Tada et al.
1
Involvement of Branched Units in Reactivity of β-D-Glucan to Antibody
2 3
Involvement of branched units at position 6 in the reactivity of a unique variety of
4
β-D-glucan from Aureobasidium pullulans to antibodies in human sera
5 6
Rui TADA,1 Asuka TANIOKA,2 Ken-ichi ISHIBASHI,1 Yoshiyuki ADACHI,1
7
Kazufumi TSUBAKI,2, 3 and Naohito OHNO1, †
8 9
1
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy,
10
Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
11
2
12
Tokyo, Japan
13
3
Advance Materials R & D Laboratory, Life Sciences Department, ADEKA Corporation,
New Business Promotion Department, ADEKA Corporation, Tokyo, Japan
14 15
Received December 4, 2008; Accepted December 17, 2008
16 17
† To whom correspondence should be addressed. Tel: +81-426-76-5561; Fax:
18
+81-426-76-5570; E-mail:
[email protected]
19 20
Abbreviations: AP-FBG, Aureobasidium pullulans-fermented β-D-glucan; BRM;
21
biological response modifier; CR3, complement receptor type 3; CSBG, 1,3-β-D-glucan
22
from Candida albicans; DB, degree of branching; DEX, Dextran T500; DP, degree of
23
polymerization; ELISAs, enzyme-linked immunosorbent assays; GRN, 1,3-β-D-glucan
24
obtained Grifola frondosa; LacCer, lactosylceramide; NMR, nuclear magnetic
25
resonance; PBS, phosphate-buffered saline; PBST, PBS containing 0.05% Tween 20;
26
SPG, sonifilan; TMB, tetramethylbenzidine
27
1
Tada et al.
1
Abstract
2 3
We recently determined the structure of a unique type of 1,3-β-D-glucan
4
obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the
5
antibodies in human sera.
6
than that of 1,3-β-D-glucan obtained Grifola frondosa (GRN) but weaker than that of
7
1,3-β-D-glucan from Candida albicans (CSBG).
8
reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in
9
human sera.
The reactivity of AP-FBG to the antibodies was stronger
Here, we demonstrated that AP-FBG
Moreover, the results of the competitive ELISAs using various glucan
10
competitors showed that these IgGs recognized branched chains at position 6.
This is
11
the first study to report that the branched chains at position 6 of β-D-glucan strongly
12
contribute to its recognition by antibodies in human sera.
13
AP-FBG to human IgG could be advantageous for the use of this glucan in medicine,
14
e.g., as an immunostimulatory agent.
This high reactivity of
15 16
Key words: Aureobasidium pullulans; β-D-glucan; antibody; human sera; structure
17
2
Tada et al.
1
β-Glucan is a well-known biological response modifier (BRM) widely
2
distributed in nature.
Various β-glucans have been isolated from different sources, e.g.,
3
fungi, plants, and seaweeds.
4
depending on their primary structure, including linkage type; degree of branching (DB);
5
degree of polymerization (DP); conformation (e.g., triple helix, single helix, and
6
random coil structure), and molecular weight.1-3)
7
significant role played by β-glucans in the treatment of cancer and infectious diseases in
8
both modern medicine and traditional oriental medicine.
9
important role as dietary substances because they lower the plasma cholesterol level,
10
enhance the hematopoietic response, and possess antitumor and immunomodulating
11
properties.4-6)
12
edodes and Schizophyllum commune, respectively,7,
13
cancer therapy in Japan.
The physicochemical properties of β-glucans differ
Recent reports have highlighted the
β-Glucans also play an
For instance, lentinan and sonifilan (SPG) obtained from Lentinus 8)
have been clinically used for
14
Recent studies have proposed that the pivotal mechanism(s) underlying the
15
immunostimulatory effects of β-glucans stems from the ability of β-glucans to induce
16
innate immune responses via specific receptors that recognize them.
