Note: Descriptions are shown in the official language in which they were submitted.
J
21~18
SPECIFICATION
METHOD FOR ASSAYING SPECIFIC ANTIBODY
Technical Field
The present invention relates to a method for high
sensitivity assay of an antibody specific to a certain antigen.
Background Art
There have been reported various methods for quantitative
assay of antibody, which are based on the principles of
immunoassay. Typical techniques include, for example,
agglutination, competitive assay and sandwich method. From the
aspect of sensitivity, it is well known that assays based on the
principles of the sandwich method can achieve their highest.
The sandwich method for specific antibody includes, for
example, a method comprising bringing a carrier bound with an
antigen into contact with a liquid sample containing the
antibody to be assayed, reacting the carrier with a labeled
anti-Ig antibody (secondary antibody~ and assaying the antibody
level from the amount of the label bound to the carrier. This
method is defective in that the non-specific antibodies
contained in the liquid sample in a large amount are also
adsorbed to the carrier to increase the background, thus making
higher detection sensitivity difficult to achieve.
Another method includes binding an anti-Ig antibody to a
carrier, bringing the carrier into contact with a liquid sample
and allowing a (labeled) antigen specific to the antibody to be
assayed, to react with the antibody. In this method, the defect
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-
lies in the fact that binding the object antibody to the
carrier is prevented by non-specific antibodies contained in
greater amounts in the liquid sample and signals cannot be
obtained in sufficient amounts. Consequently, high sensitive
assay is difficult like the above-mentioned method.
Another method includes bringing a carrier bound with an
antigen into contact with a liquid sample to allow reaction of a
labeled antigen. This method is defective in that signals
cannot be obtained in sufficient amounts in a short time due to
the low reactivity of the labeled antigen to the antibody bound
to the carrier. Again, high sensitivity assay by this method is
associated with difficulty.
As a method for highly sensitive and easy assay of an
antibody, a method has been recently reported which includes
reacting a labeled antigen and an antigen bound to hapten with
the object antibody in a liquid sample in a liquid phase,
trapping the resultant complex on a carrier using an anti-
hapten antibody, and assaying the complex [Ishikawa et al.,
Analytical Letters, vol. 21, p. 2033 (1988), DE 3705686 to
Thomas Geiger et al.]. This method is expected to achieve high
sensitive assay. However, the method is defective in that the
assay components are complicated and it rather gives lower
assay values (prozoning phenomenon) when the antibody to be
assayed is present in considerably great amounts in the liquid
sample.
Disclosure of the Invention
~. .. . .
21~31~
An object of the present invention is to develop a method
for assaying a specific antibody, which can be performed as
easily as the conventional sandwich method, has high
sensitivity and is free of the prozoning phenomenon.
The present inventors have found that the object specific
antibody can be assayed with high sensitivity and the prozoning
phenomenon can be eliminated by, in a reaction system wherein a
carrier-bound antigen, a labeled antigen, and a liquid sample
containing the specific antibody to be assayed are
simultaneously mixed to react the above-mentioned two antigens
with the above-mentioned specific antibody, forming a complex
of the carrier-bound antigen-specific antibody-labeled antigen,
separating said carrier after the reaction from the
aforementioned reaction system, and again mixing the
aforementioned separated carrier and the aforementioned labeled
antigen, which resulted in the completion of the present
invention.
That is, the present invention provides a method for
assaying a specific antibody, comprising the following steps
(A), (B) and (C):
(A) : forming a complex of a labeled antigen-a specific
antibody-a carrier-bound antigen in a first reaction system
comprising a mixture of the antigen (labeled antigen) against
the specific antibody to be assayed, the antigen being bound to
the label in advance, the antigen (carrier-bound antigen)
against the above-mentioned specific antibody, the antigen being
215531~
bound to the carrier in advance, and a liquid sample comprising
the above-mentioned specific antibody,
(B) : mixing the above-mentioned separated carrier and a labeled
antigen same as above after separating the above-mentioned
carrier after reaction from the above-mentioned first reaction
system to give a second reaction system, and
(C) : assaying, after separating the above-mentioned carrier
after reaction from the above-mentioned second reaction system,
the amount of the label bound to the above-mentioned separated
carrier.
