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High Throughput ELISAs to Measure a Unique Glycan on Transferrin in Cerebrospinal Fluid: A Possible Extension toward Alzheimer's Disease Biomarker Development

Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
International Journal of Alzheimer's Disease 08/2011; 2011:352787. DOI: 10.4061/2011/352787
Source: PubMed

ABSTRACT

We have established high-throughput lectin-antibody ELISAs to measure different glycans on transferrin (Tf) in cerebrospinal fluid (CSF) using lectins and an anti-transferrin antibody (TfAb). Lectin blot and precipitation analysis of CSF revealed that PVL (Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on "CSF-type" Tf whereas SSA (Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on "serum-type" Tf. PVL-TfAb ELISA of 0.5 μL CSF samples detected "CSF-type" Tf but not "serum-type" Tf whereas SSA-TfAb ELISA detected "serum-type" Tf but not "CSF-type" Tf, demonstrating the specificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus (iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-reactive Tf were significantly higher than in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological diseases.

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Available from: Atsushi Kuno, Jun 30, 2015
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International Journal of Alzheimer’s Disease
Volume 2011, Ar t i cle ID 352787, 5 pages
doi:10.4061/2011/352787
Research Ar ticle
High Throughput ELISAs to Measure a Unique Glycan on
Transferrin in Cerebrospinal Fluid: A Possible Extension toward
Alzheimer’s Disease Biomarker Development
Keiro Shirotani,
1
Satoshi Futakawa,
1
Kiyomitsu Nara,
1
Kyoka Hoshi,
1
Toshie Saito,
1
Yuriko Tohyama,
1
Shinobu Kitazume,
2
Tatsuhiko Yuasa,
3
Masakazu Miyajima,
4
Hajime Arai,
4
Atsushi Kuno,
5
Hisashi Narimatsu ,
5
and Yasuhiro Hashimoto
1
1
Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan
2
Disease Glycomics Team, RIKEN Advanced Science Institute, Wako 351-0198, Japan
3
Department of Neurology, Kamagaya General Hospital, Kamagaya 273-0100, Japan
4
Department of Neurosurgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
5
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba 305-8568, Japan
Correspondence should be addressed to K eir o Shirotani, keiroshi@fmu.ac.jp
Received 1 December 2010; Accepted 24 May 2011
Academic Editor: Holly Soares
Copyright © 2011 Keiro Shirotani et al. This is a n open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and r eproduction in any medium, provided the original work is properly cited.
We have established high-throughput lectin-antibody ELISAs to measure dierent glycans on transferrin (Tf) in c erebrospinal
fluid (CSF) using lectins and an anti-transferrin antibody ( TfAb). Lectin blot and precipitation analysis of CSF revealed that
PVL (Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on “CSF-type” Tf whereas SSA
(Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on “serum-type” Tf. PVL-TfAb
ELISA of 0.5 μL CSF samples detected “CSF-type” Tf but not “serum-type” Tf whereas SSA-TfAb ELISA detected “serum-type” Tf
but not “CSF-ty pe” Tf, demonstrating the s pecificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus
(iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-rea ctive Tf were significantly higher than
in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical
diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological
diseases.
1. Introduction
CSF (cerebrospinal fluid), which circulates within the ventri-
cles of the brain and subarachnoid space, reflects the phys-
iological and pathological conditions of the central nervous
system [1]. In fact, CSF proteins are used as biomarkers to
diagnose neurological diseases such as idiopathic normal
pressure hydrocephalus (iNPH) [24]andAlzheimersdis-
ease (AD) [5, 6] and may predict the disease onset in a pre-
clinical stage [7]. Since iNPH and AD show similar pheno-
types such as dementia and ventriculomegaly, it is dicult
to distinguish the two diseases especially in elderly patients.
Therefore, simultaneous measurements of a battery of iNPH
biomarkers and AD biomarkers could help exact diagnosis of
iNPH and AD.
We previously designated Tf-1 and Tf-2 as two isoforms
of transferrin in CSF which are separable by SDS-PAGE
and show ed that the Tf-2/Tf-1 ratio is higher in iNPH
patients than in non-iNPH patients [3, 4]. In that study
we used immunoblotting method to detect Tf-1 and Tf-
2, but the low throughput of immunoblotting makes it
impractical for clinical use. Although sandwich ELISA (en-
zyme-linked immunosorbent assay) or latex photometric
immunoassay is high throughput, Tf-1 and Tf-2 cannot be
distinguished by these methods because Tf-1 and Tf-2 are
dierent only in their glycan portion, and the antibodies
Page 1
2 International Journal of Alzheimer’s Disease
against the protein portion of Tf cannot distinguish the two
isoforms.
