Influence of mannosylation on immunostimulating activity of adamant-1-yl tripeptide

Article abstract of DOI:10.1002/cbdv.201200008

The mannosylated derivative of adamant-1-yl tripeptide (D-(Ad-1-yl)Gly-L- Ala-D-isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant-1-yl tripeptide (Man-OCH ₂CH(Me)CO-D-(Ad-1-yl)Gly-L-Ala-D-isoGln) is a non-pyrogenic, H ₂O-soluble, and non-toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β-D-GlcNAc-(1→4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(NH ₂)-D-Ala-D-Ala), a well-known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM. Copyright

Full text of DOI:10.1002/cbdv.201200008

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Influence of Mannosylation on Immunostimulating Activity of Adamant-1-yl
Tripeptide
a
b
b
b
a
-
by Rosana Ribic´* ), Lidija Habjanec ), Ruza Frkanec ), Branka Vranesˇic´ ), and Srdanka Tomic´ )
ˇ
a) Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000
Zagreb (phone: þ385-1-4606402; fax: þ385-1-4606401; e-mail: rribic@chem.pmf.hr)
b) Institute of Immunology, Rockfellerova 2, HR-10000 Zagreb
The mannosylated derivative of adamant-1-yl tripeptide (d-(Ad-1-yl)Gly-l-Ala-d-isoGln) was
prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated
adamant-1-yl tripeptide (Man-OCH2CH(Me)CO-d-(Ad-1-yl)Gly-l-Ala-d-isoGln) is a non-pyrogenic,
H2O-soluble, and non-toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was
tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and
peptidoglycan monomer (PGM, b-d-GlcNAc-(1!4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(eNH2)-d-
Ala-d-Ala), a well-known effective adjuvant. The mannosylation of adamantyl tripeptide caused the
amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.
Introduction. – Natural and synthetic peptidoglycans, as well as their fragments of
different molecular mass exhibit remarkable biological activities. It was established
that they, in particular, affect the immune system of mammalian hosts [1][2].
Peptidoglycan fragments of well-defined structures, the best known of which are
muramyl peptides, have been extensively studied as possible adjuvants for human and
animal vaccines [3–5]. Muramyl dipeptide (MDP, N-acetylmuramyl-l-alanyl-d-
isoglutamine) is known as the smallest synthetic adjuvant molecule capable of
replacing whole Mycobacteria in Freundꢀs adjuvant [5]. MDP is the structural fragment
of peptidoglycan monomer (PGM; 1; Fig. 1) used in this work, a disaccharide
pentapeptide b-d-GlcNAc-(1!4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(eNH2)-d-
Ala-d-Ala, originating from Brevibacterium divaricatum.
Several hundred chemically defined MDP analogs and derivatives were synthesized
to modulate the properties of the parent molecule. Replacement of the N-acetylmur-
amyl moiety with various acyl groups represents an important approach in the design of
new immunologically active MDP analogs. Up to now, our research in the field of
potential adjuvants was directed towards desmuramyl peptides, which contain
adamantylglycine and mannosylated adamantylglycine moieties bound to the essential
part of MDP, l-Ala-d-isoGln. Thus, diastereoisomers of adamant-1-yl tripeptides 2a
and 2b [6], and adamant-2-yl tripeptides 3a and 3b [7] (Fig. 1) were synthesized and
showed to exhibit adjuvant (immunostimulating) activities in vivo, in a well-defined
mouse model. Furthermore, mannosylated adamant-1-yl and adamant-2-yl tripeptides,
4 and 5 (Fig. 1), respectively, were prepared as a mixture of diastereoisomers due to the
d-l-d and l-l-d sequence in the tripeptide portion (d,l-AdGly-l-Ala-d-isoGln) [8].
Preliminary investigations of their immunostimulating activity in vivo indicated
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Structures of PGM (1), adamant-1-yl tripeptides 2, adamant-2-yl tripeptides 3, mannosylated
adamant-1-yl tripeptides 4, and mannosylated adamant-2-yl tripeptides 5
differences in their activities as a result of the introduced mannose and the chiral spacer
part of defined (R)- or (S)-configuration which connects the sugar part to the
adamantyl-tripeptide parts of the molecules.
