J. of Kyushu Univ. of Health and Welfare. 19： 87〜94，201887

Serum levels of β-1,3-1,6 glucan-specific antibodies and immune
biomarkers in normal individuals
Nobunao Ikewaki, Tohru Sonoda*, Yasushi Miyazawa**, Shin Onizuka***, Masayuki Chikamori**

Abstract
In this clinical study, we examined the serum levels of β-1,3-1,6 glucan (BG)-specific antibodies (IgG,
IgA2 and IgM) and the peripheral blood (PB) levels of immune biomarkers, including the serum total
immunoglobulin (IgG, IgA and IgM), interleukin-6 (IL-6), IL-12 and soluble-form CD44 (sCD44), in 15
normal individuals, using an originally developed enzyme-linked immunoassay (EIA). We found that thesera of even normal healthy individuals consistently contain BG-specific IgG, IgA2 and IgM, and that thetiters of these antibodies vary widely among individuals. However, the serum titers of BG-specific IgM
were strongly and significantly correlated with the serum total IgM titers (r = 0.914, P < 0.001).
Furthermore, the serum titers of BG-specific IgG were significantly correlated with the serum levels of
sCD44 (r = 0.611, P = 0.015), and the serum titers of BG-specific IgA2 were significantly correlated withthe serum levels of IL-6 (r = 0.588, P = 0.021). In addition, the serum titers of BG-specific IgG, IgA2 andIgM showed a tendency to be correlated with the serum levels of IL-12 (r = 0.449, P = 0.081; r = 0.429, P= 0.092; r = 0.473, P = 0.070, respectively). Taken together, these findings show that normal human sera
consistently and naturally contain BG-specific IgG, IgA2 and IgM. Since the titers of these BG-specificantibodies are correlated with the serum levels of some immune biomarkers, it may be surmised thatthey are closely associated with various immunological responses in the living body to maintain a normal
health condition.
Key words ：β-1,3-1,6 glucan-specific antibodies, biomarker, IL-6, IL-12, sCD44

Introductionof lymphocytes, monocytes, macrophages, granulocytes,
and natural killer (NK) cells 5). In general, the anti-

β-glucans are mainly found in the extracts of somecancer actions of β-glucans are not attributable to their

species of mushrooms and in microbes, such as black

direct actions on cancer cells, as is the case withyeast, and possess some unique immunologicalchemical anti-cancer drugs, but depends on the. β-glucans have been shown to exert

immunological enhancement of the host, e.g., by acting

accompanied

as a biological response modifier (BRM) 3). On the otherby activating the production of interleukin-2 (IL-2), IL-6,

hand, several studies have provided evidence toIL-12, interferon-γ (IFN-γ) and tumor necrosis factor-α

suggest that Dectin-1 is the β-glucan receptor 6).

activities

1, 2)

cytotoxic activity against cancer cells

3)(TNF-α) 4). These findings indicate that β-glucans

We established and succeeded in purifying β-1,3-1,6enhance the immune system underlying the activation

glucan produced by the black yeast Aureobasidium

Department of Medical Life Science, Kyushu University of Health and Welfare School of Medical Life Science, Institute of Immunology, Junsei
Educational Institution, 1714-1 Yoshino-machi, Nobeoka-city, Miyazaki, 882-8508 Japan*Department of Occupational Therapy, Kyushu University of Health and Welfare School of Health Science, Institute of Immunology, Junsei
Educational Institution, 1714-1 Yoshino-machi, Nobeoka-city, Miyazaki, 882-8508 Japan**Chikamori Hospital, 1-1-16 Okawasuji, Kochi-city, Kochi, 780-8522 Japan***Department of Orthoptics and Visual Science, Kyushu University of Health and Welfare School of Health Science, Institute of Immunology,
Junsei Educational Institution, 1714-1 Yoshino-machi, Nobeoka-city, Miyazaki, 882-8508 Japan88

九州保健福祉大学研究紀要 19： 87〜94，2018

pullulans (A. pullulans) strain AFO-202 using the latest

review boards (IRB) of Kyushu University of Healthculture technology, and examined the immunologicaland Welfare and Chikamori Hospital; the IRB numbers

actions of this β-1,3-1,6 glucan in vitro using U937, awere 15-048 and 161, respectively. Informed consent

human monocyte-like cell line, and human peripheral

was obtained from all the donors prior to theirblood mononuclear cells (PBMCs) 1).

participation in this study.

