Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
1. Title of Invention
Enzyme Immun~a 5 say
2. Technical Field
This invention relates to a novel immunoassay method
for the determination of infinitesimal amounts of substances
present in body(or biological) fl~ids, such as serum and
cerebrospinal fluid, of mammals.
The term "immunoassay" as used herein refers to a
method in which the reaction of an immunologically
(serologically) antigenic substance (i.e., a so-called
antigen) with a so-called antibody immunologically
corresponding to it is applied to the determination of the amount
or concentration of antigen or antibody present in the fluid.
3. Background Art
Conventionally, various methods such as bioassay or
enzyme activity measurement methods have been available
for the determination of infinitesimal amounts of substances
present in biological fluids. However, they are not
satisfactory because of their troublesome operations and
their inadequate sensitivity or accuracy. In contrast,
the immunoassay technique which has recently been developed
can attain significantly high sensitivity and accuracy
without requiring difficult operations. Accordingly, this
technique is now being employed in the fields of biochemistry
and clinical medicine.
Various kinds of immunoassay methods have been
proposed, among which the methods using an antigen or
antibody combined (or labeled) with a radioisotope, a
fluorescent material or an enzyme have found wide
application. Specifically further, the so-called solid-phase
modes in these immunoassay methods using the labeling
materials have been regarded as being most ad~antageous.
1~53~
In the solid-phase immunoassay method, the so-called
carrier-adsorbed reactant is used as a reaction material,
which has been prepared by adsorbing, under specific reacti~n
conditions, an antibody, a so-called antiantibody (i.e~, an
5 antibody produced by using an antibody as the antigen), or
so-called protein A (i.e., a special protein produced by
certain microorganisms and ha~ing the ability to react with
various antibodies) on a water-insoluble carrier such as
pieces of polystyrene or silicone, water-insoluble
10 polysaccharides, etc., to insolubilize the former thereby,
said carrier-adsorbed reactants in particular cases being
hereinafter referred to as antibody-on-carrier, antiantibody-
on-carrier, or protein A-on-carrier, respectively.
Now the general principle of solid-phase immunoassay
15 method using an enzyme as the labeling material is explained
hereinbelow before proceeding to the description of the
present invention.
At least one member selected from the group consisting
of an antigen, an antibody, an enzyme labeled antigen, and an enzyme labeled
20 antibody (these are referred generally to immunologically
reactive components hereinafter), or an immunological complex
thereof, is reacted immunologically with a carrier-adsorbed
reactant selected from the group consisting of an antibody-
on-carrier, an antiantibody-on-carrier, and protein A-on-
25 carrier either simultaneously ^r successively, to thereby result in an
insolubiiizea product formed by the binding of the enzyme labeled
antigen (Qr enzyme labeled antibody) or i~s immunological complex
to the carrier-adsorbed reactant together with an unbound,
soluble portion of the labeled antigen (or labeled antibody~.
30 After the unbound labeled antigen (or labeled antibody) is
separated from the insolubilized product, the activity of
the enzyme contained in the insolubilized Product
(particularly, the absorbance of the reaction mixture of the
enzyme with its substrate) is measured. Typically, the
35 same procedures as above are repeated with varying amounts
of the antigen (or antibody), and by using the measured
values thereby, a calibration curve is constructed which
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represents the relationship between the amounts of antigen
(or antibody) initially used and the activity of enzyme
attached thereto. Next, the same procedures are carried
out with a biological fluid containing an ~nknown amount of
5 the antigen (or antibody)~ to measure the activity of the
enzyme contained in the resulting insolubilized product.
Then, the amount or concentration of antigen (or antibody)
presënt in the fluid is calculated from the measured value
by reference to the calibration curve.
While the ~olid-phase enzyme immunoassay method as
above has ad~antageously high sensitivity and accuracy, it
has the disadvantage of requiring troublesome operations.
