J. Biol. Macromol., 17 (1), 15-21 (2017)

ArticleHigh-sensitivity measurement of immunoglobulin G (IgG)
using HRP-IgG-phospholipid polymer conjugate
Shujiro Sakaki1, *, Tomomichi Nishino1, Megumi Ito2, Kouichi Maruyama1 and Kai Sasaki1
1

Department of Applied Chemistry, Akita National College of Technology, 1-1 Iijima Bunkyo, Akita
011-8511
2
Technology Education Support Center, Akita National College of Technology, 1-1 Iijima Bunkyo,
Akita 011-8511
Received February 10, 2017; Accepted June 5, 2017
Key words: horseradish peroxidase, HRP, 2-methacryloyloxyethyl phosphorylcholine, MPC, ELISA
ABSTRACTThis report describes the high-sensitivity measurements of immunoglobulin (IgG) with the helpof a phospholipid polymer conjugate, 2-methacryloyloxyethyl phosphorylcholine (MPC)
polymer-IgG-horseradish
peroxidase(HRP)
conjugate.
Thephospholipid
polymer,poly(MPC-co-2-aminoethyl methacrylate) (PMAE), was synthesized. To prepare the 1-step
conjugate, IgG and PMAE were chemically modified with HRP at the same time. To prepare the2-step conjugate, PMAE was first chemically modified with HRP, and then IgG was chemicallymodified. The activities of 1-step and 2-step conjugates significantly decreased compared with thatof a conventional IgG conjugated with HRP (conventional conjugate) (p < 0.05). The detection
sensitivity of the 1-step and 2-step conjugates was also compared with that of conventional conjugate.
When 0.5 µg/mL anti(goat IgG)IgG was used as an analyte, compared with a conventional conjugate,
the values of Δ absorbance of the 1-step and 2-step conjugates were 4.8 and 9.8 times respectively.
These results demonstrate that high-sensitivity measurements are possible using HRP-IgG-PMAE
conjugate.

INTRODUCTION
Enzymes used as a label molecule is moreadvantageous than the other kinds of label
molecules
withrespectto*To whom correspondence should be addressed:
sakaki@akita-nct.jp, Tel: +81-18-847-6065

immunohistochemistry
andimmunoassay
purposes. In immunohistochemistry, an
immunoglobulin G (IgG) conjugated with an
enzyme (enzyme-IgG conjugate) permits
localization and observation of cellular antigens
by optical and electron microscopy in relation totissue structures 1–3. Immunologic measurement

16

High-sensitivity measurement using HRP-IgG-polymer conjugate

is useful for the detection or quantification of ananalyte (a very small amount of aphysiologically active molecule). The use of an
enzyme-linked immunosorbent assay (ELISA),
animmobilized
antibody,
andanenzyme-antibody
conjugateforthemeasurement of antigens is known as the
sandwich immunoassay technique 4, 5. However,for immunohistochemistry and immunoassay, ahigh-sensitivity analysis is necessary for the

For the present study, we synthesized awater-soluble MPC polymer with primaryamino groups to bind IgG and horseradish
peroxidase (HRP). We also investigated therelative enzyme activity and the chemical
modifications between the MPC polymer, IgG,
and HRP. Finally, we demonstrated ahigh-sensitivity measurement using MPC
polymer-IgG-HRP conjugate.

early detection and diagnosis of diseases.
Various enzymes can be modified
chemically by synthetic polymers having
phospholipid
polargroups.

Materials and Methods
Synthesis of water-soluble MPC polymer
A random copolymer of MPC and2-aminoethyl
methacrylate(AEMA)

2-methacryloyloxyethyl
phosphorylcholine(MPC) polymers are excellent for theenhancement of enzymatic functions andstability. Miyamoto et al. reported an MPC
polymer with a molecular weight of 5 × 103 thathydrochloride (Polysciences Inc., PA, U.S.A.)
was prepared using the conventional radical
polymerization technique in H2O with
2,2'-azobis[2-(2-imidazolin-2-yl)
propane]dihydrochloride(VA-044)
(Wako
Pure

showed no adverse effect on papain after
conjugation.6 In another study, Miyamoto et al.
compared the stability of papain–poly(MPC)and papain–PEG conjugates and reported thatthe former exhibited higher stability at 40 °C inbuffer.7 Depp et al. applied protein-initiatedatom transfer radical polymerization tosynthesize grafts of high Mw poly(MPC),
poly(N-2-hydroxypropylmethacrylamide

