1679

Sensors and Materials, Vol. 29, No. 12 (2017) 1679–1687
MYU Tokyo
S & M 1461

Semicontinuous Measurement of Mite Allergen(Der f 2) Using a Surface Acoustic Wave Immunosensor
under Moderate pH for Long Sensor Lifetime
Koji Toma, Yume Harashima,1 Naoyuki Yoshimura,2 Takahiro Arakawa,
Hiromi Yatsuda,2,3 Kiyoko Kanamori,1 and Kohji Mitsubayashi*
Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering,Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan
1
Faculty of Health Science Technology, Bunkyo Gakuin University,
1-19-1 Mukogaoka, Bunkyo, Tokyo 113-8668, Japan
2
Japan Radio Co. Ltd., 2-1-1 Fukuoka, Fujimino, Saitama 356-8510, Japan
3
OJ-Bio Ltd., NE1 4EP Newcastle upon Tyne, UK(Received April 28, 2017; accepted September 7, 2017)

Keywords: mite allergen, Der f 2, surface acoustic wave, immunosensor, repeated measurement,
regeneration

In this work, we developed a surface-acoustic-wave (SAW)-based immunosensor for themonitoring of house dust mite (HDM) allergen, Dermatophagoides farinae group 2 (Der f 2).
The SAW immunosensor was fabricated by modifying the sensor surface with a self-assembled
monolayer of ORLA85 protein and immobilized capture antibodies (cAb) for Der f 2. Becauseof the pH-insensitive property of ORLA85, the sensor surface could be used for successive
measurement and regeneration of the sensor surface. In experiments, first, the conditions
for cAb immobilization on the sensor surface were optimized, and then the sensor properties
were investigated. These revealed the limit of detection of the SAW immunosensor for Der f
2 to be 6.3 ng/mL and hence, the sensor’s high selectivity to Der f 2. Finally, the optimum pH
for regeneration, at which the regeneration efficiency and sensor output were balanced, wasinvestigated. It showed that the use of pH 4.0 HCl for regeneration allowed reproducible sensor
outputs, and less damage to immobilized cAb; thus, longer sensor lifetime could be expected.
The results allowed us to anticipate that the SAW immunosensor would be usable for sensitive,
selective, and repeated measurement of Der f 2 with a longer lifetime, and it would be a promisingsensor for HDM allergen monitoring.

1.

Introduction

The prevalence of allergic diseases has become a global health issue. In particular, IgEmediated type I allergic disease that is accompanied by asthma, rhinitis, and atopic dermatitis
are caused by environmental factors.(1,2) House dust mites (HDM) are one of the major factorsof type I allergy development,(3–6) and unlike pollens, the risk of experiencing HDM allergens
increases indoors because these mites multiply and are found in household items such as
bedding, mattresses, and sofas.(7) The most relevant HDM allergens are group 1 and group 2*

Corresponding author: e-mail: m.bdi@tmd.ac.jp
http://dx.doi.org/10.18494/SAM.2017.1634
ISSN 0914-4935 © MYU K.K.