17
that serve as β-glucan receptors have recently been reported, e.g., complement receptor
18
type 3 (CR3)9) and lactosylceramide (LacCer).10)
More recently, dectin-1 has also
19
been described as a 1,3-β-glucan receptor.11,
It is important to determine the
20
relationship between the structure and receptor-binding ability of β-glucans.13, 14)
12)
Host molecules
21
Furthermore, although β-glucans have long been considered to exhibit weak
22
antigenicity, we reported that not only innate immune responses but also acquired
23
immune responses, such as the production of antibodies against β-glucans, are involved
24
in the immunostimulatory effects of β-glucans.15, 16)
25
unique type of β-D-glucan obtained from the polymorphic fungus Aureobasidium
26
pullulans (this β-D-glucan is termed as AP-FBG) reacted to the antibodies in human
27
sera; this β-D-glucan has a mixed structure comprising a 1,3-β-D-glucan backbone with
28
single 1,6-β-D-glucopyranosyl-branching units at every 2nd residue as the major 3
Very recently, we found that a
Tada et al.
1
structure and a 1,3-β-D-glucan backbone with single 1,6-β-D-glucopyranosyl-branching
2
units at every 3rd residue as the minor structure (the ratio of the major and minor units
3
was approximately 7:3).17)
4
β-D-glucan obtained from Grifola frondosa (GRN), which comprises a 1,3-β-D-glucan
5
backbone with single 1,6-β-D-glucopyranosyl-branching units at every 3rd residue,18, 19)
6
while it was weaker than that of β-glucan from Candida albicans (CSBG), which
7
comprises a 1,3-β-D-glucan backbone with long 1,6-β-D-glucopyranosyl-branching
8
units.20)
9
human antibodies is not completely understood.
The reactivity of AP-FBG was stronger than that of
The difference between these β-D-glucans that react strongly and weakly with It is important to clearly understand
10
this difference, not only to promote the clinical application of β-D-glucans in
11
immunotherapy, but also to determine the mechanisms underlying the recognition of
12
β-D-glucans by host immune systems and the biological effects of these BRMs.
13
Therefore, in this study, we postulated that the difference in the reactivity of
14
β-D-glucans to antibodies in human sera depends on the presence of branching chains at
15
position 6 of the β-D-glucan molecule.
16
to β-D-glucans differ depending on the frequency and length of the branched chains at
17
position 6.
18
and discuss the relationship between its reactivity and primary structure.
This is because the reactivities of human sera
Here, we report the reactivity and specificity of AP-FBG to human sera
19 20
Materials and methods
21 22 23
Materials D2O (deuteration degree min, 99.96%) and Me2SO-d6 (99.96%) were
24
purchased from Merck.
CSBG, AP-FBG, and GRN were prepared as described
25
previously.17, 18, 20) Curdlan and pustulan were purchased from Sigma. Dextran T500
26
(DEX) was purchased from GE Healthcare.
27
γ-globulin preparation, was purchased from Bayer.
28
IgG was purchased from Sigma.
Polyglobin N, which is a human Peroxidase-conjugated anti-human
Peroxidase-conjugated anti-human IgG1, IgG2, IgG3, 4
Tada et al.
1
and IgG4 were purchased from Invitrogen.
2 3
Use of ELISA for detecting anti-β-glucan antibody
4
A 96-well Nunc plate was coated with β-glucan preparations in 0.1 M
5
carbonate buffer (pH 9.6) and incubated overnight at 4 °C.
The plate was washed with
6
phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and blocked with
7
1% bovine serum albumin (BSA; BPBST) at 37 °C for 60 min.
8
and incubated with polyglobin N at 37 °C for 60 min.
9
treated with peroxidase-conjugated anti-human Ig secondary antibody in BPBST, and
The plate was washed
It was then washed with PBST,
10
developed using a tetramethylbenzidine (TMB) substrate system.
Color development
11
was terminated using 1N phosphoric acid, and the optical density was measured at 450
12
nm.
13 14
Competitive ELISAs used to determine the structure required for anti-β-glucan
15
antibody binding
16
The specificity of the anti-β-glucan antibody was examined using competitive
17
ELISAs.
Competitive ELISAs were essentially conducted as described above except
18
for the following modifications.