Our longstanding interest in monosaccharide conjugates [9], and their enzymatic
transformations [10–13] and synthesis [14][15] led us to the synthesis and subsequent
biological testing of various mannosylated derivatives [8][16][17], including glyco-
tripeptide derivatives such as 4 and 5 [8], as well as mannosylated PGM [16]. The
described glycopeptides and PGM comprise in their structure mannose which is an

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1375
important sugar whose receptors, mannose receptors (MRs), present on immunocom-
petent cells (such as macrophages and dendritic cells) are considered to be pattern-
recognition receptors binding compounds with mannose, N-acetylglucosamine, or
fucose as their essential parts. Therefore, they are responsible for the binding, among
others, of mannosylated antigens or relevant biologically active molecules containing
mannose, thus affecting the immune reactions. Previous reports on conjugates of
mannosylated proteins with MDP showed enhanced immunostimulating and antitumor
properties in comparison to conjugates without mannose [18][19].
The aim of this study was to further explore the adjuvant activity of the pure
mannosylated adamant-1-yl tripeptide of d-l-d configuration on humoral IgG immune
response in mice, in comparison to its parent tripeptide 2a and PGM. In two different
mice strains, it was shown that adamant-1-yl tripeptide 2a with a d-l-d configuration
exhibited better immunomodulating activity than its isomer 2b with a l-l-d config-
uration [6]. Furthermore, our preliminary results indicated that the chirality of the
spacer connecting mannose to adamantyl tripeptide portions plays a significant role,
and compounds containing the spacer of (R)-configuration were found to exhibit
higher activities than those with (S)-configured spacers [8]. Taking this into account,
mannosylated adamant-1-yl tripeptide with a d-l-d sequence of amino acids and the
spacer of (R)-configuration was further examined as a possible promising immuno-
stimulator of choice in this class of immunomodulatory structures.
Results and Discussion. – Chemistry. Synthesis of the mixture of diastereoisomers
of mannosylated adamant-1-yl tripeptides, due to the d-l-d and l-l-d sequence in the
tripeptide portion (d,l-(Ad-1-yl)Gly-l-Ala-d-isoGln), was described in detail in [8],
and, here preparation and characterization of pure diastereoisomer 4a (with d-l-d
sequence in peptide part) is reported. The key step in the synthesis of mannosylated
adamant-1-yl tripeptide was conjugation of tert-butyl ester of (adamant-1-yl)glycyl-l-
alanyl-d-isoglutamine 6a and 6b, and perbenzylated 3-(a-mannosyloxy)-2-methylpro-
panoic acid of (R)-configuration 7, using EDC/HOBt coupling method (Scheme). tert-
Bu Group of the resulting 8a and 8b was removed by acid hydrolysis, and the mixture of
isomers 9a and 9b was obtained. After column chromatography on silica gel and
rechromatography, pure diastereoisomer 9a ((R)-a-Bn4Man-OCH2CH(Me)CO-d-
(Ad-1-yl)Gly-l-Ala-d-isoGln) was isolated from the diastereoisomer mixture 9a and
9b. Debenzylation of 9a by catalytic hydrogenation afforded the final product 4a,
1
unequivocally identified by HPLC analysis, H- and 13C-NMR spectroscopies, mass
spectrometry, and elemental analysis.
Testing of Immunostimulating Activity. Adjuvant activity was evaluated by the
immunostimulatory effect on secondary humoral response to ovalbumin (antigen) in
NIH/OlaHsd (H-2q) mice according to our previously described mouse model in vivo
[20][21]. Ovalbumin was used as a customary antigen for studying the adjuvant effect.
Anti-OVA IgG, anti-OVA IgG1, and anti-OVA IgG2a were determined in the mice
sera after the second booster. The adjuvant activity of mannosylated adamant-1-yl
tripeptide 4a in comparison to parent adamant-1-yl tripeptide 2a and PGM (1) was
studied. PGM was established as a reference for adjuvant activity in a previously well-
defined mouse model in vivo, proven suitable for comparison of the observed effects
[20][21]. The comparison of induced anti-OVA IgG levels was carried out quantita-

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme. Synthesis of Mannosylated Adamant-1-yl Tripeptide 4a
a) EDC·HCl, HOBt·H2O, Et3N, dioxane/CH2Cl2 1:1. b) CF3COOH (TFA). c) Separation of isomer 9a
from 9b. d) H2, 10% Pd/C, 50% EtOH. EDC¼1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide,
HOBt¼1-hydroxy-1H-benzotriazole.
tively, and the subclasses of IgG, IgG1, and IgG2a, as indicator of Th1 or Th2 type of
immune response, were also determined.