Recently, it was reported that human and animal
sera consistently contain β-glucan-specific antibodies 7, 8).

Processing of the blood and serum samples

However, the immunological roles of these antibodies
in the living body, in particular, the relationshipBlood samples of 15 normal individuals (5 males, age

between the β-glucan-specific antibodies and immune

38.2 ± 9.5 yr; 10 females, age 28.7 ± 5.2 yr) werebiomarkers such as various cytokines and soluble-form

obtained at Chikamori Hospital (Kochi-city, Kochi). The

molecules (CD antigens) have not yet been examined.

serum samples were prepared using the standard

In this clinical study, we examined the serum levelsof β-1,3-1,6 glucan (BG)-specific antibodies (IgG, IgA2
and IgM) and the peripheral blood (PB) levels of
immune biomarkers, including those of serum total

method and the sera were stored at -80℃ until use.

Measurement of β-1,3-1,6 glucan-specificantibodies in the sera

immunoglobulin (IgG, IgA and IgM), interleukin-6 (IL-6),
IL-12 and soluble-form CD44 (sCD44), in 15 normal

EIA plates (Sumitomo Co., Tokyo) were coated withindividuals, using an originally developed enzyme-

1 ㎍ /mL of purified β-1,3-1,6 glucan (BG) in carbonate-

linked immunoassay (EIA) in our laboratories.

bicarbonate buffer (0.010 M NaCO3 , 0.035 M NaHCO3 ,Materials and Methods
Reagents

pH9.6) for 24 hr at 4℃ . The wells were washed fourtimes with phosphate-buffered saline (PBS) containing
0.05% Tween-20 (PBST) and blocked with PBST
containing 2% BSA (BSA-PBST) for 60 min at room
temperature. Thereafter, the wells were washed three

A β-1,3-1,6 glucan was synthesized by Sophy Co.

times with PBST. The sera (x1,000 dilution for IgG, x50(Kochi-city, Kochi) from Aureobasidium pullulans (A.

dilution for IgA2 and x500 dilution for IgM with 0.1%

pullulans ) strain AFO-202 using the latest biological

BSA-PBST; 50 µL) were then added to each well,culture and preparation techniques, and is currently

followed by incubation of the plate for 60 min at room

available commercially as a health food supplement. In

temperature with shaking. Then, the wells were

this study, the purified β-1,3-1,6 glucan described above

washed five times with PBST, followed by addition of

was kindly provided to us for this study by Sophy Co.

HRPOD-conjugated rabbit anti-human IgG, HRPOD-

Enzyme-linked immunoassay (EIA) kits for measuring

conjugated mouse anti-human IgA2 mAb or HRPOD-

the blood levels of interleukin-6 (IL-6), IL-12 (p35+p40)

conjugated rabbit anti-human IgM (x5,000 dilution withand soluble-form CD44 (sCD44) were purchased from

0.1% BSA-PBST, respectively; 50 µL) to each well andDiaclone laboratories Co. (USA). Horseradish

incubation of the plate for 60 min at room temperature

peroxidase (HRPOD)-conjugated rabbit anti-human IgG,

with shaking. The wells were then washed 10 times

HRPOD-conjugated rabbit anti-human IgM andwith PBST, followed by addition of the substrate-

H R P O D - c o n j u g a t e d m o u s e a n t i - h u m a n I g A2

chromogen (TMB; Cosmo Bio Co.; 50 µL) to each well

monoclonal antibody (mAb) were purchased from MBL

and incubation of the plate for 20 min at room

Co. (Nagoya).

temperature with gentle shaking. The reaction was

Ethics statement

stopped by the addition of 0.5 M-HCl (50 µL), and theoptical density (O.D.) was read at 450 nm using a
multichannel EIA-microplate reader (TOSHO Co.). The

The study protocol was approved by the institutional

experiment was repeated five times. The specificity

Nobunao Ikewaki, Tohru Sonoda, Yasushi Miyazawa, Shin Onizuka, Masayuki Chikamori：Serum levels of β-1,3-1,6 glucan-specific antibodies in normal individuals

89

and precision were confirmed by a blocking test using

peripheral blood (PB) levels of immune biomarkers,

purified rabbit anti-β-1,3-1,6 glucan-specific IgG

including those of serum total immunoglobulin (IgG,

established in our laboratories as described previously .