19
with known quantities of various soluble glucan competitors dissolved in blocking
20
buffer. Before they were added to the wells of the microtiter plates, the competitor
21
solutions were diluted with equal volumes of polyglobin N and preincubated for 1 h at
22
37 °C.
Polyglobin N diluted with blocking buffer was mixed
23 24 25
NMR spectroscopy Exchangeable protons were removed by suspending the β-glucans in D2O and
26
lyophilizing this mixture.
This exchange process was repeated 3 times.
27
spectra were recorded in a mixed solvent of Me2SO-d6/D2O (6:1) (15 mg/mL) at 70 °C
28
by using a Bruker Avance 500 spectrometer equipped with a TXI-xyz three gradient 5
All the
Tada et al.
1
probe by using the method described by Kim et al.3)
2
dimethylsulfoxide (Me2SO) signal (δH = 2.53 ppm) for 1H as a reference and expressed
3
the chemical shift in terms of parts per million (ppm).
4
sequence was used to perform the 1D-1H experiment, and the spectral width was 5000
5
Hz in 64 K complex data points.
6
accurately achieve signal integration.
7
used 4 times, and the noise was reduced by using the TRAF function.
We used the internal
The Bruker standard pulse
A relaxation delay of 5T1 was used in order to Prior to Fourier transformation, zero-filling was
8 9
Results and discussion
10 11 12
Reactivity of AP-FBG to human γ-globulin preparation Since we had previously found antibodies against β-D-glucan in human sera,16)
13
we first examined the reactivity of AP-FBG to polyglobin N.
As shown in Fig. 1a, we
14
found that AP-FBG exhibited high reactivity to antibodies in the human sera; this
15
reactivity was stronger than that of GRN but weaker than that of CSBG.
16
reactivities differed among β-D-glucans.
Thus, the Fig. 1.
17
Next, we examined the reactivity of AP-FBG to each of the IgG subclasses.
18
This is because it is well known that the activated capacities of IgGs mediated effector
19
function through interaction with FcγRs and activation of the complement system are
20
differed with IgG subclass.21)
21
largely reactive to the IgG2 subclass.
22
IgG3 subclasses, although the extent of reactivity was substantially less than that to the
23
IgG2 subclass; the IgG4 subclass was not detected in the human sera.
24
antigen-binding IgGs such as IgG1, IgG2, and IgG3 not only lead to the activation of
25
the host immune system (immune cell activation) via suitable FcγRs but also lead to the
26
activation of the complement system.21)
27
anti-AP-FBG IgGs antibodies in human sera recognize AP-FBG and could subsequently
28
lead to the activation of the host immune system.
As shown in Fig. 1b, the β-D-glucan AP-FBG was In addition, it was also reactive to the IgG1 and
6
In general,
Together, these facts imply that the
Tada et al.
1 2 3
Structural specificity of anti-AP-FBG IgG in human sera We found that AP-FBG, a unique variety of 1,3-β-D-glucan with highly
4
branched chains at position 6, reacted with antibodies in human sera.
Its reactivity was
5
found to be stronger than that of GRN but weaker than that of CSBG (Fig. 1a).
6
exact mechanism underlying the difference in reactivity has not yet been elucidated.
7
Therefore, we focused our attention on the differences in the primary structure of 3
8
varieties of β-D-glucans, since the characteristic differences among the 3 varieties were
9
in the frequency presence and length of the branched chains at position 6.
The
Next, by
10
using competitive ELISAs, we examined the specificity of anti-AP-FBG IgGs by using
11
various glucans as competitors. Fig. 2 shows the 1D-1H NMR spectra of the various
12
glucans used in this study.
13
1,6-β-glucopyranosyl residue (δH=4.29 ppm) of CSBG and pustulan indicated the
14
existence of long 1,6-β-glucopyranosyl chain.
15
derived from 1,6-β-glucosyl residue (δH=4.25 ppm) of AP-FBG and GRN indicated the
16
existence of 1,6-β-monoglucopyranosyl chain.
17
AP-FBG and GRN is the frequencies of 1,6-β-monoglucopyranosyl chain according to
18
their signal integrities.