Chemical Industries Ltd., Osaka, Japan) as theinitiator. All other reagents were of an
extra-pure grade. To prepare a polymerization
mixture, 1187.5 µmol MPC, 62.5 µmol AEMA,
and 12.5 µmol VA-044 were dissolved in 20 mL
of H2O. After purging with Ar gas to remove
oxygen in the aqueous solution, the solutions
were put into glass tubing with sealed tubing
and then mixed. The polymerization was carried(HPMA)),
andpoly(monomethoxy-polyethyleneglycol-methac
rylate) with chymotrypsin.8 Oupicky et al.
synthesizedchymotrypsin-poly(HPMA))
conjugates that showed improved proteolytic
stability andreduced immunogenicity.9
Phospholipid polymers composed of MPC units
have
excellentbiocompatibility
andthrombogenicity properties and so areout at 50 °C for 90 h.
Thechemicalstructureofpoly(MPC-co-AEMA) (PMAE) was confirmed
using 1H-NMR (JNM-AL400; JEOL Ltd.,Tokyo, Japan) in D2O. Gel permeation
chromatography (TSKgel G5000PWXL and
TSKgel G4000PWXL; Tosoh Corp., Tokyo,
Japan) was used to determine thenumber-average molecular weight (Mn) andwell-suited as biomaterials.10

weight-average molecular weight (Mw) of

17

Sakaki et al.

Table 1 Synthetic results of poly(MPC-co-AEMA) (PMAE)
Mole fraction of MPC
In feedIn polymer a

0.95

0.99

Yield (%)

Mn b

Mw b

42

> 15× 106

> 15 × 106

a: Unit of mole fraction of MPC in the copolymer was determined by 1H-NMR

in D2O.

b: Determined by GPC with the poly(ethylene oxide) standard, Mn and Mw represent the
number-average and weight-average molecular weight, respectively.

PMAE in H2O with poly (ethylene oxide)standards. To prepare the propionic acidserum (IgG, Sigma-Aldrich Corp. LLC, MO,
U.S.A.) solution (10 mM carbonate buffer, pH

succinimide ester, 0.5 mmol of propionic acid,
0.6 mmol N,N’-carbonyldiimidazole, and 0.6mmol of N-hydroxysuccinimide were dissolvedin 1.3 mL of N,N-dimethylformamide andincubated at 25 °C for 3 h. Then, 20 mg of
PMAE was dissolved in 0.6 mL of the propionicacid succinimide ester solution and incubatedovernight at 4 °C to allow the reaction between
the amino group and propionic acid succinimide

10.0) were added to the solution. Then, thissolution was stirred slowly at 25 °C for 2 h. To

ester. After the chemical structure wasconfirmed, the MPC polymer with the aminogroup was purified by dialysis against H2O.
Figure 1 shows the chemical structure ofpoly(MPC-co-AEMA) (PMAE). Table 1
presents the synthesis results.
Preparation of conjugates

preparation of the 2-step conjugate, except that
only PMAE solution was added to form the
Schiff base. After slow stirring of the solution at
25 °C for 2 h, the IgG solution was added andstirred slowly at 25 °C for another 2 h.
The same method was also followed toprepare the conventional conjugate, except that1000 μL of a 20.0 mg/mL IgG solution wasadded to form the Schiff base.