IgA and IgM), IL-6, IL-12 and sCD44. Figure 2 shows

8)

Measurement of IL-6, IL-12 and sCD44 in theserathat the serum titers of BG-specific IgM were strongly
and significantly correlated with the serum total IgM
titers (r = 0.914, P < 0.001). Furthermore, the serum
titers of BG-specific IgG were significantly correlated

The measurement of IL-6, IL-12 and sCD44 in thewith the serum levels of sCD44 (r = 0.611, P = 0.015)

serum samples was performed using an IL-6 detection(Fig. 3), and the serum titers of BG-specific IgA2 were

EIA kit, IL-12 detection kit and sCD44 detection kit,significantly correlated with the serum levels of IL-6 (rrespectively. Each of the measurements was repeated= 0.588, P = 0.021) (Fig. 4). In addition, the serum titers

three times.

of BG-specific IgG, IgA2 and IgM showed a tendency

Measurement of serum total IgG, IgA and IgM

to be correlated with the serum levels of IL-12 (r =0.449, P = 0.081; r = 0.429, P = 0.092; r = 0.473, P =0.070, respectively) (Fig. 5).

Measurement of serum total IgG, IgA and IgM in

It is common knowledge that IgM is the firstthe samples was outsourced to a Special Reference

antibody to appear as part of the immunological

Laboratory (SRL). Each experiment was repeated three

response to initial exposure to an antigen. In this study,

times.

since the serum titers of BG-specific IgM were

Statistical analysis

strongly and significantly correlated with the serum
total IgM titers, it may be surmised that BG-specific
IgM may also play an important role in some

Data were expressed as mean ± standard deviation(SD). Statistical analysis of the correlations of theimmunobiological responses to maintain the healthcondition.

serum titers of the BG-specific antibodies (IgG, IgA2

IL-6 is a cytokine produced by T-lymphocytes,

and IgM) with the serum total immunoglobulin (IgG,

B-lymphocytes, monocytes, fibroblasts, endothelial cells,

IgA and IgM), IL-6, IL-12 and sCD44 levels was

etc., and plays some important roles in both natural

performed by the Pearson correlation test. Differences

and acquired immune responses; in particular, IL-6

at P < 0.05 were considered to indicate statisticalregulates the growth and maturation of B-lymphocytes

significance.

to produce antibodies 9). Since the serum titers ofResults and Discussion

BG-specific IgA2 were significantly correlated with theserum levels of IL-6, it is probable that BG-specificIgA2 influences IgA antibody production under IL-6

First, we examined the serum titers of BG-specificaction in the mucosal and gut immune systems.

IgG, IgA2 and IgM in 15 normal individuals using an

CD44 (molecular mass approx. 80-kDa) is expressed

originally developed enzyme-linked immunoassay

on lymphocytes, macrophages, granulocytes,(EIA). As shown in Figure 1, we consistently foundfibroblasts, endothelial cells, natural killer (NK) cells,

that BG-specific IgG, IgA2 and IgM in the sera of theetc. In particular, CD44 plays an important role innormal individuals with the titers varying widely

stimulating NK cell and lymphokine-activated killer cellamong individuals. BG-specific IgG (δOD: 0.81 ± 0.51)(LA K) f unct ions under lying I FN-γ pr oduction,

and BG-specific IgM (δOD: 0.67 ± 0.50) occurred in

activation cell surface antigen expression mediated byhigh titers, while BG-specific IgA2 (δOD: 0.09 ± 0.07)

protein tyrosine kinase (PTK) phosphorylation andoccurred in low titers (Fig. 1) in the serum samples.

intracellular Ca2+ flux 10, 11). On the other hand, there are

Next, we examined the correlations between thethree types of CD44 (CD44H, CD44E and sCD44) withserum titers of BG-specific IgG, IgA2 and IgM and thediffering biological functions. The sCD44 level in the

90

九州保健福祉大学研究紀要 19： 87〜94，2018

serum has been reported to be increased in association

Disclosure

with immunological activities. Since the serum titers ofNone of the authors has any conflict of interest to

BG-specific IgG were significantly correlated with theserum levels of sCD44, it is probable that BG-specific
IgG is related to NK cell and LAK cell functions.
IL-12 is a cytokine produced by the dendritic cells

disclose.