4. Disclosure of the Invention
We studied the conventional solid-phase enzyme
immunoassay method extensively and invented a very efficient
technique thereof, by conducting all ~he steps thereof, from the step
of the immunological reaction of immunologically reactive
component(s) or an immun~logical complex thereof with a
20 carrier-adsorbed reactant ~ough the step of mea~g the
activity of the enzyme contained in the resulting
insolubilized product,by applying chromatography with a
single column.
According to the method of the present invention, a
25 variety of antigens or antibodies can be assayed by using
an identical enzyme or insoluble carrier, and high
sensitivity and accuracy can be attained without being
affected by undesirable interfering components present in
the biological fluid, while no difficult operations are
30 required for the method. In particular, the method of the
present invention permits the highly sensitive determination
of antibodies in serum which has been resarded as being
difficult in the prior art.
By the way, besides the solid-phase enzyme immunoassay
3~ method, another enzyme immunoassay technique has hitherto
been kno~n in which an immunological reaction mixture of
immunologically reactive components is subjected to a
-~A
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physicochemical proceduxe such as gel permeation
chromatography, affinity chromatography, or the like to
separate the free labeled antigen (or labeled antibody)
therefrom, followed by measuring the activity ~f the enz~me
5 contained in the remainder to determine the concentration
of antigen or antibody initially present. However, the
known enzyme immunoassay method as above is different in
technical conception from the method of the present invention
because the chromatography of the former applies not to the
10 immunological reaction but to the physicochemical reaction.
Additionally, the method u~ing phy~icochemical chromatography
ha~, in contrast with the method of the present in~ention,
the di~advantages in that the kinds of labeling enzymes
and adsorbing materials for use therein must be carefully
15 ~elected in particular case~.
The present invention resides in a method of solid-
phase enzyme immunoassay for an antigen
(or antibody) which consi~ts of the ~tep~ of (a) reacting
immunologically either two immunologically reactive components
~elected from the group consist~ng of an antigen, an antibody,
an enzyme labeled antigen and an enzyme labeled antibody, or a reaction mixture
containing an immunological complex of said components, with a
carrier-adsorbed reactant selected from *he group consisting of
an antibody-on-carrier, an antiantibody-on-carrier and protein
A-on-carrier, simultaneously or successively, to thereby result m
an insolubilized product formed by binding of the immunologically
reactive component(s) or the immunological complex to the carrier-
adsorbed reactant together with an unbound, soluble portion of
the labeled antigen (or labeled antibody); (b) separating the
unbound labeled antigen (or labeled antibody) therefrom; (c)
measuring the activity of the enzyme contained in the insolubilized
product by using substrate solution for the enzyme; (d) repeating
the above steps (a)-(c) but with varying amounts of the anti~en
(or antibody) to thereby construct a calibration curve representing
the relati~nship between the amount of antigen (or antibody)
initially used and the activity of enzyme attached to the
lZ153Z;~
4a
insolubilized product; (e) carrying out the same procedures as
above with a biological fluid containing an unknown amount of the
antigen (or antibody), to thereby measure the activity of the
enzyme contained in the resulting insolubilized product; and (f)
calculating the amount (concentration) of antigen (or antibody)
having been present in the biological fluid by referring the
measured value in step (e) to the calibration curve, characterized
in that while all the immunolvgical reactions included in steps
(a), (d) and (e) are conducted by using purified an*igen, antibody,
antiantibody and pr~tein A, under the presence of gelatin and
sodium chloride, steps (a)-(c3 are conducted as the immunologically
reactive components or the reaction mixture co~taining the
immunological complex are/i~ passed through a column packed with
the carrier-ad~orbed reactant, whereby the insolubilized product
is produced in the co7umn while the unbound labeled antigen (or
labeled antibody) is discharged from the column, followed by
measuring the acti~ity o~ the enzyme contained in the insolubilized
product by pouring a sub~trate solution for the enzyme into the
column.
5. Brief Description of the Drawings
Fig. 1 is a diagrammatic flow sheet illustrating
various modes of operation of the immunological reaction
step (i.e., the step preceding the enzyme activity measuring
step) involved in the method of the present invention;
Fig. 2 is a graph illustrating the calibration curve
obtained in Example 1 which will be gi~en later; and
Fig. 3 is a graph illustrating the calibration curve
obtained in Example 5 which will also be given later.
