H. Kamei et al. / Carbohydrate Research 315 (1999) 243–250
249
medium (in a later experiment, DMEM
medium with a high glucose level was used
instead of RPMI medium), HAT (0.1 mM
hypoxanthine, 0.4 mM aminopterin, and 16
mM thymidine, Sigma, USA) and 20% FBS
[13]. From 2 weeks to 1 month later,
colonies grown on the agar plates were iso-
lated in liquid culture medium containing
HT (0.1 mM hypoxanthine and 16 mM
thymidine, Sigma, USA). Culture superna-
tants were assayed by ELISA for the pres-
ence of MAb that can bind to conjugate 9
and/or to fetuin. ELISA-positive clones were
selected and transfered to normal medium
for further study.
MeOH–H2O–Et3N (403 mL) was added glu-
taric acid anhydride (3.5 mg, 30 mmol) and
the mixture was stirred at room temperature.
After 20 min the mixture was concentrated
in vacuo. Purification of this residue on a
column of silica gel (3:2:1 EtOAc–MeOH–
H2O) yielded amino derivative 8 (4 mg,
59%); [h]2D5+25.5° (c 0.4, MeOH); HR-MS
m/z Anal. Calcd for C16H24N2O10Na (Na
salt): 427.1329 [M+H]+, Found: 427.1354.
Conjugate 9.—The reaction was carried
out in a centricon tube. Compound 8 (4 mg,
10 mmol) was dissolved in H2O (250 mL),
and the pH of this solution was adjusted to
6.0 by the addition of aq NaHCO3. To this
solution was added BSA (15 mg, ca 0.2
mmol) and EDC·HCl (3 mg, 16.5 mmol), and
the mixture was incubated at room tempera-
ture. After 12 h, the mixture was directly
loaded onto a gel permeation column (Sep-
hadex G-50, H2O). The fraction containing
BSA was pooled and lyophilized.
Typing of immunoglobulin subclass.—This
was done by using a Mouse MonoAB ID
Kit (Zymed, USA).
Affinity purification of IgG.—IgG in the
culture supernatant was precipitated with
ammonium sulfate at 50%, dissolved and di-
alysed against the application/binding buffer,
and applied to a protein A/G plus-agarose
affinity column (Calbiochem., USA). IgG
was eluted with acetate buffer (pH 3.0) and
glycine–HCl buffer (pH 3.0), and adjusted
to a pH of 7.0 with Tris base.
Conjugate 10.—Prior to the reaction,
KLH was dissolved in H2O (100 mg/0.5
mL), and the precipitate was removed by
filtration. The filtrate containing KLH was
lyophilized and used for the synthesis of
conjugate 10. The conditions for the reaction
were the same as those for conjugate 9.
ELISA.—A total of 100 mg of conjugate
9, or 200 mg (145 mg in a later experiment)
of fetuin containing 5.5% Neu5Ac, was dis-
solved in 1 mL of PBS; 100 mL of the solu-
tion was used to coat each well of a 96-well
test plate at room temperature for 2 h. Anti-
gen-coated plates were blocked with 1% BSA
in PBS, washed with 0.1% Tween 20 in PBS,
and used for ELISA. The culture superna-
tant of hybridoma or purified IgG, undiluted
or diluted with 0.1% BSA in PBS, was used
as the source of MAb. In some experiments,
as shown in Fig. 5, the BSA used to dilute
samples and block ELISA plates was pre-
treated with 0.1 M HCl for 1 h at 80 °C,
and neutralized and dialysed against PBS to
remove any sialic acids from sialo-com-
pounds contaminating the BSA. To test the
effects of Neu2en5Ac and Neu5Ac, they
were dissolved in PBS; and the solution was
adjusted to a pH of 7.2–7.5 with 0.1 M
NaOH by using pHBOY-P2 (Shindengen,
Japan), and diluted with PBS. Culture super-
natant, purified IgG, or their diluted solu-
tions were preincubated with various
Hybridoma.—Conjugate 10 (390 mg) was
dissolved in 1 mL of PBS and mixed into
one vial of GERBU adjuvant containing 10
mg of GMDP, 40 mg of N,N%-dimethyl-N,N-
dioctadecylammonium chloride, and 1.68 mg
of Zn-proline complex (Gerbu Biotechnik,
Germany). Female MRL/MpJ-lpr/lpr mice
[10] (Nihon Slc, Japan), 8-weeks-old, were
each immunized subcutaneously with 50 mL
of the antigen–adjuvant mixture, which con-
tained 19.5 mg of conjugate 10. The mice
were further immunized with the same dose
at 2, 5, and 8 weeks after the first immuniza-
tion. The titer of antibodies that bound to
conjugate 9, as detected by ELISA, increased
gradually after 2 weeks in all the mice im-
munized. Three or 4 days after immuniza-
tion at 5 or 8 weeks, spleen cells from two
or three mice were obtained and hybridized
with myeloma X63-Ag8.653 cells [12] by us-
ing polyethylene glycol 6000, and were
plated on 0.5% agar plates containing RPMI
.