References(DCs), macrophages and neutrophils in response to
antigenic stimulation. IL-12 plays an important role in1.

Ikewaki, N., Fujii, N., Onaka, T., et al.:the activities of NK cells and cytotoxic T-lymphocytes

Immunological actions of Sophy beta-glucan (beta-(CTLs), and directly mediates enhancement of the

1,3-1,6-glucan), currently available commercially as

cytotoxic activity of NK cells and CTLs accompanieda health food supplement. Microbiol. Immunol.

by IFN-γ production

12). Since the serum titers of

51:861-873, 2007.

BG-specific IgG, IgA2 and IgM showed a tendency to
be correlated with the serum levels of IL-12, it may be

2.

Kim, H.S., Hong, J.T., Kim, Y., et al.: Stimulatory

assumed that BG-specific antibodies also indirectly

effect of β-glucans on immune cells. Immune.

regulate NK cell and CTL functions in immune system.

Netw. 11: 191-195, 2011.

We examined the serum titers of BG-specificantibodies (IgG, IgA2 and IgM) in normal individuals.

3.

Vetvicka, V., Vetvickova, J. : Glucan

Our finding of the existence of correlations between

supplementation has strong anti-melanoma effects:

the serum titers of BG-specific antibodies and theRole of NK cells. Anticancer Res. 35: 5287-5292,

serum levels of various immune biomarkers strongly

2015.

suggest the possible involvement of BG-specific4.

Ostadrahimi, A., Ziaei, J.E., Esfahani, A., et al. :antibodies in the regulation of the immune system. The

Effect of beta glucan on white blood cell counts

detailed mechanism(s) underlying the immunologicaland serum levels of IL-4 and IL-12 in women withroles of the BG-specific antibodies in the sera of normal

breast cancer undergoing chemotherapy: aindividuals remains unclear at the present time.

randomized double-blind placebo-controlled clinical

It is possible that the intestinal bacteria flora

trial. Asian Pac. J. Cancer Prev. 15: 5733-5739, 2014.

influence the production of these antibodies. On thebasis of the relationships between the serum titers of

5.

Ding, J., Feng, T., Ning, Y., et al. : β-Glucan

BG-specific antibodies and the serum levels of someenhances cytotoxic T lymphocyte responses byimmune biomarkers, it may be considered that when

activation of human monocyte-derived dendritic

we consume BG as a health food supplement,

cells via the PI3K/AKT pathway. Hum. Immunol.

BG-specific antibodies may more effectively regulate

76: 146-154, 2015.

the immune system in the body as a biologicalresponse modifier (BRM). Further analyses are needed

6.

Kim, H.S., Park, K.H., Lee, H.K., et al.: Curdlan

to examine the detailed production mechanism(s) ofactivates dendritic cells through dectin-1 and toll-

BG-specific antibodies at the cellular and molecularlike receptor 4 signaling. Int. Immunopharmacol.

levels and the immunological roles of these antibodies

39: 71-78, 2016.

in the sera of normal individuals.

Acknowledgements

7.

Ishibashi, K., Morita, M., Motoi, M., et al.: Analysisof the titer and reactivity of antibody/ies against
fungal cell wall β-glucans in human sera. Int. J.

We wish to extend our gratitude to Onaka T and

Med. Mushrooms. 15: 115-126, 2013.

Ikeue Y of Sophy Co. (Kochi-city, Kochi) for preparing
and providing us with β-1,3-1,6 glucan.

8.

Ikewaki, K., Sonoda, T., Azuma, K.: A study of anti-

Nobunao Ikewaki, Tohru Sonoda, Yasushi Miyazawa, Shin Onizuka, Masayuki Chikamori：Serum levels of β-1,3-1,6 glucan-specific antibodies in normal individuals

β-glucan antibody in human serum. J. Kyushu

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Eur. J. Immunol. 26: 2807-2811, 1996.