In general, enhancement in total anti-OVA IgG antibody (Fig. 2,a) production was
observed in all groups, in comparison to no-adjuvant-treated (OVA alone) group.
Mannosylated adamant-1-yl tripeptide 4a and tripeptide 2a elicited better immune
response than OVA alone. A weak stimulation of total antibody production was
achieved with adamant-1-yl tripeptide 2a, whereas mannosylated adamant-1-yl
tripeptide 4a showed approximately the same immunostimulating effect as the control,
PGM-injected group. Furthermore, the measured quantity of anti-OVA IgG antibodies
was higher when compound 4a was applied, in comparison to 2a. Since mannosylated
adamantyl tripeptide 4a is more efficient in stimulating anti-OVA IgG antibody
production than non-mannosylated 2a, it can be concluded that mannosylation of
adamantyl desmuramyl dipeptide plays an important role in the stimulation of the
immune response. Mannosylation amplified the immunostimulating activity of

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 2. The effect of adamant-1-yl tripeptide and its mannosyl derivate on production of anti-OVA IgG (a)
and its subtypes, anti-OVA IgG1 (b) and anti-OVA IgG2a (c), respectively, in NIH/OlaHsd mice
immunized with OVA as an antigen. Sera were analyzed after the second booster. Experimental groups:
*
1, OVA alone; 2, OVAþ1; 3, OVAþ4a; 4, OVAþ2a. .: Group mean value; : each serum separately.
adamantyl tripeptide in such a way that the activity was comparable to that of PGM,
which is a known effective adjuvant.
Since it is well-known that vaccine adjuvants can enhance or modulate the Th1/Th2
bias of induced immune response [22][23], we were also interested in the effect of
tested adjuvants on the type of immune response. In this study, the type of generated
immune response was indirectly evaluated by quantification of OVA-specific IgG1 (for
activation of Th2 type) and IgG2a (for activation of Th1 type), and calculation of the
respective IgG1/IgG2a ratio.
When the amount of anti-OVA IgG1 antibodies was measured (Fig. 2,b), it was
observed that the highest response was provided by PGM. A slight suppression was
detected in the production of anti-OVA IgG1 antibodies, when mannosylated adamant-
1-yl tripeptide 4a or tripeptide 2a were administered, in comparison with the results
obtained for OVA alone.
When anti-OVA IgG2a antibodies were measured (Fig. 2,c), the response for
mannosylated adamant-1-yl tripeptide 4a was higher than that for PGM (1) and
adamant-1-yl tripeptide 2a.
From the IgG1/IgG2a ratio (Fig. 3), calculated for each serum (obtained after the
second booster), neither of tested compounds significantly shifted the IgG1/IgG2a
ratio in comparison to no adjuvant treated group of animals. However, it was evident
that groups treated with mannosylated adamant-1-yl tripeptide 4a and parent
tripeptide 2a had lower values than the group treated with OVA alone, indicating
the slight shift toward more pronounced Th1 type of immune response. The highest

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 3. The ratio of anti-OVA IgG1 and anti-OVA IgG2a (IgG1/IgG2a) antibodies developed in mice after
immunization and two boosters with adamant-1-yl tripeptide and its mannosyl derivate. Experimental
*
groups: 1, OVA alone; 2, OVAþ1; 3, OVAþ4a; 4, OVAþ2a. .: Group mean value; : each serum
separately.
value was obtained for the group treated with PGM, indicating that PGM stimulates
the immunomodulatory activity toward slightly more pronounced Th2 type of immune
response, in comparison to the antigen alone.
Conclusions. – In this work, mannosylated adamant-1-yl tripeptide, a-Man-
OCH2CH(Me)CO-d-(Ad-1-yl)Gly-l-Ala-d-isoGln, was prepared and characterized.