Brief Description of the Drawings
- Fig. 1 shows calibration curves of anti-thyroglobulin
antibodies in Examples 1 and 2 and Comparative Examples 1 to 3,
wherein the abscissa shows the concentration of the anti-
thyroglobulin antibody in the sample and the ordinate shows the
amount of the signal derived from the activity of the peroxidase
bound to respective solid phases.
Fig. 2 shows calibration curves of anti-thyroglobulin
antibodies in Examples 3 and 4 and Comparative Example 4,
wherein the abscissa shows the concentration of the anti-
thyroglobulin antibody in the sample and the ordinate shows the
amount of the signal derived from the activity of the ~-D-
galactosidase bound to respective solid phases.
Detailed Description of the Invention
The present invention is explained by the order of the
steps.
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(_
(A) is a step for improving sensitivity by an easy and
simple method. In (A), the amount of the labeled antigen to be-
added to the first reaction system is desirably set for such an
amount that makes the rate of the reaction between the labeled
antigen and the specific antibody in a liquid phase in the
liquid sample, and the rate of the reaction between the carrier-
bound antigen and the specific antibody almost the same. In the
case of conventional immunoassay, the molecular molar ratio of
the carrier-bound antigen to the labeled antigen is 1:1 - 50:1,
preferably 5:1 - 20:1.
In the present invention, by an antigen is meant a specific
antigen, an anti-idiotype antibody or a component such as
hapten bound to a suitable carrier, which is capable of
developing an antigen-antibody reaction with the specific
antibody to be assayed.
As the liquid sample, exemplified are body fluids such as
serum, plasma, cerebrospinal fluid, saliva and urine, and
buffers.
The specific antibody to be assayed in the present
invention is every antibody which can be substantially assayed
by conventional immunoassay if an antigen which is specifically
recognized by the antibody is used, and according to the
present invention, assay of such specific antibody is
attainable. For example, assays of autoantibodies such as assay
of anti-DNA antibody using a nucleic acid, assay of rheumatoid
21S~18
factor using an immunoaggregate, assay of anti-mitochondria
antibody using pyruvate dehydrogenase, assay of anti-microsomal
antibody using thyroid peroxidase, assay of anti-thyroglobulin
antibody using thyroglobulin, assay of anti-TSH receptor
antibody using TSH receptor, assay of anti-insulin antibody
using insulin and assay of anti-acetylcholine receptor antibody
using acetylcholine receptor; assay of antiviral antibodies such
as anti-HBV antibody and anti-HIV antibody, using virus
antigen; assay of antibodies against microorganisms; assay of
antibodies against protein preparations such as interferon and
human growth hormone; and assay of antibodies against allergens
in allergic diseases are included.
The label used for the labeled antigen which is labeled in
advance may be any substance usable for the assay in
immunological assay, and is exemplified by enzyme, radioactive
substance, luminescent substance, fluorescent substance and
metal compound. For example, enzyme is exemplified by
peroxidase, ~-D-galactosidase and alkaline phosphatase;
radioactive substance is exemplified by iodine and hydrogen;
fluorescent substance is exemplified by fluorescein
isothiocyanate; luminescent substance is exemplified by
acridinium salt; and metal compound is exemplified by europium
(Eu3+)
The aforementioned labels can be bound to antigens by any
binding method reported to be usable for binding a label to an
antibody or antigen in conventional immunological assay. When
.. ~ , .. . .
- 21~5318
the label is an enzyme, covalent linking via a suitable
multifunctional crosslinking agent is preferable. The
crosslinking reaction is advantageously carried out by utilizing
the reaction of, for example, amino group and aldehyde group
(Schiff reaction), amino group and active ester, thiol group and
maleimide group, and thiol group and pyridyl- disulfide group,
since antigen and enzyme do not lose their activity. These
reactions are all carried out by incubation in a buffer having
a pH of ~-9 at 4-37C for several minutes to several days.
When the label is a radioactive substance, l2sI can be
introduced into, for example, an antigen having a tyrosine
residue in molecule, by reacting the antigen with ['25I] sodium
iodide in the presence of an oxidizing agent such as chloramine
T at 0-10C for several to several dozen minutes.
These labels may be bound to antigens via a suitable spacer
which does not affect the antigen-antibody reactions in (A),
(B) and (C). When the antigen has a low molecular weight, the
use of such spacer is particularly preferable. Examples of the
spacer include non-specific rabbit Ig~, bovine serum albumin,
dextran and hydrocarbon (carbon number 4-10) having a suitable
length.