Univ. H. W. 16: 115-119, 2015.
11. Sun, J., Law, G.P., McKallip, R.J. : Role of CD44 in
9.

Shen, P., Fillatreau, S.: Antibody-independent

lymphokine-activated killer cell-mediated killing offunctions of B cells: a focus on cytokines. Nat. Rev.

melanoma. Cancer Immunol. Immunother. 61: 323-

Immunol. 15: 441-451, 2015.

334, 2012.

10. Galandrini, R., Piccoli, M., Frati, L., et al.: Tyrosine

12. Freeman, B.E., Raué, H.P., Hill, A.B., et al.:kinase-dependent activation of human NK cell

Cytokine-mediated activation of NK cells duringfunctions upon triggering through CD44 receptor.

viral infection. J. Virol. 89: 7922-7931, 2015.

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九州保健福祉大学研究紀要 19： 87〜94，2018

1.5
1

7
6
5
4
3
2
1
0

0

0.5 1 1.5
BG-IgG (δOD)

IL-6 (pg/mL)

0.5
0
BG-IgG

BG-IgA2

BG-IgM

Figure 1. Measurement of BG-specific IgG, IgA2 andIgM in the sera of 15 normal individuals. The
measurements were performed using an originallydeveloped EIA system (See Materials and Methods fordetails). Each measurement was repeated four times.

Serum total IgG(mg/dL)

1500
1000

r=0.124
P=0.639500
00.5 1 1.5
BG-IgG (δOD)

Serum total IgM(mg/dL)

0

300
250
200
150
100
50
0

2

Serum total IgA(mg/dL)

A

2000

350
300
250
200
150
100
50
0

B

r=0.347
P=0.205
0

0.1
0.2
0.3
BG-IgA2 (δOD)

C

r=0.914
P<0.001
0

0.5
1
1.5
BG-IgM (δOD)

2
㻲㼕㼓㻚㻞

Figure 2. Analysis of the correlations of the serum
titers of BG-specific antibodies (IgG, IgA2 and IgM) andthe serum total immunoglobulin (IgG, IgA and IgM)
titers in normal individuals (n = 15). A: Correlation
between the serum titers of BG-specific IgG and serum
total IgG titers (r = 0.124, P = 0.639) in all normal
individuals. B: Correlation between the serum titers of
BG-specific IgA2 and serum total IgA titers (r = 0.347,
P = 0.205) in all normal individuals. C: Correlation
between the serum titers of BG-specific IgM and theserum total IgM titers (r = 0.914, P < 0.001) in all
normal individuals.

A

r=0.370
P=0.174

IL-6 (pg/mL)

IL-6 (pg/mL)

2

δOD

7
6
5
4
3
2
1
0

2

7
6
5
4
3
2
1
0

r=0.588
P=0.0210r=0.233
P=0.403

0

0.5
1
1.5
BG-IgM (δOD)

B

0.1
0.2
0.3
BG-IgA2 (δOD)

C

2
㻲㼕㼓㻚㻟

Figure 3. Analysis of the correlations of the serum
titers of BG-specific antibodies (IgG, IgA2 and IgM) andthe serum levels of IL-6 in normal individuals (n = 15).
A: Correlation between the serum titers of BG-specific
IgG and the serum levels of IL-6 (r = 0.370, P = 0.174)
in all normal individuals. B: Correlation between theserum titers of BG-specific IgA2 and the serum levelsof IL-6 (r = 0.588, P = 0.021) in all normal individuals.
C: Correlation between the serum titers of BG-specific
IgM and the serum levels of IL-6 (r = 0.233, P =0.403) in all normal individuals.