1680

Sensors and Materials, Vol. 29, No. 12 (2017)of Dermatophagoides farinae (Der f 1 and Der f 2) and Dermatophagoides pteronyssinus (Derp 1 and Der p 2). Because of their strong allergenicity, most mite allergy patients produce IgE
antibody to both group 1 and group 2 HDM allergens, and a standard of 20 ng/mL for sensitizationand development of asthma was set by the World Health Organization (WHO).(8) In order toreduce the risk of sensitization to these HDM allergens, it is effective to prevent exposure tothem. In particular, airborne HDM allergen that is easily inhaled should be carefully monitored
because its concentration fluctuates over time influenced by many factors including temperature,humidity, and human activities. Previously, we proposed the concept of an airborne HDM allergenmonitoring system for the prevention of allergic diseases. Such a system would be comprised of
two parts—an airborne HDM sampler that draws air containing HDM allergens, and a biosensor
that repeatedly measures the collected HDM allergens.(9) In such a biosensor, immunoassay is aneffective method of measuring HDM allergens. Conventionally, immunoassays such as enzymelinked immunosorbent assay (ELISA) or lateral flow immunochromatographic assay are used for
one-time measurement.(10–14) Other state-of-the-art methods for measuring the allergens havealso been reported. Morris et al. reported a quartz crystal microbalance-based immunosensor
for monitoring airborne cat allergens, which allowed the measurement of cat allergen from dust
without separation.(15) Kim et al. developed a fully automated bioaerosol monitoring system using
carbon nanotube field-effect transistors.(16) It enabled in situ capture and continuous monitoringof airborne fungal particles. Kurita et al. demonstrated the measurement of mite allergen in realhouse dust using a thin layer flow cell and magnetic beads with comparative sensitivity to ELISA
within 30 min.(17) Despite such remarkable progress in the development of allergen biosensors, thedifficulties of repeatedly using the same sensor surface by regenerating it after each measurement
have been drawbacks in the development of an immunosensor for monitoring. Surface regeneration
is usually performed under harsh conditions for proteins, which causes protein degradation andthus, the loss of sensing capability. Recently, we introduced a surface-acoustic-wave (SAW)-based
immunosensor for Der f 1, and it allowed rapid and repeated (semicontinuous) measurement withhigh sensitivity and selectivity.(18) Although ten repetitions of measurement were demonstrated
with high reproducibility, the regeneration solution used was pH 1.0 HCl, and there was potential
damage of the immobilized capture antibody (cAb) on the sensor surface. Considering thatfrequent exchange of a sensor chip in the airborne allergen monitoring system is laborious, thelifetime of the sensor must be prolonged. This could be achieved if surface regeneration could beaccomplished using a relatively moderate pH solution that reduces the risk of damage to proteins.
In addition, it would make the monitoring system more powerful if the system could be used tomonitor not only Der f 1 but also other various airborne allergens concurrently.
Therefore, in this work, we first developed a SAW immunosensor for Der f 2, and characterized
the sensor properties, including the sensitivity and selectivity to Der f 2. Then, the influence of theregeneration solution pH on the degree of surface regeneration (regeneration rate) was investigatedbecause the balance between measurement reproducibility and damage reduction is important inachieving monitoring with a long sensor lifetime. Finally, repeated measurement of Der f 2 wasdemonstrated using a selected pH for the regeneration in order to assess the sensor capability forthe semicontinuous measurement of Der f 2 and the possibility of implementation in the airborne
HDM allergen monitoring system.

Sensors and Materials, Vol. 29, No. 12 (2017)

2.

Materials and Methods

2.1

Reagents and chemicals

1681

Ethanolamine and β-mercaptoethanol (BME) were purchased from Sigma-Aldrich.   Sodium
dodecyl sulfate (SDS), sodium acetate and acetic acid were from Wako. Der f 2 antigens (Mw
14 kDa), and the capture (cAb) and detection (dAb) antibodies for Der f 2, were from Shibayagi.
Coarse extraction solutions of another mite allergen from Der p 1 and ragweed-pollen allergen(Amb a 1) were from ITEA. A cross-linker, PEGylated bis(sulfosuccinimidyl)suberate [BS(PEG)5],
was from Thermo Fisher Scientific. Orla IgGBinder-G kit, including a mixed solution of protein
G-fused Orla protein (ORLA85 protein) and 11-mercaptoundec-11-ylhexaethylene glycol (HS-C11EG6, PEG-thiol), tris(2-carboxyethyl)phosphine (TCEP), and Tris-HCl buffer solution (50 mM, pH7.5), was from Orla Protein Technologies. Phosphate-buffered saline (PBS) with Tween 20 (PBS-T)
was prepared by mixing Tween 20 [0.05% (v/v)] with PBS solution (10 mM phosphate buffer,
140 mM NaCl, 3 mM KCl, pH 7.4). Acetate-buffered saline (ABS) with Tween 20 (ABS-T) was
prepared by a similar protocol to that of PBS-T: acetic acid solution (10 mM) was added to sodium
acetate solution (10 mM) until the desired pH was obtained, and then this ABS solution and Tween
20 [0.05% (v/v)] were mixed.
2.2

