Screening of antigenically reactive fragments of aSi-casein (asx-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with aSi-CN and complete Freund&apos;s adjuvant, and 14 days later, all the mice were boosted with as^CN and incomplete Freund&apos;s adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti as^CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. aSi-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti aSj-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199)  were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of aSj-CN by the direct and simple detection of specific antigen-antibody interaction

Akio Ametani

Kunio Yamauchi

Makoto Sfflmizu

Shuichi Kaminogawa

CiteSeerX

/ . Biochem. 102, 42M25 (1987)
Rapid Screening of Antigenically Reactive Fragments of
asi-Casein Using HPLC and ELISA
Akio AMETANI, Shuichi KAMINOGAWA, Makoto SfflMIZU,
and Kunio YAMAUCHI
Department of Agricultural Chemistry, The University of Tokyo,
Bunkyo-ku, Tokyo 113
Received for publication, March 6, 1987
Screening of antigenically reactive fragments of aSi-casein (asx-CN), the major
casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent
assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with
aSi-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted
with as^CN and incomplete Freund's adjuvant. Twenty-one days after the 1st
immunization, the mice were bled and antiserum was separated. Anti as^CN
antibody fraction was obtained by precipitation from the antiserum with 50%
saturated ammonium sulfate. aSi-CN was digested with trypsin and chymotrypsin,
and 35 peptides were purified from the digests by reversed-phase HPLC with ODS
(octadecylsilica) columns. Reactivity of peptides with the antibody were examined
by ELISA. The solid phase in the wells of the polystyrene microtiter plate was
coated with peptides, and the plate was successively incubated with anti aSj-CN
antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP)
and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151)
and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in
an ELISA test. These five fragments would correspond to four antigenic sites.
We could thus find antigenically reactive fragments of aSj-CN by the direct and
simple detection of specific antigen-antibody interaction.
The antiserum against a protein antigen contains case of myoglobin (I). So far, the antigenic resi-
a group of antibodies which can specifically bind dues in only a small number of proteins have been
the protein antigen at some definite positions, the determined (2). The investigation of antigenic
antigenic determinants. It takes many years to structures is expected to be useful in connection
complete studies on the antigenic structures of a with studies of protein-protein interaction, protein
protein, e.g., 11 years have been required in the folding, and topographical structure (3-6), as well
as vaccine synthesis, detecting gene products and
Abbreviations: ELISA, enzyme-linked immunosorbent isolating proteins (7). Although there have been
assay; oSj-CN, asj-casein; PBS, phosphate-buffered recent advances in protein chemistry, it remains
saline; ODS, octadecylsilica; ALP, alkaline phospha- difficult to determine antigenic residues with any
tase; Ig, immunoglobulin. precision. Therefore, methods to determine the
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422 A. AMETANL. S. KAMINOGAWA, M. SHIM1ZU, and K. YAMAUCHI
antigenic structures of many proteins simply and
rapidly should be developed. We attempted to
identify antigenically reactive peptides of bovine
asj-casein (aSx-CN), since caseins in bovine milk
are known to have high allergenicity (8). aSx-CN,
which is the major casein in bovine milk and is
absent in human milk, may be one of the main
allergens. Although studies on the antigenic struc-
tures of aSi-CN have already been started (9-11),
the antigenic determinants have still not been
revealed in detail. We describe here the identi-
fication of four antigenically reactive regions as a
result of the separation of proteolytic peptides by
reversed-phase high-performance liquid chromatog-
raphy (reversed-phase HPLC) and enzyme-linked
immunosorbent assay (ELJSA). A smaller amount
of protein antigen, and less time and effort are
necessary with our method than with other methods
such as an assay using immunoprecipitation or