There is no particular limitation imposed on the carrier as
long as it does not impair the object of the invention, and
those used in conventional immunological assay suffice for use.
For example, polystyrene, polyacryl, Teflon, paper, glass and
agarose can be used. The shape thereof is not particularly
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limited.
An antigen can be bonded to a carrier by a known method for
forming a carrier in immunological assay. In general terms,
physical adsorption utili~ing a hydrophobic interaction between
carrier and antigen is applied when an antigen is a protein
having a relatively large molecular weight. Generally, a
carrier is immersed in a buffer (in the range of from pH 7 to pH
9.5) containing an antigen at a concentration of several dozen -
several hundred mg/O, at 4-37C for several hours to several
days. When the antigen has a low degree of hydrophobic
interaction with a carrier, a functional group such as amino and
carboxyl is introduced in advance into the carrier and the
antigen is covalently linked via a crosslinking agent such as
glutaraldehyde, or the antigen is mixed with a suitable monomer
and immobilized by polymerization using r ray and the like.
When the antigen has a low molecular weight, it is bonded to a
carrier via a spacer which does not affect the steps of (A) to
(C). Examples of the spacer include non-specific rabbit IgG,
bovine serum albumin, dextran, hapten and antibody, and biotin
and avidin.
In (A), the first reaction system is obtained by mixing a
specific antibody in a liquid sample, a labeled antigen and a
carrier-bound antigen, after which a complex of the labeled
antigen-specific antibody-carrier-bound antigen is formed in
this first reaction system under the conditions generally used
for conventional antigen-antibody reaction. In general, a
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-
complex is formed at 0-45C for several hours-several dozens of
hours, preferably at 20-37C for 1-6 hours of reaction.
(B)
(B) is a step for eliminating the prozoning phenomenon.
When the amount of the specific antibody to be assayed in a
liquid sample is present in excess in comparison with the amount
of the antibody that an antigen bound to a carrier can trap,
the steps of (A) and (C) alone results in an excess amount of
specific antibody binding not to the carrier-bound antigen but
to labeled antigen to form an incomplete complex of labeled
antigen-specific antibody. The excess specific antibody
consumes labeled antigen necessary for forming a complete
complex, causing insufficient level of the labeled antigen in
comparison with the amount of specific antibody bound to the
carrier-bound antigen, which can sometimes result in partial
formation of an incomplete complex of specific antibody-carrier-
bound antigen. Consequently, the assay values showing the
amount of the antibody bound to the carrier-bound antigen
becomes lower than those corresponding to the amount of the
antibody actually bound (prozoning phenomenon). The instant
step supplements the labeled antigen consumed by the excess
antibody in the liquid sample and allows the labeled antigen to
be bound to the incomplete complex of the specific antibody-
carrier-bound antigen to form a complete complex of labeled
antigen-specific antibody-carrier-bound antigen, which enables
compensation of the lowered signal values caused by the
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prozoning phenomenon.
The method for separating the aforementioned carrier after
reaction from the aforementioned first reaction system, namely,
a method for separating, in the first reaction system after (A),
an incomplete complex of labeled antigen-specific antibody,
unreacted specific antibody and unreacted labeled antigen, from
a complete complex of labeled antigen-specific antibody-
carrier-bound antigen, an incomplete complex of specific
antibody-carrier-bound antigen and unreacted carrier-bound
antigen, can be any method which is employed in conventional
immunological assay using carrier.
- Then, the carrier separated from the above-mentioned first
reaction system is again mixed with a labeled antigen to give a
second reaction system. When the carrier separated from the
first reaction system comprises an incomplete complex of
specific antibody-carrier-bound antigen, the carrier separated
from the first reaction system is preferably washed before
mixing with the labeled antigen to ensure binding of the labeled
antigen to the incomplete complex. The carrier is washed under
- the conditions conventionally used in sandwich method. In
general, a solution containing a mild surfactant such as Tween
20 is brought into contact with the carrier separated from the
first reaction system for a certain time and the solution and
the carrier are separated. This step is repeated several times.
While the amount of the labeled antigen to be mixed in tB)
is not subject to any particular limitation, mixing of the
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antigen in excess increases non-specific binding of the labeled
antigen to the carrier after separation, which in turn
increases background and lowers sensitivity. Accordingly, the
antigen is desirably used in an amount necessary and sufficient
to complete the reaction. Generally, the amount is from 1/10
to the same amount as the labeled antigen used in (A).