Fabrication of SAW immunosensor

The SAW device was composed of an input or output transducer, a SAW propagation path (sensingarea), and a reflector. The device was fabricated by depositing 90-nm-thick gold on a 36Y-X
quartz substrate, followed by a lift-off process. SAW excited at the input transducer with a center
frequency of 250 MHz propagated on the propagation path to the reflector and then reflectedback to the output transducer [Fig. 1(a)]. Here, a shear horizontal (SH)-SAW was adopted because
it propagated along a path with less attenuation into a liquid; thus, accurate measurement waspossible. In the SAW immunosensor, Der f 2 was detected as a mass and/or viscoelasticity changeon the sensing area, which induced velocity and attenuation changes in the SH-SAW.
The SAW immunosensor was fabricated as follows. The sensing area surface was modified bya self-assembled monolayer of ORLA85 and PEG-thiol; the binding of cAb and its immobilization
by cross-linking on the sensing area surface took place [Fig. 1(b)].(18–20) The surface modificationof the sensing area with the SAM was carried out as per the manufacturer’s protocol: the surface
was initially rendered hydrophilic in order to prevent nonspecific hydrophobic adsorption of
ORLA85 by incubating the sensing area with 1% (v/v) BME in ultrapure water for 5 min.(21)Afterwards, in order to form the Au-S bond between ORLA85 and the gold on the SAW device,disulfide bonds of ORLA85 were reduced by mixing the protein solution with TCEP solution(TCEP 7.5 mg/mL in Tris-HCl) and letting stand for 30 min. Then the mixed solution was applied
to the sensing area, and incubated for 10 min to form the SAM. Noncovalently adsorbed ORLA85
proteins and PEG-thiols were removed by washing the surface with 1% (w/v) SDS in ultrapure
water. This SAM formation process was repeated twice to increase the surface density.
As the last process in SAW immunosensor fabrication, cAb was bound and immobilized on thesensing area surface modified with ORLA85 SAM. To bind cAb to ORLA85, 100 mg/mL cAb
in a buffer solution at an optimum pH was applied and left for 10 min. After removing excess

1682

Sensors and Materials, Vol. 29, No. 12 (2017)

(a)

(b)

Fig. 1. (Color online) (a) Schematic diagram of the SAW immunosensor chip. SH-SAWs propagate along
a propagation path and reflect back to the transducer.   (b) Steps for (i), (ii) SAW chip fabrication and (iii), (iv)
repeated measurement of Der f 2. cAb, capture antibody; dAb, detection antibody.

and unbound cAb, a cross-linker, 0.5 mM BS(PEG)5 in PBS-T, was applied and left for 10 min to
cross-link the cAb and protein G through a reaction of N-hydroxysuccinimide ester groups withlysine and N-terminal amino groups. Finally, 100 mM HCl and PBS-T were successively added
to remove non-cross-linked cAb from the surface. This cross-linking process was repeated threetimes to improve the immobilization rate, that is, the ratio of cross-linked cAb to bound cAb. At
the end of the cross-linking process, unreacted active esters were passivated by ethanolamine (1 M,
pH 8.4).
2.3

Characterization of Der f 2 SAW immunosensor

For the evaluation of the sensitivity of the SAW immunosensor, two measurement methods werecompared.   In one method, a phase shift from the baseline after dAb binding, ΔPdAb, was used
as a sensor output; in the other, a differential method was used to shorten the measurement time,
where the slope of the phase change during dAb binding, ΔPdAb/Δt, was used as the output.(19) The
experimental procedures of these two methods were as follows. Der f 2 at a given concentration
in PBS-T was initially applied to the sensing area and left for 10 min. After rinsing with PBS-T,
20 mg/mL dAb in PBS-T was applied and left for another 10 min, followed by rinsing with PBS-T
to determine the sensor output ΔPdAb; in the differential method, the incubation time of dAb was
3 min instead of 10 min.   Then, the surface was rinsed.   The slope ΔPdAb/Δt was obtained bydifferentiating the phase change of dAb binding for 30 s by time.
To assess the selectivity of the sensor, two other environmental allergens, Der p 1 from miteand Amb a 1 from ragweed pollen, in mixed solutions with (three allergens) and without (twoallergens) Der f 2 were measured by the differential method at the sample concentration of 100
ng/mL in PBS-T.
2.4

Semicontinuous measurement of Der f 2 under moderate condition

To extend the sensor lifetime as well as retain the capability of semicontinuous measurement of
Der f 2 in the SAW immunosensor, the optimum pH for regeneration, at which the reproducibility