This glycopeptide comprises in its structure mannose, an important sugar whose
receptors on immunocompetent cells are considered to be pattern-recognition
receptors binding compounds comprising mannose or some other specific saccharides.
Mannose is connected to adamant-1-yl tripeptide through a chiral linker of (R)-
configuration.
The immunostimulating activity of mannosylated adamantyl tripeptide 4a was
compared to peptidoglycan monomer 1, an efficient adjuvant and the parent adamantyl
tripeptide 2a. In experiments in vivo, 4a exhibited higher adjuvant activity than 2a. Its
adjuvant effect is comparable to that of PGM (1). The obtained results indicate that the
introduction of the mannosyl moiety plays a significant role in stimulation of the
immune response. It should be also noted that the tested 4a is a stable, non-pyrogenic,
H2O-soluble, and non-toxic compound. Such characteristics render mannosylated
adamant-1-yl tripeptide 4a potentially applicable as an adjuvant for vaccines. There is
no clear evidence so far that mannosylated tripeptide binds to mannose receptors
(MRs), but the possibility of effecting the immune response by binding mannose to
MR is not excluded. Therefore, further research on this topic is required.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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We wish to thank the Ministry of Science, Education and Sports of the Republic of Croatia for support
of this work (projects 119-1191344-3121 and 021-0212432-2431).
Experimental Part
General. Bovine serum albumin (BSA), Tween 20, monoclonal anti-chicken egg albumin (clone
OVA-14, mouse IgG1 isotype), avidin-peroxidase, and o-phenylenediamine dihydrochloride (OPD)
were from Sigma (USA). Ovalbumin (OVA) was purchased from Serva, Germany. Horseradish
peroxidase conjugated goat anti-mouse IgG (HRP-anti-mouse IgG) was from Bio-Rad Laboratories,
USA. Biotin-conjugated rat anti-mouse IgG1 and anti-mouse IgG2a monoclonal antibodies, and
streptavidin-peroxidase were purchased from PharMingen, Becton Dickinson (USA). Chemicals for
buffers and solns. were from Kemika, Croatia, unless stated otherwise. Peptidoglycan monomer was
prepared in PLIVA, Chemical and Pharmaceutical Works (HR-Zagreb), according to the procedure
described in [24]. Adamant-1-yl tripeptide 2a [6] and N-[(R)-3-(2,3,4,6-tetra-O-benzyl-a-d-mannopyr-
anosyloxy)-2-methylpropanoyl]-l(and d)-(adamant-1-yl)glycyl-l-alanyl-d-isoglutamine tert-butyl esters,
(8a and 8b, resp.) [8], were prepared as described earlier. Chemical reagents used in syntheses were
obtained from Fluka and Aldrich. All org. solvents were purified using standard procedures.
Column chromatography (CC, solvents and proportions as given in the text): Merck silica gel 60
(SiO2; 70–230 mesh ASTM). TLC: Fluka SiO2 (60 F254) plates (0.25 mm) were used; with UV light,
ninhydrin, and 10% H2SO4. HPLC: Waters HPLC system equipped with 2996 PDA detector and
Empower software (Milford, MA, USA); a LiChrosorb RP-18 column (244 mmꢀ4 mm; 5 mm) and a
LiChrospher guard column 100 RP-18 (5 mm; Merck, D-Darmstadt); flow rate, 1.0 ml/min at r.t.; the
eluate was monitored at 200 nm; the gradient solvent system used, MeCN containing 0.035%
trifluoroacetic acid (TFA), and H2O containing 0.05% TFA; the percentage of MeCN at 0, 15, and
20 min was 10, 30, and 10, resp., and a running time was 25 min; MeCN and TFA were of HPLC-grade
from Merck (D-Darmstadt); a daily supply of H2O was obtained from Millipore Simplicity – Personal
ultra pure water system (Bedford, MA, USA). NMR Spectra: Bruker Avance (300 MHz) spectrometer;
d in ppm rel. to Me4Si as internal standard, J in Hz. MS: Waters MS-Quattro micro instrument; in m/z. C,
-
ˇ
´
H, and N analysis: Analytical Services Laboratory of the Ruder Boskovic Institute, Zagreb.