To establish a high throughput method to distinguish Tf-
1 and Tf-2, we developed ELISAs using a combination of
lectins and an anti-Tf antibody (TfAb). Tf-1 has a “CSF-
type” biantennary asialo- and agalacto-complex type N-
glycans with bisecting β1,4-N-acetylglucosamine (GlcNAc)
and core α1,6-Fucose [3, 8], whereas Tf-2 in CSF has a
“serum-type” biantennary N-glycans with α2,6-N-acetyl-
neuraminic acid (NeuAc) [3, 9]. Because of their distinct
terminal sugars, that is, GlcNAc on Tf-1 and α2,6-NeuAc
on Tf-2, we chose PVL (Psathyrella velutina lectin) and SSA
(Sambucus sieboldiana agglutinin) to detect Tf-1 and Tf-2
respectively. Our data showed that the lectin-TfAb ELISAs
distinguish the two isoforms to be quantified and that the
amounts of SSA-reactive Tf were higher in iNPH patients
than in non-iNPH patients. Our newly established lectin-
TfAb ELISAs are high throughput methods to measure
glycoforms” of transferrin which might be practical for
clinical use.
2. Materials and Methods
2.1. Patients. This study included 28 iNPH patients compris-
ing 14 males and 14 females aged 75.2
± 6.1 years (mean ±
SD) and 18 non-iNPH patients comprising 10 males and 8
females aged 74.9
± 5.2 years [3]. The iNPH patients were
diagnosed using the clinical guidelines for iNPH issued by
the Japanese Society of NPH [10]. A bolus infusion test and
the tap test were performed routinely. Patients whose gait
disturbance improves after the tap test, which removes 30 mL
of CSF via a lumbar puncture, were treated with a shunt
operation. Those who showed symptomatic improvement
1 month after the shunt operation were defined as iNPH
patients while t hose who d id not were defined as non-iNPH
patients. In addition, those who did not show improvement
after the tap test were classified as non-iNPH patients. The
study was approved by the ethics committee of Fukushima
Medical University (No. 613), which is guided by local policy,
national law, and the World Medical Association Declaration
of Helsinki.
2.2. Immunoblotting and Lectin Blotting. CSF samples were
dissolved in Laemmli buer, boiled for 3 min, and loaded
onto SDS-polyacrylamide gels (SuperSep Ace; Wako Pure
Chemical Industries, Osaka, Japan). After SDS-PAGE, the
proteins were tr ansferred to a nitrocellulose membrane (Bio-
Rad Laboratories, Hercules, Calif, USA). The membrane
was blocked in 3% skim milk
incubated sequentially
with an anti-transferrin antibody (Bethyl Laboratories,
Montgomery, Tex, USA) and a horseradish peroxidase-
labeled anti-goat IgG (Jackson ImmunoResearch Laborato-
ries, West Grove, Pa, USA), and developed using a Super
Signal West Dura Extended Duration Substrate (Pierce
Biotechnology, Rockford, Ill, USA). For lectin blotting,
the transferred membrane was blocked in 1% BSA, incu-
bated with a biotinylated PVL or biotinylated SSA (Seika-
gaku Corporation, Tokyo, Japan) followed by a horseradish
peroxidase-labeled streptavidin (Takara, Shiga, Japan), and
developed.
2.3. Lectin Precipitation. CSF was incubated with S SA-aga-
rose (Seikagaku Corporation), and the bound proteins were
precipitated by centrifugation. The unbound proteins were
further incubated with PVL-agarose (Seikagaku Corpora-
tion), and the bound proteins were precipitated.
2.4. Purification of Tf-1. Tf-1 was purified from human CSF
as described before [3]. Briefly, CSF was applied to a HiTrap
Blue HP column (GE Healthcare, Buckinghamshire, UK).
The unbound proteins were applied to a HiTrap Q HP
column (GE Healthcare). The bound proteins were eluted
with a linear gradient o f NaCl from 0 to 300 mM. Tf-1 was
eluted at 130 mM NaCl. Tf-1 was further purified by rechro-
matography with a HiTrap Q HP column. The concentration
of the purified Tf-1 was determined by immunoblot analysis
with commercially available human Tf (Sigma-Aldrich, S t.
Louis, Mo, USA) as the standard.