6. Best Modes for Carrying Out the Invention
Firstly, the immunological reaction step, i.e., the
step preceding the enzyme activity measuring step, involved
in the method of the present invention can be carried out
in various modes of operation. These modes of operation
are explained hereinbelow by reference to the diagrammatic
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4~
flow sheet shown in Fig. 1. It is to be understood however
that, in the following description, no mention is made of
the immunological reaction products containing no labeling
enzyme because their presence does not exert any influence
on the immunoassay in accordance with the present invention.
1,;2~53ZZ
Where mention is made of a smaller or greater amount of an
immunologically reacti~e component, it means an amount
smaller or greater, respectively, than the reaction
equivalent to the given amount of the immunol~gically
5 reactive component to be reacted therewith.
Mode (a): A predetermined amount of a labeled antibody
2-4 is reacted with a smaller amount of an antigen 1 to result in
an immunological complex 1-2-4 consisting of the labeled
antibody 2-4 and of the antigen 1 together with an unbound,
10 free portion 2-4 of the labeled antibody. The reaction
mixture is passed through a c~lumn packed with a carrier 5
having a greater amount of the antibody 2 ads~rbed thereon,
whereby an insolubilized product formed by binding of the
labeled antibody-antigen complex 1-2-4 to the antibody-on-
15 carrier 5-2 is produced, while the unbound labeled antibody
2-4 is discharged from the colùmn.
Mode (b): Contrary to the mode (a), a predetermined
amount of a labeled antigen 1-4 is reacted with a smaller
amount of an antibody 2 to result in a labeled antigen-antibody
20 complex 2-1-4 and an unbound portion 1-4 of the labeled antigen.
The reaction mixture is passed through a column packed with a
carrier 5 ha~ing a greater amount of an antiantibody or protein
A 3 adsorbed thereon, whereby an insolubilized product formed
by binding of the labeled antigen-antibody comple~ 2-1-4 to the
25 antiantibody-on-carrier or protein A-on-carrier 5-3 is produced,
while the unbound labeled antigen 1-4 is discharged from the
column.
Mode (c): This mode is the same as the mode (b) above,
except that an antigen 1 is added to the reaction system in
30 an amount equivalent to or different from that of the labeled
antigen 1-4 (hereinafter referred to briefly as a different
amount),whereby the immunological reaction is effected to
result in a labeled antigen-antibody complex 2-1-4 and an unb~und,
free portion 1-4 of the labeled antigen. Tha reaction mixture
35 is passed *hrough a column packed with a carrier 5 having
a greater amount of an antiantibody or protein A 3 adsorbed
thereon. Thus, an insolubilized product formed by binding
~2~S3Z;~
of the labeled antigen-antibody complex 2-1-4 to the
antiantibody-on-carrier or protein A-on-carrier 5-3 is
produced, w~ile the unbound labeled antigen 1-4 is discharged
from the column~
Mode (d): A predetermined amount of a labeled antigen
1-4 is mixed ~-ith a different amount of an unlabeled antigen
1, and the mixture is passed through a column packed with
a carrier 5 having a smaller amount of an antibody 2 adsorbed
thereon. Thus, an insolubilized product formed by binding
of the labeled antigen 1-4 to the antibody-on-carrier 5-2
is produced, while the unbound labeled antigen 1-4 is
discharged from the column.
M~de (e): A smaller amount of an antigen 1 is
~assed through a column packed ~ith a carrier ~ ha~ing a
15 greater amount of an antibody 2 adsorbed thereon to result in
antigen-antibody-on-carrier 5-2-1. Subsequently, a greater
amount of a labeled antibody 2-4 is passed through the column,
w~ereby an insolubilized product formed by binding of the
labeled antibody 2-4 to the antigen-antibody-on-carrier 5-
20 2-1 is produced, while the unbound labeled antibody 2-4 is
discharged from the column.
