
group are recognized by the antibodies ABY-KLH-2
(IC50>500 mM).
There title was rather low (by a factor of 5) compared
to the related ABM-KLH-2 sera of mice which were
immunized by the same conjugate (Fig. 3, compare ꢀ
to ꢁ)1 but the total amount of immunoglobulins pro-
duced was nevertheless by far, largely superior.
We have previously reported1 similar behavior of
ABM-KHL-2 and ABM-OVA-1 antibodies derived
from mice towards substrates 6a–e, 6c% and 7 (Fig. 2),1
whose structures are somewhat related to that of the
b-ketoalcohol (1R)-4.
We also observed, using an inhibition ELISA,¶ that the
b-ketoalcohol (1R)-4 did not inhibit the fixation of
ABY-KHL-2 antibodies to the BSA-2 antigen coated on
the plate (IC50:>500 mM) and this is also the case of (i)
the O-unsubstituted oxime 6a, which possesses all the
functional groups present on the hapten (Fig. 4, " and
ꢂ).
In conclusion, we have reported that antibodies har-
vested from hens (IgY) possess a recognition aptitude,
for various conjugates, very closely related to antibod-
ies harvested in a different organism (mice, IgG). All
these antibodies proved to be highly specific to the
whole structure of the hapten including the side chain.
This is quite remarkable and also quite unusual.
Alkoximinoalcohols (1R)-6 (6b R=Et, 6c R=n-Bu, 6d
R=n-Hex, 6e R=(CH2)4ꢀCO2Me; Fig. 2) which pos-
sess the bicyclic structure as well as a side chain related
to the linker bind quite efficiently (Fig. 4). Recognition
proved to be the best for 6c (R=Bu, IC50: 16 mM, ꢃ)
which probably possess a side chain of the critical
We are pursuing our work to understand the reasons
behind this unusual behavior. Nevertheless, we have
clearly shown that chickens are a valuable substitute to
mammals for antibodies production.
Figure 4. ELISA involving IgY collected from KLH-2 and (1R)-4 and 6a–d.
‡ Preparation of the affinity gel:8
Activation step: Divinylsulphone (2 ml) is added to the well stirred suspension of thoroughly washed Sepharose 4B-CL (20 g). After reaction (20°C,
2 h) the resulting gel is collected on a sintered-glass filter and washed with water. The activated gel is stored in water at 4°C until use. Coupling:
20 g of vinylsulphone-activated Sepharose, pre-washed with an aqueous solution of sodium carbonate (0.5 M), is mixed to a solution of
2-mercaptonicotinic acid (1.5 mmol) dissolved in aqueous solution of NaOH (0.5 M, 20 ml). The mixture was shaken (20°C, 1 h), glycerol (5
ml) was added and after 6 h the resulting gel slurry was transferred to a glass filter, washed with water and suspended in sodium phosphate
buffer solution (25 mM, pH 7.4) before packing.
§
General procedure for the indirect ELISA:9 BSA-2-conjugate (1 mg/ml) is coated (4°C, 24 h) on microtiter plates 96 wells. After blocking
unoccupied adsorption sites on the polymer surface, IgY serum is added in various dilutions to the wells and incubated (1 h, 37°C). The wells
are washed and rabbit anti-chicken IgG labeled to horseradish peroxidase 1:1000 is used as the secondary antibody. Color development (‘key
blue’ reagent) is stopped after 0.2 h by adding an H3PO4 aqueous solution to the wells, and the absorbance is read at 450–630 nm.
¶ General procedure for the inhibition ELISA:9 The procedure is the same as described for the ELISA except that increasing concentration of
inhibitors is mixed to the antibodies samples before incubation.