The reaction between the labeled antigen and the incomplete
complex bound to the carrier (carrier-bound antigen-specific
antibody) in the second reaction system is carried out under the
conditions used for conventional antigen-antibody reaction.
The reaction generally proceeds at 0-45C for several dozen
minutes to several hours, preferably 20-37C for 30 minutes to
2 hours to give a complete complex (labeled antigen-specific
antibody-carrier-bound antigen).
(C)
In (C), the method for separating the carrier after
reaction from the aforementioned second reaction system and the
method for washing the carrier after separation may be any
method employed in conventional immunological assay using
carrier. Washing is preferably performed by adding a washing
solution to the reaction system, leaving the mixture for several
minutes and removing the washing solution. The amount of the
label bound to the carrier is determined by detection of
radiation, detection of fluorescence, detection of absorbance,
spectrometry, atomic absorption analysis and the like,
depending on the peculiar nature of the label introduced into
~ 2155318
the antigen in (A).
For determination of radiation, a ~ counter or liquid
scintillation counter is used for several dozen seconds to
several minutes. When an enzyme is used as a label, a substrate
capable of emitting a detectable signal in response to the
action of the enzyme is added and the signal is detected by the
detection step as mentioned above. For example, when the enzyme
is horseradish peroxidase, the substrate can be, for example,
H202 and-o-phenylenediamine (detection of absorbance) or H202
and o-hydroxyphenylpropionic acid (fluorescence detection).
When the enzyme is, for example, ~-D-galactosidase, the
substrate can be, for example, p-nitrophenyl-~-D-
galactopyranoside (detection of absorbance) or 4-
methylumbelliferyl-~-D-galactose (fluorescence detection).
For example, when the enzyme is alkaline phosphatase, the
substrate can be, for example, ~-nitrophenylphosphate (detection
of absorbance) or 4-methylumbelliferylphosphate (fluorescence
detection). These reactions are carried out at 20-37~ for
several minutes to several hours.
The present invention is described in more detail by
referring to Examples, to which the invention is not limited.
Example 1
Purification of thyroglobulin
Human thyroglobulin (10 mg, UCB Bioproducts, Belgium) was
purified by a method using DE-52 cellulose (Whatman, Kent, UK)
column [Otaki et al., Journal of Clinical Endocrinology and
21~53~ ~
Metabolite, vol. 52, p. 239 tl981)].
The above-mentioned purified thyroglobulin (7.0 mg) was
eluted with 0.1 M sodium phosphate buffer, pH 7.0, using rabbit
(anti-human-IgG r -chain) IgG immobilized on Sepharose 4B
column (l.Ox 4.5 cm), and subjected to gel filtration using the
same buffer and Ultrogel AcA22 (1.6x 45 cm, LKB, Stockholm,
Sweden). The purity of the purified product was confirmed by
SDS-polyacrylamide gel electrophoresis. The concentration of
the thyroglobulin was determined from absorbance at 280 nm
using absorption coefficient of 1.0 0/g- cm.
Preparation of thyroglobulin-peroxidase (labeled antigen)
1. Preparation of mercaptosuccinyl-thyroglobulin
Thiol groups were introduced into the purified
thyroglobulin by a known method [Ishikawa et al., Journal of
Immunoassay, vol. 4, p. 209 (1983)] using S-acetylmercapto-
succinic anhydride. The number of the thiol groups introduced
was 3.8 per 1 molecule of the thyroglobulin.
2. Preparation of maleimide-peroxidase
Maleimide groups were introduced into horseradish
peroxidase by a known method [Hashida et al., Journal of
Applied Biochemistry, vol. 6, p. 56 (1984)] using N-
succinimidyl-6-maleimidohexanoate. The number of the maleimide
groups introduced was 1.3 per 1 molecule of the peroxidase.
3. Preparation of thyroglobulin-peroxidase
0.1 M Sodium phosphate buffer (70 ~0, pH 6.0) containing
mercaptosuccinyl-thyroglobulin (310 ~g) and 5 mM EDTA, and 0.1
1 3
.