Mode (f): A smaller amoun~ of an antibody 2
is passed through a column packed with a carrier 5 having
a greater amount of an antiantibody or protein A 3 adsorbed
25 thereon to result in antibody-antiantibody or protein A-on-
carrier 5-3-2. Subsequently, a greater amount of a labeled
antigen 1-4 i~ passed through the column, whereby an
insolubilized product formed by binding of the labeled
antigen 1-4 to the antibody-antiantibody-on-carrier or
3 antibody-protein A-on-carrier 5-3-2 is produced, while the
unbound labeled antigen 1-4 is discharged from the column.
Secondly, according to the method of the present
invention, the enzyme activity measuring step following
after the immunological reaction step as explained above,
35 is carried out as follows.
After the immunological reaction step has been
accomplished by any one of the modes of operation (a)-(f),
~'
~2~5;~Z
the column is washed with a buffer solution, and the acti~ity
of the labeling enzy~e 4 contained in the insolubilized
product which has remained in the column is measured
by passinS through the column a solution containing a
substrate for the enzyme. Typically, the foregoing
procedures are pre~iously repeated with ~arying amounts of
the antigen 1 or antibody 2, and the measured values thereby
are pl~tted against the amounts of antigen 1 or antibody 2
initially used to construct a calibration cur~e. Next, the
10 same procedures as abo~e are conducted by using a biological
fluid containing an ul~nown amount of the antigen 1 or
antibody 2. Then, the concentration of the antigen 1 or
antibody 2 in the fluid is calculated from the measured
value by reference to the calibration curve.
In the practice of the present in~ention, the
immunological reaction, the reaction of an anti~en or antibody
with a labeling material, and the adsorption of an antibody,
an antiantibody, or protein A on an insoluble carrier are
att~ined at a temperature ranging from about 4C to about
20 40 C, as is usual with biochemical reactions. In most cases,
the time required to complete these reactions is 10 minutes
or more.
The antigen 1 to be assayed by the method of the
present in~ention may be any of the hormones and proteins
25 contained in various biological fluids, the functions of some
of said proteins being not elucidated. Specific examples
of the antigen include insulin, triiodothyronine, thyroxine,
calcitonin, a-fetoprotein, S-100 protein, enolase, calmodulin,
secretory immunoglobulin A, and the like. In addition, the
30 blood le~els of externally administered drugs, such as
antibiotics, can also be determined. The antibody 2 for use
in the method of the present in~ention is an antibody
corresponding to the aforesaid antigen 1. Most of these
antigens 1, antibodies 2, antiantibodies 3, or biolo~ical
35 fluids containing them are commercially available in `
purified form, and such commercial preparations were used in
the examples of the present in~ention gi~en later~
~21~3~
Specific examples of the labeling en3yme for use in
the method of the present invention include ~-D-galactosidase,
alkaline phosphatase, peroxidase, glucose oxidase, malate
dehydrogenase, and the like. Such an enzyme 4 may be co~pled
5 to an antigen 1 or an antibody 2 by any con~entional
procedures. That is, this can be accomplished by using a
bi~unctiOnal reagent as the coupling agent such as,
glutaraldehyde, carbodiimide, N,N-o-phenylenedimaleimide,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, or the like.
1~ Specific examples of the insoluble carrier 5 for use
in ~he method of the present invention include polysaccharides
s~ch as agarose, dextran, oellulose, etc.; synthetic resins
such as polystyrene, polyacrylamide, etc.; and pieces of
glass. In carrying out the method of the present in~ention,
1~ it is desirable that the carrier-adsorbed reactant consisting
of an antibody 2, antiantibody 3 or protein A 3 adsorbed on
the insoluble carrier 5 is in finely spherical or finely
fibrous form, so long as the rate of flow of the
immunological reaction mixture through the column is not
20 reduced to an undue extent.