To prepare the 1-step conjugate, 1000 L of
a 10 mg/mL horseradish peroxidase (HRP,
Wako Pure Chemical Industries Ltd., Osaka,

reduce the Schiff base, 100 L of 10 mg/mL
sodium tetrahydroborate aqueous solution wasadded and then stirred slowly at 4 °C for 2 h in
the dark. For purification, the solution was
dialyzed against 10 mM sodium phosphatebuffer (pH 7.0) at 4 °C for 24 h.
The same method was used for the

Schiff base, 20 L of a 200 mM carbonate
buffer (pH 10.0), 500 L of a 1.0 mg/mL
PMAE solution (10 mM carbonate buffer, pH

The 1-step, 2-step, and conventional
conjugates were isolated from unreacted IgG
and HRP with the help of size exclusion
chromatography using Bio-Gel P-100 Gel(Bio-Rad Laboratories Inc., CA, U.S.A.), (15 ×260 mm, flow rate 6.0 mL/h, 10 mM sodium
phosphate buffer (pH 7.0) as the mobile phase).
A schematic representation of the synthesis of
the 1-step conjugate and 2-step conjugate is10.0) and 500 L of a 4.0 mg/mL IgG from goatshown in Figure 2.

Japan) aqueous solution and 200 L of 250 mM
sodium periodate aqueous solution were mixed
and stirred slowly at 25 °C for 20 min. Then,they were dialyzed against 1.0 mM acetate
buffer (pH 4.5) at 4 °C for 24 h. To form a

High-sensitivity measurement using HRP-IgG-polymer conjugate

18
CH3
n H2C

CH3

C

m H2C

+

C

O

C

O

C

O(CH2)2OPO(CH2)2N+(CH3)3

O

O(CH2)2NH2·HCl

O2-methacryloyloxyethyl
phosphorylcholine (MPC)

2-aminoethyl methacrylatehydrochloride (AEMA)

CH3
H2C

CH3

C
n
C O

H2C

C

Measurement
activitym
C O

OO(CH2)2OPO(CH2)2N+(CH3)3 O(CH2)2NH2·HCl
Opoly (MPC-co-AEMA) (PMAE)

Figure 1 Chemical structure of poly(MPC-co-AEMA)

CH2OH

CH2OH

O
HRP

O

OH HO

OH

NaIO4

HRP

NH2

NH2
&

O

n
PMAE:

OH
CH
O
CH2OH

HC
O

1-step

IgG:

HRP

OH

2-step

NaBH4

NH2

HC
N

CH
N

NH2
n

n
PMAE:

sample was electrophoresed onpolyacrylamide gel (7.5%T) for 80
min at 20 mA. A staining solution(EzStain Aqua; Atto Corp., Tokyo,
Japan) was added to the gel toascertain the location of the 1-step,
2-step or conventional conjugates.

IgG:

1-step conjugate or
2-step conjugateofrelative enzyme

Each solution of the 1-step,
2-step,andtheconventional
conjugates were diluted to 0.1 µg/mL
HRP using 10 mM sodium phosphatebuffer (pH 7.0). To measure therelative enzyme activity, 50 µL ofthese diluted solutions and 100 µL of
ABTS 1-Component Microwell
Peroxidase Substrate (Kirkegaard &Perry Laboratories Inc., SF, U.S.A.)
solution were mixed at 25 °C for 10
min. The absorbance at 405 nm wasmeasured (Model 680 Microplate
Reader; Bio-Rad Laboratories Inc.,
CA, U.S.A.).

Figure 2 Schematic representation of synthesisof 1-step and 2-step conjugates

Measuring sensitivity for immunoassay
Molecular weight evaluation using SDS-PAGE
The molecular weight of the 1-step, 2-step,and conventional conjugates were evaluatedusing SDS-polyacrylamide gel electrophoresis(SDS-PAGE). The samples were prepared bydirect mixing of each solution of the 1-step,
2-step, or conventional conjugates and the
Sample Treatment for Tris solution (Cosmo Bio
Co. Ltd., Tokyo, Japan). Then, they were kept

Figure 3 shows the procedure for measuring
sensitivity. To immobilize rabbit anti (goat IgG)
antibody (Rockland Immunochemicals Inc., PA,
U.S.A.) on the surface of an ELISA plate H(Sumitomo Bakelite Co. Ltd., Tokyo, Japan),
100 µL of 0, 0.5 and 1.0 µg/mL rabbit anti (goatIgG) antibody solution (Dulbecco’s phosphatebuffer saline, D-PBS) were pipetted into a well.
Then, the ELISA plate was sealed with an

at 100 °C for 5 min. The ratio of each solution
to the Tris SDS solution was 1:1 (vol/vol). Eachadhesive strip and incubated at 4 °C for 24 h

19

Sakaki et al.