synthesized peptides. Our method can detect
specific antigen-antibody interaction directly and
simply.
MATERIALS AND METHODS
Preparation of Peptides Derived from asv
Casein by Proteinases—Bovine aSx-casein-B was
prepared according to the method of Zittle et al.
(12) and purified by column chromatography on
DEAE-Cellulofine AM (Seikagaku Kogyo Co.,
Ltd.). A solution of 10 mg of as rCN in 0.1 M
NH4OH/HCOOH buffer (pH 8.5) at a concen-
tration of 0.2% was treated with 0.1 mg of L-(1-
tosylamide-2-phenyl)ethyl chloromethyl ketone-
treated trypsin (Cooper Biomedical). Further, 10
mg of os^CN dissolved in 0.1 M NH4OH/HCOOH
buffer (pH 8.0) at a concentration of 0.2% was
treated with 0.1 mg of 7V-/>-tosyl-L-lysine chloro-
methyl ketone-treated chymotrypsin (Sigma Chem-
ical Co.). Both solutions were incubated for 24 h
at 37°C.
Each digest was subjected to HPLC using
LiChrosorb RP-18 (Merck), Vydac C18 (the Sepa-
rations Group), and Finepak SIL C18 (Japan
Spectroscopic Co., Ltd.). The amino acid compo-
sition of each peptide purified by HPLC was ana-
lyzed after acid hydrolysis.
Preparation of Mouse Anti-as,-CN Antibody—
Six-week-old female mice (BALB/c, Charles River
Japan) were injected intraperitoneally with 100
Hg of aSj-CN as a 100 (i\ of emulsion consisting
of equal volumes of protein solution dissolved
in 0.11 M phosphate buffer (pH 7.1) containing
0.04 M NaCl (PBS) and complete Freund's adju-
vant (Difco Laboratories). Fourteen days later,
all the mice were boosted with 100 /<g of asrCN
as an emulsion in incomplete Freund's adjuvant
(Difco Laboratories). Twenty-one days after the
first immunization, all the mice were bled from
the tail and the antisera were separated and pooled.
Portions of 1 ml of the antiserum were each mixed
with 1 ml of saturated ammonium sulfate solution.
The precipitates were dissolved in 1 ml of PBS
and the solution was dialyzed against PBS.
ELISA—The peptides were dissolved in 500
/*1 of PBS. One hundred microliters of the antigen
solution was added to each well of a Petra plastic
microtiter plate (Sanko Junyaku Co., Ltd.), and
the plate was incubated for 2 h at room temper-
ature. After removal of the antigen solution, the
wells were washed three times with 125 /<1 of PBS
containing 0.05% Tween-20 (PBS-Tween). Then
100̂ *1 of the antibody diluted 100 times with
PBS-Tween was added, and the plate incubated
for 2 h. Following removal of the solution and
triple washing, 100 fi\ of bovine intestinal mucosa
alkaline phosphatase (Sigma)-labeled goat anti-
mouse Ig (Cappel) diluted with PBS-Tween was
added, and the plate incubated for another 2 h.
After removal of the solution and washing, 100
ft] of a solution of p-nitrophenylphosphate, diso-
dium salt in 1 M diethanolamine-HCl buffer (pH
9.8) containing 0.01% MgCl,-6H2O, and 0.02%
NaN3 was added. Thirty minutes later, the reac-
tion was stopped by adding 20 //I of 6 M NaOH
and the absorbance of each well was determined
at 405 nm.
RESULTS
Purification of Fragments of as^CN—as^CN
was exhaustively digested by trypsin or chymo-
trypsin, and the digests were fractionated by HPLC
using an ODS column of LiChrosorb RP-18 (Figs.
1 and 2). Each fraction was chromatographed on
the Vydac C18 column and subjected to amino
acid analysis. The amino acid compositions of
the peptides in each fraction were compared with
those of fragments which were expected to be
derived from the aSx-CN sequence by considering
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ANTIGENICALLY REACTIVE FRAGMENTS OF aSx-CASEIN 423
0.064AU
20
T-7
16
17
feikl
25
20 40
Time(min)
Fig. 1. The first step of purification of tryptic peptides
by HPLC. One milligram of peptides derived from
aSi-CN was applied per run. Mobile phase, 0.1%
trifluoroacetic acid/95% CH.CN; column, LiChrosorb
RP-18 (4.6mm i.d.x25cm); detection, ultraviolet ab-
sorbance at 230 nm. After rechromatography with
Vydac C18, peptides were identified by amino acid
analysis (see Fig. 3).
22
0.064 AU
111
20 40 60
Time(min)
Fig. 2. The first step of purification of chymotryptic
peptides by HPLC. Two milligrams of peptides derived
from aSx-CN was applied per run. Mobile phase,
0.1% trifluoroacetic acid/95% CH,CN; column, LiChro-
sorb RP-18; detection, ultraviolet absorbance at 230
nm. Peptides C-23 and C-24 (rechromatographed with
Vydac C18 and Finepak C18) and other peptides (re-
chromatographed with Vydac C18) were identified by