1683

Sensors and Materials, Vol. 29, No. 12 (2017)of the measurement could be balanced with the damage to cAb on the sensor, was investigated.
HCl with pH 1.0–4.0 was used to regenerate the sensor surface after successive bindings of
100 ng/mL Der f 2 and 20 mg/mL dAb, and the regeneration rate ΔPRe/ΔPdAb3, defined as theratio between the phase shift after 3 min of dAb binding, ΔPdAb3, to the phase recovery throughregeneration, ΔPRe, was determined. These regeneration rates indicated degrees of surface
regeneration at various pH values when using the differential method.
After optimizing the HCl pH for surface regeneration, the sensor capability for thesemicontinuous measurement of Der f 2 was assessed by conducting five repetitions of themeasurement of 100 ng/mL Der f 2 by the differential method, and the sensor outputs of thesemeasurements were compared to assess the reproducibility.

3.

Results and Discussion

3.1

Optimizing conditions for SAW immunosensor fabrication

In order to maximize the cAb density on the sensor surface, the relationship between the cAb
solution pH and the phase shift induced by cAb binding, ΔPcAb, was investigated with ABS-T (pH
4.0–5.0) and PBS-T (pH 6.0–8.0). Figure 2(a) shows the phase change when 100 mg/mL cAb in
PBS-T at pH 6.0 was applied. A phase decrease as a response to cAb binding and phase recovery
upon rinsing nonbound cAb were observed.  The degree of phase shift from cAb binding, ΔPcAb,
was determined after rinsing, and these at different pH were plotted in Fig. 2(b). Throughout theentire pH range, the use of PBS-T resulted in larger phase shifts ΔPcAb than those with ABS-T.
For PBS-T, the shift ΔPcAb was maxium at pH 5.0 and 6.0, and was about 1.9-fold larger than thatin the case of ABS-T at pH 6.0. This was probably attributed to the optimum pH of IgG binding
to protein G being a slightly acidic condition.(22) Considering that the buffer capacity of PBS-T
ranges from pH 5.8 to 8.0, we used pH 6.0 in the subsequent experiments.

(a)

(b)

(c)

Fig. 2. (Color online) (a) Typical time course of phase during cAb binding.   (b) Effects of buffer and pH on
cAb binding to ORLA85 protein.   Note that the error bars in this figure and all the others indicate the standard
deviations. (c) Cross-linking between cAb and protein G with BS(PEG)5. The cross-linking process was repeatedthree times to increase the immobilization rate Δa/Δb. CL, cross-linking; Ri, rinsing; Re, regeneration with HCl;
EA, ethanolamine terminating the reaction.

1684

Sensors and Materials, Vol. 29, No. 12 (2017)

Figure 2(c) shows the time course of the phase change during the binding and cross-linking of
cAb. After the first cross-linking, HCl of pH 1.0 was applied to the sensor surface to remove noncross-linked cAb, and the immobilization rate Δa1/Δb1 was 90.3%. a1 and Δb1 were defined asphase shifts before and after applying HCl; thus the immobilization rate indicated the degree ofsuccessfully cross-linked cAb on the surface among all bound cAb. The immobilization rate was
improved to 99.3% by repeating the cross-linking three times.
3.2

Characterization of Der f 2 SAW immunosensor

Figure 3(a) shows the time course of the phase during Der f 2 measurement conducted by two
different methods. The employment of the differential method resulted in a 10 min shorteningof the total measurement time to 20 min because it took only 3 min for dAb binding comparedwith the 10 min needed in the phase shift method. In addition, although the phase shift method
required rinsing in order to determine the sensor output ΔPdAb before surface regeneration, it was
not needed in the differential method, and dAb was removed only with HCl, which also allowed
further shortening of the measurement time. Figure 3(b) shows the calibration curves of the two
methods for Der f 2. The plots were fitted by the following equations in the range of 1.0–3000
ng/mL with coefficients of correlation of 0.997 and 0.996, respectively:

(a)

(b)

Fig. 3. (Color online) (a) Time courses of phase during Der f 2 measurement by phase shift (upper) anddifferential (lower) methods.  (b) Calibration curves of the phase shift method (●) and differential method (△) for
Der f 2. ΔPdAb, phase shift after dAb binding; ΔPdAb/Δt, slope of phase change during dAb binding; ΔPdAb3, phaseshift after 3 min of dAb binding; ΔPRe, phase recovery via regeneration.