: rabbit anti(goat IgG)antibody

in H2O. The PMAE chemical structure
was confirmed using 1H-NMR, i.e., the: blocking reagent (BSA)

HRP : 1-step, 2-step or conventional conjugates

ELISA plate

Step I

Step II
Substrate

HRP

Step III

HRP

Step IV

Figure 3 Procedure for measuring sensitivity

Product

peaks assigned to -CH3 (0.95 ppm),
-CH3 (2.0 ppm), -N+(CH3)3 (3.1 ppm),-CH2N- (3.3 ppm), -CH2PO2- (3.6 ppm), –OCH2- (4.3 and 4.7 ppm) were observed.
The mole fraction of the MPC unit in thepolymer was 0.99, which was determinedwith the help of NMR spectroscopy,
specifically taking into account the ratio of(step I). After incubation, the rabbit anti (goatIgG) antibody solution was aspirated from thewell. To remove the unimmobilized antibody,the well was washed three times with 200 µL of
D-PBS. After washing, 200 µL of 1% bovine
serum albumin (BSA, Sigma-Aldrich Corp.
LLC, MO, U.S.A.) and D-PBS were pipetted
into the well. Then, the ELISA plate was sealedwith an adhesive strip and incubated at 4 °C for

integration of -N+ (CH3)3 (3.1 ppm) peakand -CH2N- (3.3 ppm) peak.

24 h (step II). After incubation, the BSA
solution was aspirated from the well. Eachsolution of the 1-step, 2-step, and conventional
conjugates was diluted to 20.0 µg/mL IgG using
1% BSA solution, and D-PBS was pipetted into
the well. Then, the ELISA plate was sealed withan adhesive strip and incubated at 4 °C for 24 h
(step III). After incubation, each solution was
aspirated from the well. Then, the wells were

solution. The authors could not use theprecipitate modified conventional conjugate in
the following procedures in this experiment.
The fractions containing the 1-step, 2-step,and conventional conjugates were collected
before the fraction containing unreacted IgG
and HRP. In order to measure the HRP and the
IgG concentration of each conjugate,
absorbance at 280 nm and 403 nm waswashed three times with 200 µL of D-PBS.
Later, 100 µL of ABTS 1-component microwell
peroxidase substrate solution was pipetted into
the well of the ELISA plate and incubated at
25 °C for 10 min (step IV). Absorbance at 405
nm was measured using the microplate reader.

measured using a UV–VIS spectrophotometer(UV-VIS Spectrophotometer V-560, JASCO
Corp.) as absorption coefficients (mg-1 cm2);
HRP (280 nm) = 1.209, HRP (403 nm) = 2.275,
IgG (280 nm) = 1.285, IgG (403 nm) = 0.000.
Concentration of HRP and IgG was 0.29 and0.14 mg/mL, 0.29 and 0.12 mg/mL, and 0.27
and 0.93 mg/mL for 1-step conjugate, 2-step
conjugateandconventional
conjugate,

Results and Discussion
Synthesis of water-soluble MPC polymer
Copolymerization proceeded homogeneously

Preparation of conjugates
The 1-step, 2-step, and conventional
conjugates did not precipitate. Furthermore, toprepare the modified conventional conjugate,
1000 µL of a 2.0 mg/mL IgG solution was usedinstead of 1000µL of a 20.0 mg/mL IgG

respectively.

20

High-sensitivity measurement using HRP-IgG-polymer conjugate

Molecular weight evaluation using SDS-PAGE
Figure 4 shows an SDS-PAGE image. The
molecular weights of 1-step, 2-step, andconventional conjugates were over 400,000
because the stacking gel was 5%T. The
SDS-PAGE result indicates that the 1-step,
2-step, and conventional conjugates wereisolated from unreacted IgG and HRP. In case ofmolecular weight measurement by gel
permeation chromatography, the peak of the
1-step and 2-step conjugates was out of thecalibration curve because the size of theseconjugate was too large to permeate the pores(Date not shown).
Measurement of relative enzyme activity
As the absorbance of native HRP was 100%,
the relative enzyme activities (%) of 1-step,

conjugate and conventional conjugate.