2.5. Lectin-Antibody ELISAs. For PVL-TfAb ELISA, a 96-
well C8 Maxisorp Nunc immuno module plate (Nunc,
Roskilde, Denmark) was coated with 2.5 μg PVL (Seikagaku
corporation) at 4
C overnight and blocked with 0.4% Block-
Ace (Dainippon Sumitomo Pharma, Osaka). Purified Tf-1
was used as the standard. The standards and CSF samples
were appropriately diluted w ith PBST (phosphate-buered
saline/0.05% Tween-20), applied to the plate, and incubated
at 4
C overnig ht. After three washes with PBST, the plate
was incubated sequentially with anti-Tf antibody (Bethyl
laboratories, Montgomery, Tex, USA) and horseradish
peroxidase-labeled anti-goat IgG (Jackson ImmunoResearch
Laboratories, West Grove, Pa, USA). After three washes with
PBST, the wells were incubated with TMB solution (Wako,
Osaka, Japan), and 1 N HCl was added to stop the reaction.
Absorbances at 450 nm were measured by a plate reader (Bio-
Rad Laboratories). CV (Coecientofvariation)ofPVL-
ELISA was 5.36%.
For SSA-TfAb ELISA, anti-Tf antibodies (Cappel; ICN
Pharmaceuticals, Aurora, Ohio, USA) were pretreated with
1.4 mM sodium periodate to abolish SSA epitopes on the
antibody and coated on a 96-well plate. Human reference
serum (Bethyl laboratories) containing 3 mg/mL serum Tf
was used as the standard. The standards and CSF samples
were appropriately diluted with TBS (Tris-buered saline)
containing 0.05% Tween 20 and 0.5 mM EDTA and applied
to the plate and incubated at 4
Covernight.Afterthree
washes with TBST, the plate was incubated sequentially with
a biotin-SSA (Seikagaku Corporation) and a horseradish
peroxidase-labeled streptavidin (Takara). After three washes
with TBST, the plate was incubated with the TMB substrate,
and the absorbances at 450 nm were measured. CV of SSA-
ELISA was 3.65%.
2.6. Statistical Analysis. Data were analyzed with SPSS ver-
sion 17 (SPSS, Chicago, Ill, USA). Amounts of SSA-Tf and
Page 2
International Journal of Alzheimer’s Disease 3
PVL SSA
Tf-2
Tf-1
75 kDa
Anti-Tf
(a)
SSA-
PVL-
agarose
agarose
Tf-2
Tf-1
Input
Bound
Unbound
Bound
(b)
Figure 1: PVL and SSA specifically detect Tf-1 and Tf-2, respectively. (a) CSF was electrophoresed, blotted, and stained with anti-Tf antibody
(left panel), PVL (center panel), and SSA (right panel). (b) CSF (input) was sequentially precipitated by SSA-agarose and PVL-agarose. The
bound and unbound proteins were electrophoresed and immunoblotted by anti-Tf antibody.
PVL-Tf were analyzed by the Student’s t-test and Mann-
Whitney U test, respectively.
3. Results
To establish high throughput lectin-TfAb ELISAs that dis-
tinguish “CSF-type” Tf-1 and “serum-type” Tf-2, we first
examined whether PVL and SSA specifically detect g lycans
on Tf-1 and Tf-2, respectively, by lectin-blotting. As we
reported previously by immunoblotting, Tf-1 and Tf-2 in
CSF were separated on SDS-gel (Figure 1(a) left). When PVL
was used as a probe, a band with similar mobility to Tf-
1wasdetectedinCSF(Figure 1(a) center), suggesting that
PVL specifically detects the terminal GlcNAc on Tf-1 but not
sugars on Tf-2. In contrast, when SSA was used as a probe,
a band with similar mobility to Tf-2 was detected in CSF
(Figure 1(a) right), suggesting that SSA, detects the terminal
α2,6-NeuAc on Tf2 but not sugars on Tf-1. These band
signals detected by PVL and SSA (Figure 1(a) center and
right) were depleted by TfAb (data not shown), suggesting
that the glycan epitopes detected by PVL and SSA reside on
the Tf core protein. Moreover, when CSF was precipitated
sequentially by SSA- and PVL-agarose, Tf-2 was specifically
recognized by SSA, and Tf-1 was recognized by PVL
(Figure 1(b)). Taken together, PVL and SSA can recognize
Tf-1 and Tf-2, respectively, and distinguish the two Tf glyco-
isoforms.
Next we investigated whether Tf-1 and Tf-2 were specif-
ically detected by PVL-TfAb ELISA and SSA-TfAb ELISA
systems, respectively (Figures 2(a) and 2(b)). The purified
Tf-1wasusedasastandardtomeasureTf-1inCSFbyPVL-
TfAb ELISA, whereas Tf in serum was used as a standard
to measure Tf-2 by SSA-TfAb ELISA, since serum contains
only “serum-type” Tf with very similar glycans to Tf-2 [3].