amino acid analysis (see Fig. 3).
the substrate specificities of the proteinases. The
amino acid analyses showed that the fractions cor-
responding to the peaks of C-23 and C-24 were
not pure, and consequently rechromatography for
further purification was run on Finepak SIL CIS.
Thirty-five peptides were purified. The amount
10 20 30 10
RPKHPIKH9GLPQEVLNEJILLRFFVAPFPQVFGKEKVNEL
T-8 T-22 T-13
C-5 C-26r C-28 C-23
C-15
50 60 70 80
SKDIG£ESTEDQAHED1KQHEAESISSSEEIVPNSVEQKH
T - 1 6 T - 1 5
JC-20
90 100 110 120
IQKEDvPSERYLGYLEQURLKKYKVPGI_EIVPN£AEERL
T -7 T-10 T-24 T-20
C-12 T-17 '
3
C-10 C-30 C-24
130 110 150 160
HSMKEGIHAQGKEPMIGVNQELAYFYPELFRQFYQLDAYP
T-12 T-9 T-25 T-23
C-8 C-6 C-14 C-29 C-13 C-22
ZC
C-18
170 180 190
SGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEKTTMPLW
T-21
C-17 C-31
C-16
Fig. 3. Primary structure of as^CN and location of
peptides tested for antigenic reactivities. White and
black squares show peptides negative and positive for
reaction with anti as^CN antibody by ELISA, respec-
tively. ' |S" indicates a phosphoserine residue.
of each peptide obtained from 10 mg of as^CN
ranged from 10 to 200 ng. The identities of the
peptides are shown in Fig. 3.
ELISA— The solid phase in the wells of the
microtiter plate was coated with peptides dissolved
at a concentration of 10 to 100 //g/ml in PBS. An
ovalbumin solution in PBS was added to the wells
for blocking after washing the peptide-coated wells,
and the plate was successively incubated with the
antibody, conjugate of anti-Ig with alkaline phos-
phatase (ALP) and substrate of ALP. In this
experiment, absorbance data which were expected
to show the same value varied widely from well
to well and the reproducibility was poor. If the
antibody solution diluted with PBS was added to
uncoated wells, color was developed in the wells
at the last stage of ELISA. The control experi-
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424 A. AMETANT, S. KAMINOGAWA, M. SHIMIZU, and K. YAMAUCHI
ment,- in which the antibody solution was diluted
with PBS-Tween and added to the uncoated wells,
resulted in no coloration. This indicates that in
the case of no antigen coating and no blocking,
the antibody in PBS was adsorbed on the solid
phase, but that the antibody in PBS-Tween was
not adsorbed. Reliable data were obtained by a
procedure which involved incubation with an anti-
body solution diluted 100 times with PBS-Tween
without blocking after the peptide coating. Thirty-
five peptides derived from as^CN were examined
for reactivity with the antibody in mouse sera.
No coloration was observed in wells coated with
any peptide and oSj-CN in an ELISA test using
sera before immunization. Two tryptic fragments
(T-20 (residues 104-119) and T-25 (133-151)), and
three chymotryptic fragments (C-23 (33-54), C-24
(105-121), and C-31 (174-199)) were positive in an
ELISA test (Fig. 3) using anti-aSj-CN antibody
solution. The absorbance of the well coated with
aSi-CN at 405 nm was outside the scale range,
and those of these five peptides ranged from 0.1
to 0.5. The ELISA in which aSi-CN was added
to the antibody solution showed that bindings of
anti-aSi-CN antibody with as^CN and these five
peptides coated to the wells were inhibited by
aSi-CN dissolved in the solution.
DISCUSSION
Proteolytic fragments were purified by HPLC. The
amino acid analysis showed that purification on
two or three columns could exclude peptide con-
taminants. One tryptic fragment of "Phe-"Lys
and some chymotryptic fragments were missing in
the elution system of trifiuoroacetic acid/CH,CN
and LiChrosorb RP-18. To avoid loss of these
peptides, another elution system and/or column
could be adopted.
The proteolytic fragments purified by HPLC
were coated on the solid phase in the wells of
microtiter plates. Blocking by a method such as
incubating with ovalbumin solution is generally
done after antigen coating, but blocking was un-
necessary to obtain reliable data in our ELISA
procedure, for peptide antigens containing about
ten to twenty residues. When the plate was incu-
bated with the ovalbumin solution, the ovalbumin
was presumed to have been adsorbed on peptide-
free regions of the solid phase and to have influ-
enced the specific interaction between the antigen
and the antibody. The antibody was diluted with
PBS containing the nonionic detergent, Tween-20,
which is known to inhibit nonspecific interaction