0.5
1
1.5
BG-IgG (δOD)
300
250
200
150
100
50
0

2

r=0.075
P=0.790
0

A

250
200
150

100
0

0.1
0.2
0.3
BG-IgA2 (δOD)

r=0.449
P=0.081

50
0

0.5 1
1.5
BG-IgG (δOD)

C

r=0.050
P=0.858
0

0.5
1
1.5
2
BG-IgM (δOD)

2

200

150
100

r=0.429
P=0.09250
00Figure 4. Analysis of the correlations of the serum
titers of BG-specific antibodies (IgG, IgA2 and IgM) andthe serum levels of sCD44 in normal individuals (n =15). A: Correlation between the serum titers of
BG-specific IgG and the serum levels of sCD44 (r =0.611, P = 0.015) in all normal individuals. B:
Correlation between the serum titers of BG-specificIgA2 and the serum levels of sCD44 (r = 0.075, P =0.790) in all normal individuals. C: Correlation between
the serum titers of BG-specific IgM and the serum
levels of sCD44 (r = 0.050, P = 0.858) in all normal
individuals.

0.1
0.2
0.3
BG-IgA2 (δOD)

C

200
150
100

r=0.473
P=0.070

50
0

㻲㼕㼓㻚㻠

93

B

250

250
IL-12 (pg/mL)

0B

IL-12 (pg/mL)

r=0.611
P=0.015

300
250
200
150
100
50
0

IL-12 (pg/mL)A
sCD44 (ng/mL)

300
250
200
150
100
50
0

sCD44 (ng/mL)

sCD44 (ng/mL)

Nobunao Ikewaki, Tohru Sonoda, Yasushi Miyazawa, Shin Onizuka, Masayuki Chikamori：Serum levels of β-1,3-1,6 glucan-specific antibodies in normal individuals

0

0.5 1 1.5
BG-IgM (δOD)

2
㻲㼕㼓㻚㻡

Figure 5. Analysis of the correlations of the serum
titers of BG-specific antibodies (IgG, IgA2 and IgM) andthe serum levels of IL-12 in normal individuals (n = 15).
A: Correlation between the serum titers of BG-specific
IgG and the serum levels of IL-12 (r = 0.449, P =0.081) in all normal individuals. B: Correlation between
the serum titers of BG-specific IgA2 and the serum
levels of IL-12 (r = 0.429, P = 0.092) in all normal
individuals. C: Correlation between the serum titers of
BG-specific IgM and the serum levels of IL-12 (r =473, P = 0.070) in all normal individuals.

94

九州保健福祉大学研究紀要 19： 87〜94，2018

健常人におけるβ-1,3-1,6 glucan特異抗体と免疫バイオマーカーの血清レベル
池脇

信直，園田

徹 *，宮澤靖 **，鬼塚信 ***，近森正幸 **九州保健福祉大学生命医科学部生命医科学科・順正学園免疫学研究所〒882-8508

宮崎県延岡市吉野町1714-1*九州保健福祉大学保健科学部作業療法学科・順正学園免疫学研究所〒882-8508

宮崎県延岡市吉野町1714-1**近森病院〒780-8522

高知県高知市大川筋1-1-16***九州保健福祉大学保健科学部視機能療法学科・順正学園免疫学研究所 〒882-8508 宮崎県延岡市吉野町1714-1

要旨
健常人（15名）におけるβ-1,3-1,6 glucan（BG）特異抗体と免疫バイオマーカーの血清レベルを自主開発した酵素抗体法（enzyme immunoassay：EIA）を用いて検討した。その結果、健常人血清中には BG特異
IgG、IgA2および IgMが恒常的に存在することが分かった。次に、BG特異抗体と免疫バイオマーカーとの相関性を検討したところ、BG特異 IgMは血清総 IgMと強い相関（r=0.914、P<0.001）を示した。また、BG
特異 IgGは可溶性 CD44
（sCD44）と有意な相関（r=0.611、P=0.015）を示し、BG特異 IgA2は IL-6と有意な相関（r = 0.588、P = 0.021）を示した。さらに、BG特異 IgG、IgA2および IgMと IL-12との相関は有意傾向（r=0.449、P=0.081；r=0.429、P=0.092； r=0.473、P=0.070）を各々示した。以上の結果は、BG特異 IgG、IgA2および IgM が生体恒常性維持のための免疫応答に密接に関与することを示唆している。
キーワード：BG 特異抗体、バイオマーカー、インターロイキン 6、インターロイキン 12、可溶性 CD44