As shown in Figure 2(c), the purified Tf-1 was successfully
detected in a dose-dependent manner (4–64 ng/mL) by PVL-
TfAb ELISA, but no significant signals were detected with the
serum Tf, indicating that the PVL-TfAb ELISA specifically
detects Tf-1. Moreover, SSA-TfAb ELISA detected serum
Tf (4–64 ng/mL) but not the purified Tf-1 (Figure 2(d)),
suggesting specificity of the SSA-TfAb ELISA for serum Tf
and Tf-2. Tfs which were detected by PVL-TfAb ELISA and
SSA TfAb ELISA were designated as PVL-Tf and SSA-Tf,
respectively .
Finally we measured the concentrations of PVL-Tf and
SSA-Tf in CSF from iNPH and non-iNPH patients. As
shown in Figure 3, amounts of SSA-Tf were significantly
increased in iNPH patients compared to non-iNPH patients
while amounts of PVL-Tf were not significantly dierent
suggesting that the SSA-Tf might be an iNPH marker. The
SSA-Tf/PVL-Tf ratio were also increased in iNPH patients
(not shown), which is consistent with our previous data
[3].
4. Discussion
In this study we have developed the high throughput lectin-
TfAb ELISAs to measure Tf isoforms that have dierent
terminal sugars. PVL-TfAb ELISA successfully detected Tf-1
but not Tf-2 whereas SSA-TfAb ELISA detected Tf-2 but not
Tf-1, demonstrating that the lectin-TfAb ELISAs distinguish
the two Tf isoforms to be quantified. Application of the
method to iNPH patients revealed that amounts of SSA-
Tf were significantly higher in patients with iNPH than
in those without, suggesting that the lectin-TfAb ELISAs
are promising high throughput methods for diagnosing
iNPH. It would be interesting if the SSA-Tf or PVL-Tf is a
diagnostic marker for AD or the Tfs can distinguish AD and
iNPH.
The lectin-TfAb ELISAs have two dierences compared
to our previously developed immunoblotting method. First,
the lectin-TfAb ELISA is more suitable for clinical use
because the ELISA can process more samples. Second,
the lectin-TfAb ELISAs detect amounts and structures
of glycans on Tf while the immunoblot detects Tf core
protein. The immunoblot is a well-established method for
discovering biomarkers, but most glycoforms of CSF pro-
teins, in contrast with Tf glycoforms, are not separable by
Page 3
4 International Journal of Alzheimer’s Disease
Tf-2
sTf
TfAb
PVL
Tf-1
PVL
(a)
Tf-1
Tf-2
sTf
TfAb
SSA
SSA
(b)
OD 450 nm
Tf (ng/mL)
0.5
1
1.5
2
2.5
0 10203040506070
Tf-1
sTf
(c)
OD 450 nm
Tf (ng/mL)
0.5
1
1.5
2
2.5
0 10203040506070
Tf-1
sTf
(d)
Figure 2: PVL-TfAb ELISA and SSA-TfAb ELISA specifically detect Tf-1 and Tf-2/serum Tf (sTf), respectively. (a) and (b). Schematic
representation of lectin-TfAb ELISAs. PVL-TfAb ELISA (a) detects o nly Tf-1 while SSA-TfAb ELISA (b) detects only Tf-2/sTf. Closed
triangles and rectangles represent “CSF-type” and “serum-type” glycans on Tf, respectively. (c) and (d) both the purified Tf-1 and serum
Tf were measured in PVL-Tf ELISA (c) and SSA-TfAb ELISA (d). ODs at 450 nm were plotted at each concentration of each Tf. Closed and
opened circles show the Tf-1 and serum Tf, respectiv ely.
SDS-PAGE (unpublished observation). In such cases, the
lectin-antibody ELISAs are more useful to detect specific
glycoforms than immunoblotting. Based on the concept
that searches for biomarkers should involve not only “pro-
teomics but also “glycoproteomics” [11, 12], we are cur-
rently trying to develop ELISAs using various combinations
of lectins and antibodies and to find new glycobiomarkers
for iNPH as well as other neurological diseases such as
AD.
5. Conclusion
We have developed the PVL-TfAb ELISA and SSA-TfAb
ELISA to measure Tf-1 and Tf-2, respectively. Amounts of
the SSA-reactive Tf were significantly higher in CSF of iNPH
patients than in non-iNPH patients, suggesting that the lec-
tin-TfAb ELISAs are promising high throughput methods
for diagnosing iNPH. The lectin-antibody ELISAs might be
useful for a CSF biomarker study of neurological diseases.