19
the results of the competitive ELISA experiments, it was apparent that 1,6-β-D-glucans
20
such as pustulan inhibit the binding of anti-AP-FBG IgGs to AP-FBG to a greater extent
21
than the 1,3-β-D-glucans such as curdlan do (Fig. 3).
22
inhibitory effect between AP-FBG and GRN might be reflected in their frequencies, that
23
is thickly condition, of 6-branced side chain units.
24
AP-FBG is stronger than CSBG.
25
anti-CSBG antibodies are higher, because the long 6-branched side chain units within
26
CSBG could be attributed to its binding amount, but binding affinity (avidity) is lower
27
than anti-AP-FBG antibodies, because the density of 6-branched side chain units could
28
be attributed to its affinity.
Fig. 2.
Briefly, the down-fielded signals derived from
On the other hand, up-fielded signals
In addition, the difference between
This structural information was summarized in Table 1.
From
In addition, the difference of
Besides, the inhibitory effect of
This result may indicate that the binding amounts of
These facts strongly suggest that the reactivity of 7
Table 1.
Tada et al.
1
β-D-glucans to IgG in human sera depends on the branched chains at position 6.
It is
2
well known that the 1,6-β-D-glucopyranosl side chains extend from the outer side of the
3
1,3-β-D-glucopyranosl backbone.22, 23)
4
1,6-β-D-glucopyranosl side chains on the reactivity of β-D-glucans to IgG in human
5
sera.
This may explain the strong influence of the
6 7 8 9
Conclusion In the present study, we demonstrated that a unique type of A. pullulans β-D-glucan
that
comprises
a
1,3-β-D-glucan
backbone
and
single
10
1,6-β-D-glucopyranosyl side chains at every 2nd or 3rd residues reacted with IgG
11
antibodies, especially those of the IgG2, IgG1, and IgG3 subclasses, in human sera.
12
Moreover, we found that these IgGs recognized the β-D-glucan molecule on the basis of
13
the branched chains at position 6.
14
position 6 of β-D-glucan might be responsible for recognition via the acquired immune
15
systems, such as the recognition by antibodies, in humans.
16
antibodies to highly 6-branced β-glucan might be contributed to activate immune cell
17
activations such as FcγR-mediated effects by complexes of antibodies and β-glucans.
18
This characteristic of AP-FBG could be an advantage for its use in medicine, e.g., as an
19
immunostimulatory agent.
These data imply that the branching chains at
Thus, these recognitions of
20 21
Acknowledgments
22 23
This work was supported by a grant for private universities provided by the
24
Ministry of Education, Culture, Sports, Science, and Technology and Japan Private
25
School Promotion Foundation.
26
8
Fig. 3.
Tada et al.
1
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Akira, S., Adachi, Y., Ohno, N., Kinjo, T., Nakamura, K., Kawakami, K., Iwakura,
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Ohno, N., Characterization of beta-glucan recognition site on C-type lectin, dectin 1.
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barley beta-D-glucan to the beta-glucan recognition molecule dectin-1. J. Agric.
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Food Chem., 56, 1442-1450 (2008).
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15. Harada, T., Nagi-Miura, N., Adachi, Y., Nakajima, M., Yadomae, T., Ohno, N.,
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Antibody to soluble 1,3/1,6-beta-D-glucan, SCG in sera of naïve DBA/2 mice. Biol.
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Pharm. Bull., 26, 1225-1228 (2003).
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16. Ishibashi, K., Yoshida, M., Nakabayashi, I., Shinohara, H., Miura, N.N., Adachi, Y.,
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Ohno, N., Role of anti-beta-glucan antibody in host defense against fungi. FEMS
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Y., Yamazaki, M., Tsubaki, K., Ohno, N., Structural characterization and biological
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activities of a unique type beta-D-glucan obtained from Aureobasidium pullulans.
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21. Heyman, B., Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu. Rev. Immunol., 18, 709-737 (2000).
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22. McIntire, T.M., Brant, D.A., Observations of the (1-->3)-beta-D-glucan linear triple
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helix to macrocycle interconversion using noncontact atomic force microscopy. J.