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M sodium phosphate buffer (5 ~0, pH 6.0) containing maleimide-
peroxidase (93 ~g) and 5 mM EDTA were reacted at 4C for 20
hours. The reaction mixture was subjected to gel filtration
using ultrogel AcA22 column (1.6x 45 cm) and 0.1 M sodium
phosphate buffer, pH 6.~. The number of the peroxidase
introduced was 1.7 per 1 molecule of the thyroglobulin.
Preparation of thyroglobulin-bound solid phase (carrier-bound
antigen)
Using a solution of the puri~ied thyroglobulin (O.Ol g/0),
thyroglobulin was bound to the surface of each well of a
microplate [33 mm2x 11.3 mm, Maxisoap F8 (Nunc, Denmark)] by a
known method [Ishikawa et al., Scandinavian Journal of
Immunology, vol. 8 (Suppl. 7), p. 43 (1978)] utilizing physical
adsorption.
Assay of human anti-thyroglobulin antibody
Test samples (0.05 ml) obtained by diluting sera from
patients with Basedow's disease, the anti-thyroglobulin
antibody concentration thereof having been identified in
advance, to various concentrations with sera of healthy humans,
and 0.01 M sodium phosphate buffer (0.1 ml, pH 7.0) containing
thyroglobulin-peroxidase (100 fmol), 0.55 M sodium chloride,
0.1% bovine serum albumin and 0.1~% Tween 20 were placed in each
well of the thyroglobulin-bound plate (solid phase), and the
plate was left standing at room temperature for 2 hours to allow
reaction. Each well was washed three times with 0.01 M sodium
phosphate buffer (0.3 ml, pH 7.0) containing 0.1 M sodium
.. .. . . . .
- ~ 215~18
.
chloride and 0.1~ Tween 20. Then, 0.01 M sodium phosphate
buffer (0.15 ml, pH 7.0) containing thyroglobulin-peroxidase
(50 fmol), 0.1 M sodium chloride, 0.1% bovine serum albumin and
0.1% Tween 20 was added, and the plate was left standing at room
temperature for 1 hour to allow reaction. Each well was washed
three times with the above-mentioned washing solution and the
activity of peroxidase bound to the plate was assayed as in the
following.
Assay of peroxidase activity
A substrate solution (150 ~, 0.05 M sodium phosphate-
citrate buffer, pH 4.8, containing 0.017% hydrogen peroxide and
0.6 mg/ml o-phenylenediamine) was added to each well and the
plate was left standing at room temperature for 30 minutes. 2N
Sulfuric acid (50 ~) was added to each well to stop the
reaction. The activity of peroxidase which formed a complex
was determined by measuring absorbance at 492 nm of the reaction
mixture in each well. The results are shown in Fig. 1.
A conventional assay method (Comparative Example 1) wherein
the target specific antibody is sandwiched by carrier-bound
antigen and labeled anti-Ig antibody and a conventional assay
method (Comparative Example 2) wherein the target specific
antibody is sandwiched in two steps by carrier-bound antigen and
labeled antigen are shown in the following as comparative
examples for the present invention.
Comparative Example 1
Purification of thyroglobulin, preparation of
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thyroglobulin-bound solid phase and assay of peroxidase
activity were performed according to the method of Example 1.
Assay of human anti-thyroglobulin antibody
Test samples (0.05 ml) obtained by diluting sera from
patients with Basedow's disease, the concentration of the anti-
thyroglobulin antibody thereof having been identified in
advance, to various concentrations with sera of healthy humans,
and 0.01 M sodium phosphate buffer (0.1 ml, pH 7.0~ containing
0.55 M sodium chloride, 0.1% bovine serum albumin and 0.15%
Tween 20 were placed in each well of the thyroglobulin-bound
plate (solid phase), and the plate was left standing at room
temperature for 2 hours to allow reaction. Each well was washed
three times with 0.01 M sodium phosphate buffer (0.3 ml, pH
7.0) containing 0.1 M sodium chloride and 0.1% Tween 20. 0.01
M Sodium phosphate buffer (0.15 ml, pH 7.0) containing
peroxidase-labeled affinity purified goat anti-human IgG (7.5
ng, Cappel, Maryland), 0.1 M sodium chloride, 0.1% bovine serum
albumin and 0.1% Tween 20 was added, and the plate was left
standing at room temperature for 2 hours to allow reaction.
Each well was washed three times with the above-mentioned
washing solution and the activity of peroxidase bound to the
solid phase was assayed. The results are shown in Fig. 1.