Page 4
International Journal of Alzheimer’s Disease 5
60
40
20
0
iNPHNon-iNPH
SSA-Tf (µg/mL)
(a)
2
1
0
iNPHNon-iNPH
PVL-Tf (µg/mL)
(b)
Figure 3: The SSA-Tf is increased in iNPH patients. Concentrations o f SSA-Tf (a) and PVL-Tf (b) were measured in CSF of non-iNPH
(n
= 18) and iNPH (n = 28) patients, and box plots were shown. An asterisk indicates significantly dierent (P<0.05).Anopenandclosed
circle represent an outlier and an extreme value, respectively.
Acknowledgments
This work was supported by the New Energy and Industrial
Technology Development Organization (NEDO) of Japan.
Yasuhiro Hashimoto was the recipient of a grant from the
Ministry of Health, Labor, and Welfare of Japan (Grant
no. 2006-Nanchi-Ippan-017); the Ministry of Education,
Science, Sports, and Culture of Japan (Grant no. 20023023);
the Naito Foundation. We thank Professor Tatsuya Okada for
advice on the statistical analysis, Dr. Kenneth Nollet for edi-
torial advice, and Ms. Kaori Hagita and Yukari Saitou for sec-
retarial assistance. We sincerely acknowledge our colleagues
in the Hashimoto laboratories for valuable discussions.
References
[1] P. Davidsson and M. Sj
¨
ogren, The use of proteomics in
biomarker discovery in neurodegenerative diseases, Disease
Markers, vol. 21, no. 2, pp. 81–92, 2005.
[2]X.Li,M.Miyajima,R.Mineki,H.Taka,K.Murayama,and
H. Arai, Analysis of potential diagnostic biomarkers in cere-
brospinal fluid of idiopathic normal pressure hydrocephalus
by proteomics, Acta Neurochirurgica, vol. 148, no. 8, pp. 859–
864, 2006.
[3] S. Futakawa, K. Nara, M. Miyajima et al., A unique N-glycan
on human transferrin in CSF: a possible biomarker for iNPH,
Neurobiology of Aging. In press.
[4] Y. Hashimoto, A unique N-glycan in cerebrospinal fluid: a
possible biomarker for idiopathic normal pressure hydro-
cephalus, in Proceedings of the 2nd Conference on Asian Com-
munications of Glycobiology and Glycotechnology, Taipei, Ta i-
wan, October 2010.
[5] L. M. Shaw, H. Vanderstichele, M. Knapik-Czajka et al., “Cer e-
brospinal fluid biomarker signature in alzheimer’ s disease
neuroimag ing initiative subjects, Annals of Neurology,vol.65,
no. 4, pp. 403–413, 2009.
[6] H. Hampel, Y. Shen, D. M. Walsh et al., “Biological m arkers
of amyloid β-related mechanisms in Alzheimer’s disease,
Experimental Neurology, vol. 223, no. 2, pp. 334–346, 2010.
[7]J.Q.Trojanowski,H.Vandeerstichele,M.Koreckaetal.,
“U pdate on the biomarker core of the Alzheimer’ s disease
neuroimag ing initiative subjects, Alzheimer’s and Dementia,
vol. 6, no. 3, pp. 230–238, 2010.
[8] A. Homann, M. Nimtz, R. Getzla, and H. S. Conradt,
“’Brain-type’ N-glycosylation of asialo-transferrin from hu-
man cerebrospinal fluid, FEBS Letters, vol. 359, no. 2-3, pp.
164–168, 1995.
[9] G.Spik,B.Bayard,B.Fournet,G.Strecker,S.Bouquelet,and
J. Montreuil, “Studies on g lycoconjugates. LXIV. Complete
structure of two carbohydrate units of human serotransferrin,
FEBS Letters, vol. 50, no. 3, pp. 296–299, 1975.
[10] M.Ishikawa,H.Oowaki,A.Matsumoto,T.Suzuki,M.Furuse,
and N. Nishida, “Clinical significance of cerebrospinal fluid
tap test and magnetic resonance imaging/computed tomog-
raphy findings of tight high convexity in patients with pos-
sible idiopathic normal pressure hydrocephalus, Neurologia
Medico-Chirurgica, vol. 50, no. 2, pp. 119–123, 2010.
[11] S. Miyamoto, “Clinical applications of glycomic approaches
forthedetectionofcancerandotherdiseases,Current Opin-
ion in Molecular Therapeutics, vol. 8, no. 6, pp. 507–513, 2006.
[12]H.J.An,S.R.Kronewitter,M.L.A.deLeoz,andC.B.
Lebrilla, “Glycomics and disease markers, Current Opinion in
Chemical Biology, vol. 13, no. 5-6, pp. 601–607, 2009.
Page 5
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