General Procedure for Hydrolysis of tert-Butyl Esters. The mixture 8a/8b [8] (300 mg; 0.28 mmol)
was dissolved in CF3COOH/H2O 95 :5 (3.3 ml). The resulting soln. was stirred at r.t. for 2 h, and then
concentrated in vacuo. The residue was purified by flash chromatography (FC; SiO2; CHCl3/MeOH
5 :1), and the mixture 9a/9b was obtained as white solid foam.
Pure diastereoisomer 9a (67 mg) was separated from the mixture by CC (SiO2; CHCl3/MeOH 3 :1)
and rechromatography.
N-[(R)-3-(2,3,4,6-Tetra-O-benzyl-a-d-mannopyranosyloxy)-2-methylpropanoyl]-d-(adamant-1-yl)-
1
glycyl-l-alanyl-d-isoglutamine (9a). Rf (CHCl3/MeOH 3 :1) 0.56. H-NMR (CDCl3): 7.34–7.13 (m, 20
arom. H); 4.91 (s, HꢁC(1)); 4.84–4.47 (m, 3 HꢁC(a) of AdGly, Ala, isoGln, 5 H of PhCH); 4.64 (d,
Jgem ¼11.7, 1 H of PhCH2); 4.51 (d, Jgem ¼11.9, 1 H of PhCH2); 4.48 (d, Jgem ¼11.0, 1 H of PhCH2); 3.94–
3.24 (m, HꢁC(2,3,4,5), CH2(6), OCH2); 2.84–2.70 (m, CH); 2.18–2.04 (m, HꢁC(g) of isoGln); 1.96–
1.83 (m, HꢁC(g), CH2(b) of isoGln, 3 CH of Ad); 1.61–1.51 (m, 6 CH2 of Ad); 1.42 (d, J¼6.9, Me of
Ala); 0.99 (d, J¼7.2, Me). 13C-NMR (CDCl3): 175.66, 170.72, 170.65, 170.15, 169.18 (C¼O); 138.49,
138.31, 138.29, 138.15 (arom. C); 128.29–127.38 (arom. CH); 98.83 (C(1)); 80.36, 74.90, 72.00
(C(2,3,4,5)); 74.99, 73.45, 72.63, 72.06, 69.39 (C(6), 4 PhCH2); 58.94 (OCH2); 50.70 (CH(a) AdGly);
43.81 (CH(a) isoGln); 41.06 (CH(a) Ala); 39.85 (CH); 38.80, 38.66, 36.59 (CH2(g) isoGln, CH2 Ad, C
Ad); 29.68 (CH2(b) isoGln); 28.12 (CH Ad); 14.16 (Me Ala); 13.66 (Me). ESI-MS: 1017.50 ([MþH]þ
,
C58H73N4Oþ12 ; calc. 1017.52).
General Procedure for O-Debenzylation. To a soln. of 9a (50 mg, 0.066 mmol) in a 3 :1 mixture
CHCl3/MeOH (5 ml), 25 mg of 10% Pd/C and 20 ml of 50% EtOH in H2O were added. The mixture was
subjected to H2 under 4 bars at r.t. and stirred for 24 h. Then, catalysts were filtered, and the filtrates were
concentrated under reduced pressure. The residue was purified by CC (SiO2; CHCl3/MeOH 1:1) to yield
4a (43 mg, 99%). RP-HPLC: tR(4a) 22.10 min.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
N-[(R)-3-(a-d-Mannopyranosyloxy)-2-methylpropanoyl]-d-(adamant-1-yl)glycyl-l-alanyl-d-iso-
1
glutamine (4a). Rf (CHCl3/MeOH 1:1) 0.34. H-NMR (CD3OD): 8.11 (d, <1 H, J¼8.3, NH); 7.89 (d,
<1 H, J¼7.4, NH); 7.61 (d, <1 H, J¼8.7, NH); 7.29 (pt, <1 H J¼7.5, J¼11.0, NH); 4.77 (s, HꢁC(1));
4.44 (s, HꢁC(a) of AdGly); 4.24 (dd, J¼4.6, 7.5, HꢁC(a) of isoGln); 4.14 (q, J¼7.0, HꢁC(a) of Ala);
4.09–3.41 (m, HꢁC(3,4,5), CH2(6), OCH2); 4.03 (d, J(2,3)¼3.9, HꢁC(2)); 2.79–2.67 (m, CH); 2.20–
2.06 (m, 2 H, CH2(g) and CH2(b) isoGln); 1.86 (br. s, 3 CH of Ad); 1.66–1.53 (m, 6 CH2 of Ad); 1.28 (d,
J¼7.3, Me of Ala), 1.14 (d, J¼7.1, Me ) . 13C-NMR (CD3OD): 177.37, 176.46, 175.18, 172.44 (C¼O); 102.16
(C(1)); 74.69, 72.63, 71.98, 68.82 (C(2,3,4,5)); 71.12 (C(6)); 62.99 (CH(a) AdGly); 62.80 (OCH2); 53.70
(CH(a) isoGln); 51.34 (CH(a) Ala); 42.08 (CH); 39.94; 37.93; 37.49; 31.60 (CH2(g) isoGln, CH2 Ad, C