The adsorbing reaction of an antibody 2, antiantibody
3 or protein A 3 on an insoluble carrier 5 may be performed
by any conventional procedures. For example, this can be
accomplished by acti~ating an insoluble polysaccharide with
25 an activating agent such as cyanogen bromide, sodium
periodate, epichlorohydrin, 1,1'-carbonyldiimidazole,
p-toluenesulfonyl chloride or the like, followed by adding
an antibody or the like to the activated polysaccharide.
The amount of antîbody 2, antiantibody 3, or protein A 3
3~ to use for this purpose is suitably in the range of 0.1 to
20 mg per milliliter of the insoluble carrier.
The antibody 2, antiantibody 3 or protein A 3 for use
in the method of the present in~ention may be so-called
active fragments isolated therefrom. In the case of an
35 anttbody 2 or an antiantibody 3, for example, the active
fragments thereof can be isolated by treatment with a protease
such as papain, pepsin or the like.
5~Z;Z
In view of the small sample ~olumes usually available
for immunoassay, and of the simplification Of operati~ns, and
of the attainment of a reasonably short operating time, it
is desirable that the capacity of the column f~r use in the
5 method of the present inve~tion is not greater than 1 ml.
~ urthermore, in carrying out the method of the present
invention, any interference with the immunological reaction
can be prevented by the technique preYiously proposed by us,
that is, by the addition of a hydrophobic protein (e.s.,
ge}atin) together with a salt (e.g., s~dium chloride) to the
immunological reaction system.
The method according to the present in~ention i5
more specifically illustrated by the following examples
without however limiting it in any way.
15 Example 1 Assay of insulin
(1) Preparation of an antibody and a labeled antibody
A commercial preparation of swine insulin having
been extracted from the pancreas of swine was used as the
20 antigen. A commercial preparation of serum containing an
antibody having been produced by injecting the antigen into
a guinea pig was treated according to conventional
procedures, such as fractionation with ammonium sulfate
and DEAE-cellulose chromatography, to isolate the antibody
25 therefrom. The antibody thus obtained was split by addition
of pepsin and then subjected to column chromatography using
"Sephadex G-150'' whereby the active fragments of the antibody
were isolated. These active fragments were reduced by
2-mercaptoethylamine in the usual manner and then reacted
30 with N,N'-o-phenylenedimaleimide to join it to the reduced
active fragments. Thereaft,er, the reaction product was
further reacted with a commercial preparati,on of ~
galaotosidase to prepare the active fragments labeled with
~-D-galactosidase (hereinafter referred to simply the labeled
35 antibody).
The aforesaid antigen (i.e., s~ine insulin) and its
corresponding antibody have generally the ability to react
* Trade~ark. "Sephadex" is the trademark for a highly cross-linked
-~ dextran, in the form of macroscopic beads. The dextran chains ha~Tefunctional ionic groups attached thereto by ether linka~es.
S3:~2
immunologically with other serum insulins (as antigens)
originating from a wide variety of animal species ( including
human) and their corresponding antibodies, ~o that they can
be used in the assay of insulins (as antigens) or their
5 corresponding antibodies contained in biological fluids of
many animal species (e.g., human).
(2) Preparation of an antibody-on-carrier
~ n antibody-on-carrier was prepared by adsorbins
the antibody obta~ned in item (1) above on a water-insoluble
10 carrier(C~Br-activated'Sepharose)" in the usual manner.
(3) Co~struction of a calibration curve
To l-ml portions of a buffer solution containing
100 ~U/ml of the labeled antibody in the above item (1)
were added 0.1 ml each of aqueous solutions containing
15 smaller amounts (i.e., ~ to 320 ~U/ml) of the antigen, and
the mixtures were incubated at 37C for
2 hours to result in a labeled antibody-antigen complex
together with an unbound portion of the(labeled antibody.