Comparative Example 2
Purification of thyroglobulin, preparation of
thyroglobulin-peroxidase, preparation of thyroglobulin-bound
solid phase and assay of peroxidase activity were performed
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according to the method of Example 1.
Assay of human anti-thyroglobulin antibody
Test samples (0.05 ml) obtained by diluting the sera from
patients with Basedow's disease, the concentration of the anti-
thyroglobulin antibody thereof having been identified in
advance, to various concentrations with sera of healthy humans,
and 0.01 M sodium phosphate buffer (0.1 ml, pH 7.0) containing
0.~5 M sodium chloride, 0.1% bovine serum albumin and 0.15%
Tween 20 were placed in each well of the thyroglobulin-bound
plate (solid phase), and the plate was left standing at room
temperature for 2 hours to allow reaction. Each well was washed
three times with 0.01 M sodium phosphate buffer, pH 7.0,
containing 0.1 M sodium chloride and 0.1% Tween 20. 0.01 M
Sodium phosphate buffer (0.15 ml, pH 7.0) containing thyro-
globulin-peroxidase (100 fmol), 0.1 M sodium chloride, 0.1%
bovine serum albumin and 0.1% Tween 20 was added, and the plate
was left standing at room temperature for 2 hours to allow
reaction. Each well was washed three times with the above-
mentioned washing solution and the activity of peroxidase bound
to the solid phase was assayed. The results are shown in Fig.
1.
An example using a different carrier-bound antigen is given
as another example of the present invention.
Example 2
Purification of thyroglobulin, preparation of
thyroglobulin-peroxidase, assay of human anti-thyroglobulin
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antibody and assay of peroxidase activity were performed
according to the method of Example 1.
Preparation of solid phase bound to dinitrophenyl-
thyroglobulin-bound rabbit (anti-dinitrophenyl-bovine serum
albumin) IgG (carrier-bound antigen)
1. Preparation of solid phase bound with rabbit (anti-
dinitrophenyl-bovine serum albumin) IgG
Sodium sulfate (0.747 g) was portionwise added to rabbit
(anti-dinitrophenyl-bovine serum albumin) antiserum (4.23 g,
Seikagaku Kogyo, Tokyo), and the mixture was stirred at room
temperature for 30 minutes and centrifuged at 10,000x g for 15
minutes. The precipitate was dissolved in 0.0175 M sodium
phosphate buffer (4.0 ml, pH 6.3) and dialyzed against the same
buffer. The dialysate was subjected to anion exchange
chromatography using DE-52 cellulose (Whatman, Kent, UK) column
(1.6x 8.0 cm) with the linear concentration gradient of sodium
chloride. Using this solution, rabbit (anti-dinitrophenyl-
bovine serum albumin) IgG was bound to the surface of each well
of a microplate [33 mm2x 11.3 mm, Maxisoap F8 (Nunc, Denmark)]
by a known method [Ishikawa et al., Scandinavian Journal of
Immunology, ibid.] utilizing physical adsorption.
2. Preparation of dinitrophenyl-thyroglobulin
(1) Synthesis of succinimidyl-2,4-dinitrophenyl-~ -caproic acid
2,4-Dinitrophenyl-~ -caproic acid (Sigma, Missouri) and N-
hydroxysuccinimide (Wako Pure Chemical Industries Ltd., Osaka)
were condensed by a known method [F. Levi-Schaffer et al.,
1 8
.. . . , , . . ~ . . . . . . . ; . . . .
2~ 5~1 8
American Journal of Tropical Medicine and Hygiene, vol. 32, p.
343 (1983)] using dichlorocarbodiimide (Wako Pure Chemical
Industries Ltd.) to synthesize succinimidyl-2,4-dinitrophenyl-
~ -caproic acid. The acid was purified by silica gel (40 g)
column in a chloroform/methanol [40/1 (v/v)] system, and the
structure was confirmed by NMR (nuclear magnetic resonance) and
mass spectrum.