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Am. Chem. Soc., 120, 6909-6919 (1998).
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23. Okobira, T., Miyoshi, K., Uezu, K., Sakurai, K., Shinkai, S., Molecular dynamics
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studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution.
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Biomacromolecules., 9, 783-788 (2008).
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24. Natarajan, K., Dimasi, N., Wang, J., Mariuzza, R.A., Margulies, D.H., Structure and
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25. Giammatteo, P.J., Bolton, P.H., Stipanovic, A.J., Characterization of conformational
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Mater. Sci. Eng., 54, 650-655 (1986).
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26. Ochiai, M., Ashida, M., A pattern-recognition protein for beta-1,3-glucan. The
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25 26
Figure legends
27 28
Figure 1. Reactivity of β-D-glucans to antibodies in human sera 11
Tada et al.
1
(a) The reactivity of AP-FBG (▲), CSBG (♦), and GRN (■) was tested by
2
ELISA.
3
blocked and incubated with human sera.
4
anti-human IgG and developed using a TMB substrate system.
Color development
5
was terminated and the optical density was measured at 450 nm.
(b) The reactivity of
6
AP-FBG to each IgG subclass in the human sera was tested by using ELISA.
7
96-well plate was coated with the glucan preparation.
8
incubated with human sera.
9
and developed using a TMB substrate system.
10
A 96-well plate was coated with the glucan preparations.
The plate was
It was then treated with antibodies for
A
The plate was blocked and
It was then treated with antibodies to the Ig subclasses Color development was terminated and
the optical density was measured at 450 nm.
11 12 13 14
Figure 2. 1D-1H NMR spectra of various glucans used in this study Stacked plots of the 1D-1H NMR spectra (from top to bottom: AP-FBG, GRN, curdlan, CSBG, pustulan, and DEX).
15 16 17
Figure 3. Structural specificity of anti-AP-FBG IgG antibodies in human sera The structural specificity of the anti-AP-FBG IgG antibodies was tested by
18
using competitive ELISAs.
A 96-well plate was coated with the glucan preparation.
19
The plate was blocked and incubated with a mixture of human sera and competitive
20
glucans (left to right columns describe the concentration of the competitors: 0.0977,
21
0.195, 0.391, 0.781, 1.56, 3.13, and 6.25 µg).
22
antibody to anti-human IgG in BPBST and developed using a TMB substrate system.
23
Color development was terminated and the optical density was measured at 450 nm.
24
The data are expressed as percentage of inhibition against the control.
25
12
The plate was then treated with the
Tada et al.
1
Table 1. Primary structure of polysaccharides analyzed in this study Polysaccharide
Source
Primary structure
Reference
AP-FBG
Aureobasidium pullulans
1,6-β-monoglucopyranosyl branched 1,3β-glucan
17
GRN
Grifora frondosa
1,6-β-monoglucopyranosyl branched 1,3β-glucan
18, 19
Curdlan
Alcaligenes faecalis
Linear 1,3-β-glucan
24
CSBG
Candida albicans
1,6-β-long glucopyranosyl branched 1,3-βglucan
20
Pustulan
Umbilicaria papullosa
Linear 1,6-β-glucan
25
Dextran T500
Leuconostoc dextranium
1,3-α-glucopyranosyl branched 1,6-αglucan
26
Table 1. Tada et al.
13
Tada et al.
a)
b)
0.45 0.4
2
Optical density at 450 nm
0.35 Optical density at 450 nm
1
1.5
1
0.3 0.25 0.2 0.15 0.1
0.5
0.05 0
0 64000
32000
16000
8000
4000
2000
1000
500
IgG1
Serum dilution
IgG2
IgG3
IgG subclass
Figure 1. Tada et al.
14
IgG4
Tada et al.
1
α-
1,3-β-
1,6-β-
AP-FBG GRN Curdlan CSBG Pustulan DEX
Figure 2. Tada et al.
15
Tada et al.
1
100
Percentage inhibition
80
60
40
20
0 AP-FBG
GRN
Curdlan
CSBG
-20 Competitor
16
Pustulan
DEX