In this and other examples, "U" represents the unit amount
20 of a hormone having been adopted in the standard bioassay
method. Then, each of the reaction mixtures was passed
through a column packed with a greater amount of the
antibody-on-carrier prepared in item (2) above,whereby
the insolubilized product formed by binding of the labeled
25 antibody-antigen complex to the antibody-on-carrier is
produced,while the unbound labeled antibody was discharged
from the column. After the column was washed with a buffer
solution, 0~1 ml of an a~ueous solution containing 10 mg/ml
of o-nitrophenyl-~-D-galactoside ~hereinafter referred to
30 o-NPG) was poured into the column, which was then incubated
- at 37C for 2 hours to prodùce o-nitrophenol. The column
was washed with 2 ml of a 0.1 M solution of Na2C03 and the
absorbance at ~20 1~ of the filtrate was measured to
determine the acti~ity of enzyme ha~ing been contained
thereinD The measured ~alues were plotted against the
amounts of antigen initially used to construct a calibration
curve as shown in Fi~. 2.
* Trademark for agarose.
1;~153Z2
11
On the other hand, the above procedure was modified by
preparing a series of solutions containing the antigen in the
same amounts as before and passing each of these solutions through
a column packed with the antibody-on-carrier. After the c~lumn
. . .
was washed, the same amount of the labeled antibody was passed
therethrough, followed by measurement of the absorbance of
filtrate. In this manner, a calibration curve quite similar to
that of Fig. 2 was obtained.
(4) Determination of insulin in human serum
A human serum sample having an unknown concentration (but
falling in the range of 0-320 ~U/ml as defined in item (3); herein-
after the same~ of insulin was used as the antigen, and the same
procedure as in the former part of item (3) was carried out. That
is, 0.1 ml of the sample was reacted with the labeled antibody,
and the reaction mixture was passed through a column packed with
the antibody-on-carrier. After o-NPG was poured into the column
and incubated, the absorbance of the column filtrate was measured.
When calculated from the measured value by reference to the calibration
curve shown in Fig. 2, the concentration of insulin in the sample
was found to be 92 ~U/ml. For comparison, the same sample was
subjected to the similar mode in the conventional solid-phase
radioimmunoassay (hereinafter referred to RIA) using a radioisotope
as the labeling material, so that the concentration of insulin in
the sample was estimated to be 102 ~U/ml.
On the other hand, the same sample was treated following the
same procedure as in the latter part of item (3~. Thus, the
concentration of insulin in the sample was found to be 95 ~U/ml.
For comparison, the same sample was subjected to the similar mode
in the conventional RIA, so that the concentration of insulin in
the sample was estimated to be 105 ~U/ml.
Example 2 Assay of anti-thyroxine antibody
(1) Preparation of a labeled antigen and an antibody
A commercial preparation of thyroxine having been
~21:~3;~Z
isolated from an extract of swine th3~roidglands was used as
the antigen. The amino group of this antigen was coupled
with the SH group of B-D-galactosidase in the usual manner
to form a labeled antigen. On the other hand, a commercial
5 preparation of serum containing an antibody having been
produced by injecting the antigen into a rabbit was
immediately ~sed as the antibody.
(2) Preparation of an antiantibody-on-carrier
Generally, immunoglobulin G (herei~fter referred to
~0 IgG), which is a kind of protein inherently present in the
serum of animals, has the ability to react with antigenic
substances originating from a ~ariety of animal species
and hence the properties of an antibody corresponding to
them. Accordingly,IgG has been used widely for the
15 preparation of an antiantibody.
In this example, a commercial preparation of serum
containing an antiantibody having been produced by injecting
rabbit IgG into a ~oat was treated in the same manner as
in item (1) of Example 1 to isolate the antiantibody
20 therefrom. By using the antiantibody so isolated, the same
procedure as in item (2) of Example 1 was carried out to
prepare an antiantibody-on-carrier.
(3) Construction of a calibration cur~e
To 0.5-ml portions of a dilution of the labeled
25 antigen prepared in item (1) abo~e were added 3 ,ul each of
serum dilutions containing smaller amounts of the antibody,
and after completion of the reaction, each of the reaction
mixtures was passed through a column packed with a greater
amount of the antiantibody-on-carrier prepared in item (2).
30 Thereafter, the procedure in item t3) of Example 1 was
followed to construct a calibration cur~e.