2-step, and conventional conjugates were
determined. To determine if the two sets of data
were significantly different from each other, thestudent t-test was used, and the p-value wasobtained from relative enzyme activity. Figure 5
presents the relative enzyme activity results. In
comparison with native HRP, the relative
enzyme activities of the 1-step and 2-step andconventional conjugates were in the range ofAs the absorbance of 0 µg/mL rabbit anti
(goat IgG) antibody solution was blank, Δabsorbance of the 1-step, 2-step, andconventional conjugates were determined.
Figure 6 presents results of the sensitivity of the
1-step, 2-step, and conventional conjugates. The
Δ absorbance values of the 1-step conjugate and2-step conjugate, using 1.0 µg/mL anti (goatIgG) antibody, were 2.8 and 6.5 times higher

60-77% because of chemical modification using
sodium periodate and sodium tetrahydroborate.
No significant difference was recorded (p >0.05) between 1-step conjugate and 2-step
conjugate, but a significant difference wasobserved (p < 0.05) between 1-step or 2-stepthan that of the conventional conjugate. The Δ
absorbance values of the 1-step and 2-step
conjugate, using 0.5 µg/mL anti (goat IgG)
antibody, were 4.8 and 9.8 times higher thanthat of the conventional conjugate.

Measuring sensitivity for immunoassay

21

Sakaki et al.

0.35

Average ± SD (n=8)

*

80

*
**

60

40

20

0

0.25

0.20
0.15
0.10
0.05
0.00

conventional
conjugate

1 stepconjugate

2 stepconjugate

Figure 5
Relative enzyme activity of
conventional, 1-step and 2-step conjugates.
Absorbance of Native HRP is 100%.
*: p < 0.05, **: p > 0.05 (Student t-test)

The periodic acid-Schiff reaction was used forthesynthesisof
HRP-IgG-poly(MPC-co-AEMA) (PMAE) conjugates. The
enzyme-IgG-PMAE conjugate is superior toconventional enzyme-IgG conjugate withrespect to high-sensitivity measurements.

0.5 1.0
0.5 1.0
conventional
1-stepconjugate conjugate

0.5 1.0
2-stepconjugate

Rabbit anti(goat IgG) antibody (μg/mL)

Conjugate nameCONCLUSIONSAn amphiphilic phospholipid polymer with
an amino group was synthesized using
conventional radical polymerization technique.

Average ± SD (n=4)

0.30

D absorbance at 405 nm

Relative enzyme activity (%)

100

Figure 6
Measuringsensitivityofconventional, 1-step and 2-step conjugates.
The absorbance of 0 µg/mL rabbit anti(goatIgG) antibody solution is blank.

3.
4.

Orci, L. Diabetologia 28, 528-546 (1985).
Anderton, B. H. & Thorpe, R. C. Immunol.

5.

Today 2, 122-127 (1980).
Wisdom, G. B. Clin. Chem. 22, 1243-1255(1976).

6.

Miyamoto, D., Watanabe, J. & Ishihara, K.

7.

J. Appl. Polym. Sci. 91, 827-832 (2004).
Miyamoto, D., Watanabe, J. & Ishihara, K.

8.

Biomaterials 25, 71-76 (2004).
Depp, V., Alikhani, A., Grammer, V. & Lele,

9.

S. B. Acta Biomaterialia 5, 560-569 (2009).
Oupicky, D., Ulbrich, K. & Rihova, B. J.

ACKNOWLEDGMENTS
The authors are grateful to NOF Corp. for

Bioactive Compat. Polym. 14, 213-231(1999).

their assistance with regard to the provision ofmaterials. We also thank Dr. Katsuhiko
Takenaka of the Nagaoka University ofTechnology for his aid in relation to 1H-NMR
analysis.

10. Ishihara, K. Trend. Polym. Sci. 5, 401(1997).

REFERENCES
1. Osborn, M., Webster, R. E. & Weber, K., J.
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Cell Biol. 77, R27-R34 (1978).
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Communicated by Mihara Hisaaki