(2) Preparation of dinitrophenyl-thyroglobulin
N,N-Dimethylformamide (60 ~l) containing succinimidyl-2,4-
dinitrophenyl-~ -caproic acid (66 nmol) prepared in the above
(1) was added to 0.1 M sodium phosphate buffer (0.6 ml, pH 7.0)
containing 0.5 mg of purified thyroglobulin dissolved therein,
and the mixture was reacted at 30C for 30 minutes. After the
reaction, the reaction mixture was subjected to gel filtration
using 0.1 M sodium phosphate buffer, pH 7.0, and Sephadex G-25
(Pharmacia, Sweden) column (l.Ox 30 cm). The number of the
dinitrophenyl groups introduced was 11 per 1 molecule of the
thyroglobulin.
The dinitrophenyl groups were quantitatively determined
from the absorbance at 360 nm using an absorption coefficient
of 17400/M- cm.
3. Preparation of solid phase bound with dinitrophenyl-
thyroglobulin-bound rabbit (anti-dinitrophenyl-bovine serum
albumin) IgG
0.01 M Sodium phosphate buffer (0.15 ml, pH 7.0) containing
dinitrophenyl-thyroglobulin (500 fmol) prepared in the above
.. . . . .... . . .. . .
5 3 1 ~
2., 0.1 M sodium chloride, 0.1% bovine serum albumin and 0.1%
sodium azide was added to the plate (solid phase) bound with
rabbit (anti-dinitrophenyl-bovine serum albumin) IgG, prepared
in the above 1., and the reaction was carried out at 4C for 16
hours. After the reaction, the plate was washed with the same
buffer, and stored with 0.3 ml of the buffer added thereto.
Assay of human anti-thyroglobulin antibody
In the same manner as in Example 1 except that the
thyroglobulin-bound plate (solid phase), i.e. a carrier-bound
antigen, was replaced with a plate (solid phase) bound with
dinitrophenyl-thyroglobulin-bound rabbit (anti-dinitrophenyl-
bovine serum albumin) IgG, the activity of peroxidase bound to
the plate was assayed. The results are shown in Fig. 1.
A conventional assay (Comparative Example 3) wherein the
target specific antibody is sandwiched by hapten-bound antigen
and labeled antigen, and trapped by a carrier bound with an
anti-hapten antibody is given as a comparative example for the
present invention.
Comparative Example 3
Purification of thyroglobulin, preparation of
thyroglobulin-peroxidase and assay of peroxidase activity were
performed according to the method of Example 1, and preparation
of dinitrophenyl-thyroglobulin and a solid phase bound with
rabbit (anti-dinitrophenyl-bovine serum albumin) IgG were
performed according to the method of Example 2.
Assay of human anti-thyroglobulin antibody
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Test samples (0.05 ml) obtained by diluting the sera from
patients with Basedowrs disease, the concentration of the anti-
thyroglobulin antibody thereof having been identified in
advance, to various concentrations with sera of healthy humans,
and 0.01 M sodium phosphate buffer (0.1 ml, pH 7.0) containing
thyroglobulin-peroxidase (100 fmol), dinitrophenyl-thyroglobulin
(100 fmol), 0.55 M sodium chloride, 0.1% bovine serum albumin
and 0.15% Tween 20 were placed in each well of a plate (solid
phase) bound with anti-dinitrophenyl-bovine serum albumin IgG,
and the plate was left standing at room temperature for 3 hours
to allow reaction. Each well was washed three times with 0.01 M
sodium phosphate buffer (0.3 ml, pH 7.0~ containing 0.1 M
sodium chloride and 0.1% Tween 20, and the activity of the
peroxidase bound to the solid phase was assayed. The results
are shown in Fig. 1.
As is evident from the results of Examples 1-2 and
Comparative Examples 1-3 as shown in Fig. 1, the target specific
antibody was assayed with high sensitivity which was about 100
times greater in comparison with Comparative Example 1, and
about 10 times greater in comparison with Comparative Example 2,
and the problem of prozoning phenomenon which occurred in
Comparative Example 3 could be resolved. As shown in Example
2, the use of a carrier-bound antigen different from that in
Example 1 also led to the above-mentioned effects.
In the following, the above-mentioned Examples 1 and 2
wherein different labeled antigens were used are described as
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~ 21~53~8
Examples 3 and 4.
Example 3
Purification of thyroglobulin, preparation of
thyroglobulin-bound solid phase and assay of human anti-
thyroglobulin antibody were performed according to the method
of Example 1.