Ad); 29.83 (CH Ad); 28.01 (CH2(b) isoGln); 17.35 (Me Ala); 14.54 (Me). ESI-MS: 657.40 ([MþH]þ
,
C30H49N4Oþ12 ; calc. 657.33). Anal. calc. C30H48N4O12 (656.72): C 54.87, H 7.37, N 8.53; found: C 54.75, H
7.14, N 8.28.
Experiments in vivo. Experiments in vivo were performed on NIH/OlaHsd (H-2q) inbread mice
strains. All mice used were females 2–2.5 months old. Commercial food and water were provided ad
libitum. During the experiments, animals were kept at the animal facility at the Institute of Immunology,
and all experiments were performed according to the Croatian Law on Animal Welfare (The Official
Gazette ꢃNNꢀ 135/06).
Exper. groups of five mice were immunized and boosted two times subcutaneously (s.c.) into the tail
base at 21-day intervals. Mice were anesthetised prior to blood collection on 7th day after the second
booster. Sera were collected, decomplemented at 568 for 30 min, and stored at ꢁ208 until testing.
The dose of OVA (antigen) was 10 mg per mouse. The dose of PGM and tested compounds was
200 mg per mouse. OVA and tested substances were dissolved in saline, and the injection volume in all
exper. groups was 0.1 ml per mouse.
Enzyme Immunoassays for Qual. and Quant. Determination of OVA-Specific IgG (anti-OVA IgG) in
Mice Sera. Enzyme immunoassays (ELISA) were performed on flat-bottomed high binding microtitre
plates (Costar, USA) according to the procedures described in [16][25]. The relative quantities of anti-
OVA IgG were determined by parallel line assay comparing each serum to monoclonal anti-chicken egg
albumin, declared as standard preparation, to which 20,000 arbitrary units per ml (AU/ml) were
voluntarily assigned [26].
Enzyme Immunoassays for Qual. and Quant. Determination of OVA-Specific IgG Subtype Anti-OVA
IgG1 and Anti-OVA IgG2a, Resp., in Mice Sera. ELISA for determination of anti-OVA IgG1 and anti-
OVA IgG2a were performed as described in [21]. The relative quantities of antibody subtypes were
determined by parallel-line assay using appropriate standard preparation. The monoclonal anti-OVA
IgG1 was a standard for relative quantification of anti-OVA IgG1 to which 400,000 AU/ml was assigned,
while polyclonal mouse serum containing high levels of anti-OVA IgG2a was used as a standard for
relative quantification of IgG2a specific antibodies with voluntarily assigned 20,000 AU/ml. The ratio of
anti-OVA IgG1 and anti-OVA IgG2a (IgG1/IgG2a) was used as indication of the Th1/Th2 bias of
induced immune response.
Statistics. Statistical analyses were performed using Statistica 6.0 for Windows, StatSoft Inc. The
significant difference between experimental groups was evaluated by KruskalꢁWallis ANOVA, followed
by multiple MannꢁWhitney U-nonparametric tests. A probability values less than 0.05 (p<0.05) were
considered significant.
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Received January 9, 2012