1685

Sensors and Materials, Vol. 29, No. 12 (2017)

ΔPdAb (deg) = A1 + (B1 − A1) / {1 + ([Der f 2] / C1)D1},(1)

ΔPdAb /Δt (deg/s) = A2 + (B2 − A2) / {1 + ([Der f 2] / C2)D2},(2)

where A1 = 0.3, B1 = 4.5, C1 = 181, and D1 = −1.2, and A2 = 1.3 × 10−3, B2 = 1.4 × 10−2, C2 = 316,
and D2 = −1.1 are the coefficients, and [Der f 2] is the concentration of the applied Der f 2 in
ng/mL. The limit of detection (LOD) of each method was 15.1 ng/mL (phase shift, 1.1 nM) and 6.3
ng/mL (differential, 0.4 nM), encompassing a sensitization risk level of 20 ng/mL.(23) This LOD
was determined to be the concentration at which the mean phase shift, or the slope for a blanksample, plus three times the standard deviation σ intersected the fitted curve. This result revealed
that using the differential method improved the sensitivity by a factor of 2.4 along with shortening
the measurement time by 10 min, and suggested that the differential method was more suited to themonitoring system.
The result of sensor selectivity evaluation is shown in Fig. 4. Almost no response was observed
from samples without Der f 2, which validated the high selectivity of the SAW immunosensor to Der f 2.
It suggested that parallel measurement of different HDM allergens, Der f 1 and Der f 2, would bepossible by combining it with the previously developed Der f 1 SAW immunosensor, because theDer f 1 sensor also showed high selectivity to Der f 1.(18)
3.3

Semicontinuous measurement of Der f 2

Prior to demonstrating semicontinuous measurement of Der f 2, the influence of the pH of theregeneration solution on the regeneration rate ΔPRe/ΔPdAb3 was investigated. The results showedthat the regeneration rate was almost 100% for pH 1.0–2.0, and it gradually declined above pH 2.0 (Fig.
5). When using pH 4.0, the regeneration rate dropped to about 48%. In the monitoring system, it

Fig. 4. (Color online) Sensor responses to various
allergens and their mixture for the assessment ofsensor selectivity. Hatched bars are sensor outputs
from the samples containing Der f 2.

Fig. 5. Regeneration rate ΔPRe/ΔPdAb3 for various
pH of regeneration solution.

1686

Sensors and Materials, Vol. 29, No. 12 (2017)

(a)

(b)

Fig. 6. (Color online) (a) Phase change over five repeated measurements of Der f 2 and relative sensor output at
each measurement.  (b) 1st (●), 3rd (■), and 5th (▲) measurements taken from (a).

is convenient for users if a sensor chip need not be exchanged for a long period of time, and it wasconsidered that using a moderate pH solution for surface regeneration would lead to such a longsensor lifetime. Therefore, in the subsequent experiment, pH 4.0 HCl was chosen for the surface
regeneration despite its low regeneration rate.
Figure 6(a) shows the time course of the phase during five repetitions of the measurementof 100 ng/mL Der f 2, and the relative sensor output ΔPdAb/Δt for each measurement. It wasobserved that the baseline gradually declined with repeated measurement because of the imperfectregeneration and accumulation of residual molecules on the sensor surfaces, as indicated in Fig.
5, which shows a regeneration rate of 48% at pH 4.0. However, the coefficient of variation (C.V.)of the sensor outputs in the five measurements was 4.4%, which indicated a high reproducibilityof measurement. Additionally, overlaid phase changes for the 1st, 3rd, and 5th measurements
in Fig. 6(b) were well matched, which also supports the high reproducibility. The reason for thehighly reproducible output despite the decline of the baseline was considered to be that adequate
binding sites still remained for Der f 2; thus, the influence of the occupation of the binding site on
the reproducibility for five repetitions of measurement was negligible. These results indicate thepossibility of semicontinuous measurement of Der f 2 using the SAW immunosensor even with aregeneration solution at a moderate pH.

4.