Preparation of thyroglobulin-~-D-galactosidase (labeled
antigen)
1. Preparation of maleimide-thyroglobulin
0.55 mM N-Succinimidyl-6-maleimidohexanoate (0.2 ml)
dissolved in dimethylformamide was added to purified
thyroglobulin (1.08 mg) dissolved in 0.1 M sodium phosphate
buffer (2.0 ml, pH 7.0), and the mixture was allowed to react
at 30C for 30 minutes. After the reaction, the reaction
mixture was subjected to gel filtration using Sephadex G-25
column (lx 30 cm) equilibrated with 0.1 M sodium phosphate
buffer, pH 6.0, containing 5 mM EDTA, to give maleimide-
thyroglobulin. The number of the maleimide groups introduced
was 4 per 1 molecule of the thyroglobulin.
2. Preparation of thyroglobulin-~-D-galactosidase
~ -D-Galactosidase (0.45 mg) dissolved in 0.1 M sodium
phosphate buffer (85 ~0, pH 6.0) containing 5 mM EDTA was added
to maleimide-thyroglobulin (0.55 mg) dissolved in said buffer
(50 ~) containing 5 mM EDTA, and the mixture was allowed to
react at 4C for 2 hours. The reaction mixture was subjected to
gel filtration using ultrogel AcA22 column (1.6x 70 cm)
- ~ 215531~
equilibrated with 0.01 M sodium phosphate buffer, pH 7.0,
containing 0.1 M sodium chloride, 0.1% bovine serum albumin,
0.1 mM MgCl2 and 0.1~ NaN3, to give thyroglobulin-~-D-
galactosidase. The number of the ~-D-galactosidase introduced
was 1 per 1 molecule of the thyroglobulin.
Assay of human anti-thyroglobulin antibody
In the same manner as in Example 1 except that the
thyroglobulin-peroxidase was replaced with thyroglobulin-~-D-
galactosidase, the activity of ~-D-galactosidase bound to the
plate was assayed.
Assay of ~-D-galactosidase activity
The ~-D-galactosidase activity was determined using 4-
methylumbelliferyl ~-D-galactoside as a substrate after the
reaction at room temperature for 30 minutes [Imagawa et al.,~
Annals Clinical Biochemistry, vol. 21, p. 310 tl984)]. The
fluorescent intensity was corrected based on the fluorescent
intensity of 0.1 M glycine-NaOH buffer, pH 10.3, containing
10-8 M 4-methylumbelliferone (4 MU) dissolved therein as being
100. The results are shown in Fig. 2.
Example 4
Purification of thyroglobulin and assay of human anti-
thyroglobulin antibody were performed according to the method
of Example 1, preparation of a solid phase bound with
dinitrophenyl-thyroglobulin-bound rabbit (anti-dinitrophenyl-
bovine serum albumin) IgG were performed according to the method
of Example 2, and preparation of thyroglobulin-~-D-
~ . . . . . . . ..
- 2 ~ ~ ~ 3 ~ ~
galactosidase and assay of ~-D-galactosidase activity were
performed according to the method of Example 3.
Assay of human anti-thyroglobulin antibody
In the same manner as in Example 3 except that the
thyroglobulin-bound solid phase was replaced with a solid phase
bound with dinitrophenyl-thyroglobulin-bound rabbit (anti-
dinitrophenyl-bovine serum albumin3 IgG, the activity of ~-D-
galactosidase bound to the plate was assayed. The results are
shown in Fig. 2.
An example wherein, in the assay of the above-mentioned
Comparative Example 2, the same labeled antigen as in the
above-mentioned Examples 3 and 4 was used, is shown as a
comparative example.
Comparative Example 4
Purification of thyroglobulin and preparation of
thyroglobulin-bound solid phase were performed according to the
method of Example 1, and preparation of thyroglobulin-~-D-
galactosidase and assay of ~-D-galactosidase activity were
performed according to the method of Example 3.
Assay of human anti-thyroglobulin antibody
In Comparative Example 2, thyroglobulin-peroxidase was
replaced with thyroglobulin-~-D-galactosidase. The activity
of ~-D-galactosidase bound to the plate was assayed in the
same manner as in Example 3. The results are shown in Fig. 2.
As is evident from the results of Examples 3-4 and
2 4
.
- ~ 215531~
-
Comparative Example 4 as shown in Fig. 2, it was confirmed that
the target specific antibody could be assayed with high
sensitivity, even when a labeled antigen different from those
used in Examples 1 and 2 was used.
2 5