between the solid phase and immunoglobulin (13).
Our results by ELISA revealed some of the
antigenic structures of as^CN. The antibody
against native as^CN reacted specifically with five
peptides derived from native as rCN by protease
treatment and adsorbed on the solid phase. Those
peptides that were negative in the ELISA test
either were not adsorbed on the solid phase or
did not contain antigenic determinants. The se-
quences of some peptides which were negative in
the ELISA test overlap with those of the peptides
which were positive (Fig. 3). T-8, T-21, and C-15
are regarded as not being adsorbed on the solid
phase, whereas T-23 is regarded as not containing
a whole antigenic determinant. The C-terminal
residues of T-23 seem to be significant for the
antigenic determinant. If T-20 and C-24 are con-
sidered to contain the same antigenic sites, the
five fragments would correspond to four antigenic
sites, although there is a possibility that other
antigenic sites exist. Louvard et al. found a linear
relationship between the logarithm of molecular
weight of globular proteins and the logarithm of
the number of antigenic determinants (14). From
their relationship and the molecular weight, the
number of determinants of aSj-CN was estimated
to be four. In the present experiment, 20 mg of
aSj-CN was hydrolyzed for ELISA, but the experi-
ment could be performed with a smaller amount
of protein antigen. This procedure using HPLC
and ELISA should be applicable to identify the
antigenic regions in other proteins.
Otani et al. have studied the localization of
the antigenic determinants of as rCN (9-11). They
found one antigenic site in each region of residues
1 to 54, 124 to 135, and 136 to 196, and two sites
in the region of 61 to 123, using six fragments
obtained by cyanogen bromide treatment and sepa-
rated by open column chromatography. The posi-
tions of these sites were thus defined rather broadly,
and a more detailed study is required to clarify
whether their results agree precisely with ours.
They used outbred rabbit antiserum and mainly
employed a quantitative immunoprecipitation in-
hibition test. We used inbred mouse antiserum
and detected binding of the antibody to peptides
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ANTIGENICALLY REACTIVE FRAGMENTS OF oSj-CASEIN 425
by using ELISA with direct coating of the pep-
tides. At present, the reason for the differences
between the results is not clear. There have also
been differences between the results of studies on
myoglobin antigenic determinants by Atassi and
Twining (1, 15) and by Rodda et al. (16). Atassi
and Twining used inhibition of immunoprecipita-
tion and absorption of the antibody, whereas Rodda
et al. used the ELISA procedure, in which the pep-
tides were still bound to the solid phase used for
peptide synthesis. Rodda et al. suggested that the
method of Atassi may detect low-affinity interactions
which occur under conditions of peptide excess.
The molecular nature of the antigenic struc-
ture of proteins, especially globular proteins having
a compact structure, has been widely discussed
(17-20), but without general agreement. Two
kinds of predictions of antigenic determinants from
amino acid sequences of globular proteins have
been applied to as^CN: one was by Welling et al.
(2) and the other was by Hopp and Woods (21).
The former prediction was that T-20 (C-24) con-
tained short regions with relatively high values,
but that T-25 and C-31 did not contain such
regions. The latter prediction was that C-23, T-20
(C-24), and C-31 contained regions with high
antigenicity values, but that T-25 did not contain
such regions. The antigenic regions thus pre-
dicted do not coincide with all the positions of
the antigenic determinants that we have deduced
from the ELISA results. It is known that aSi-CN
has a rather unstable structure, which may ap-
proach a random coil behavior (22). Elucidation
of the antigenic structures of as^CN with precision
may give a new insight into the nature of protein
antigenicity.
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Rapid Screening of Antigenically Reactive Fragments of asi-Casein Using HPLC and ELISA

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Rapid Screening of Antigenically Reactive Fragments of asi-Casein Using HPLC and ELISA

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