Conclusions

In this study, we developed a SAW immunosensor for semicontinuous measurement of the
HDM allergen, Der f 2. The employment of the differential method allowed us to not only
shorten the measurement time by 10 min compared with the phase shift method (30 min) but
also improve the sensitivity by a factor of 2.4 (LOD of 6.3 ng/mL). The SAW immunosensor
also showed high selectivity to Der f 2. The demonstration of five repetitions of measurement
revealed that reproducible semicontinuous measurement was feasible even with a regeneration

1687

Sensors and Materials, Vol. 29, No. 12 (2017)

solution at a relatively moderate pH (pH 4.0). These results allowed us to confirm that the Der f 2
SAW immunosensor could be used in airborne HDM allergen monitoring in conjunction with thepreviously reported Der f 1 SAW immunosensor.

Acknowledgments
This work was supported by Japan Radio Co. Ltd., the Japan Society for the Promotion of
Science (JSPS) Grant-in-Aid for Scientific Research System, the Japan Science and Technology
Agency (JST), the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
Special Funds for Education and Research “Advanced Research Program in Neo-Biology”, and the
Research Center for Biomedical Engineering.

References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

R. Beasley, U. Keil, E. von Mutius, and N. Pearce: Lancet 351 (1998) 1225.
J. Douwes, P. Thorne, N. Pearce, and D. Heederik: Ann. Occup. Hyg. 47 (2003) 187.
T. J. Nuttall, J. R. Lamb, and P. B. Hill: Res. Vet. Sci. 71 (2001) 51.
A. P. Jackson, A. P. Foster, B. J. Hart, C. R. Helps, and S. E. Shaw: Vet. Dermatol. 16 (2005) 32.
M. A. Calderon, A. Linneberg, J. Kleine-Tebbe, F. de Blay, D. Hernandez Fernandez de Rojas, J. C. Virchow, and P.
Demoly: J. Allergy Clin. Immunol. 136 (2015) 38.
S. Dreborg: Allergy 53 (1998) 88.
M. Cinteza and C. Daian: Mædica 9 (2014) 313.
I. W. Report: Bull. World Health Organ. 66 (1988) 769.
H. Saito, Y. Suzuki, T. Gessei, K. Miyajima, T. Arakawa, and K. Mitsubayashi: Sens. Mater. 26 (2014) 121.
E. R. Tovey, C. Almqvist, Q. Li, D. Crisafulli, and G. B. Marks: J. Allergy Clin. Immunol. 122 (2008) 114.
N. J. Custis, J. A. Woodfolk, J. W. Vaughan, and T. A. E. Platts-Mills: Clin. Exp. Allergy 33 (2003) 986.
E. R. Tovey, D. Liu-Brennan, F. L. Garden, B. G. Oliver, M. S. Perzanowski, and G. B. Marks: PLoS One 11 (2016)
1.
A. Tsay, L. Williams, E. B. Mitchell, and M. D. Chapman: Clin. Exp. Allergy 32 (2002) 1596.
R. Polzius, T. Wuske, and J. Mahn: Allergy 57 (2002) 143.
D. R. P. Morris, J. Fatisson, A. L. J. Olsson, N. Tufenkji, and A. R. Ferro: Sens. Actuators, B 190 (2014) 851.
J. Kim, J. H. Jin, H. S. Kim, W. Song, S. K. Shin, H. Yi, D. H. Jang, S. Shin, and B. Y. Lee: Environ. Sci.
Technol. 50 (2016) 5163.
R. Kurita, H. Yanagisawa, and O. Niwa: Biosens. Bioelectron. 48 (2013) 43.
K. Toma, D. Miki, C. Kishikawa, N. Yoshimura, K. Miyajima, T. Arakawa, H. Yatsuda, and K. Mitsubayashi:Anal. Chem. 87 (2015) 10470.
K. Toma, M. Horibe, C. Kishikawa, N. Yoshimura, T. Arakawa, H. Yatsuda, H. Shimomura, and K.
Mitsubayashi: Sens. Actuators, B 248 (2017) 924.
K. Toma, D. Miki, N. Yoshimura, T. Arakawa, H. Yatsuda, and K. Mitsubayashi: Sens. Actuators, B 249 (2017)
685.
S. Terrettaz, W.-P. Ulrich, H. Vogel, Q. Hong, L. G. Dover, and J. H. Lakey: Protein Sci. 11 (2002) 1917.
B. Akerstrom and L. Bjorck: J. Biol. Chem. 261 (1986) 10240.
K. Y. Jeong, H. S. Jin, S. H. Oh, C. S. Hong, I. Y. Lee, H. I. Ree, and T. S. Yong: Allergy 57 (2002) 29.