On the other hand, the above procedure was modified
by preparing a series of serum dilutions (0.5 ml each)
35 containing the antibody in the same amounts as before and
passing each of the dilutions through a column packed with
f~
~53;~Z
13
ehe antibody-on-carrier. After the column was washed, the same
amount of the albeled anti8en was passed therethrough, followed by
~easurement of the absorbance of the column filtrates. In this
manner, a quite similar calibration curve to the above-described
was obtained.
(4) Determination of anti-thyroxine antibody in rabbit serum
A rabbit serum sample of unkno~n concentration of anti-
thyroxine antibody was treated following the same procedure as
in the former part of item (3~ to measure the absorbance of the
filtrate. When calculated from the measured value by reference
to the calibration curve constructed in item (3) above, the
concentration of anti-thyroxine antibody in the sample was fount
to be 1.05U/ml. Similarly, the same sample was treated in the
same manner as in the latter part of item (3), and the anti-
thyroxine antibody concentration was found to be 1.10 U/ml.
Example 3 Assay of anti-insulin antibody.
(1) Preparation of a labeled antigen
A commercial preparation of swine insulin was used as the
antigen. The same procedure as in item (l) of Example 1 was
carried out to label the ~-D-galactosidase to the antigen. On
the other hand, a commercial preparation of serum containing an
antibody having been produced by iniecting the antigen into a guinea
pig was immediately used as the antibody.
(2) Preparation of an antiantibody-on-carrier
A commercial preparation of serum containing an antiantibody
(i.e., antibody to guinea pig IgG) having been produced by injecting
guinea pig IgG into a rabbit was treated in the usual manner to
isolate the antiantibody therefrom. By using the antiantibody,
the procedure in item (2) of Example 2 was followed to prepare
an antiantibody-on-carrier.
(3) Construction of a calibration cur~e
To l-ml portions of a buffer solution containing the
labeled antigen prepared in item (l) were added a series of
serum dilutions containing smaller amounts of the antibody, and
after completion of the reaction,-each of the reaction mixtures
was passed through a column packed with a greater amount of
the antiantibody-on-carrier prepared in item (2).
~2~53Z2
mereafter~ the column was treated follcw~g the ~ pro~re as in
the former part of item (3) of Example 2 to mea~ure the
absorbance of the column filtrate. The measured ~alues
were plotted against the amounts of antibody initially used
5 to construct a calibration curve.
(4) Determination of anti-insulin antibody in guinea pig
serum
A guinea pig serl2m sample with an unknl)wn concentration
of anti-insulin antibo~ was tr~3ated following the s~ne proced~re
10 as in item (3) above to measur0 the absorbance of the
filtrate. When calculated from the measured ~alue by
reference to the calibration curve in item (3) abo~e, the
concentratiOn of anti-insulin antibody in the sample was
found to be o.76 ~U/ml.
15 Example 4 Assay of a-fetoprotein,
(1) Preparation of a labeled antibody
A commercial preparation of a-fetoprotein ha~ing been
isolated from a h~ Yerum was used as the antigen. A
20 commercial preparation of serum containing an antibody having
been produced by injecting the antigen into a goat was treated
in the usual manner to isolate the antibody. By using the
antibody, the procedure in item (1) of Example 1 was followed
to prepare a labeled antibody.
25 (2) Preparation of an antibody-on-carrier
By using Toyopearl HW-55 (trademark) as the insoluble
carrier, an antibody-on-carrier was prepared in the usual
manner.
(3) Construction of a calibration cur~e
To 1-ml portions of a solution containing the labeled
antibody prepared in item (1) were added smaller amounts of
the antigen, and after completion of the reaction, each of
the reaction mixtures ~-as passed through a column packed
with a greater amount of the antibody-on-carrier prepared in
35 item (2). Thereafter, the absorbance of the column filtrate
was measured. The measured ~alues were plotted against the
C amounts of antigen initially used to construct a calibration
curve.
(4) Determination of a-fetoprotein in human serum
A human serum sample of unknown concentration of
a~fetoprotein was treated in the same procedure as in item
5 (3) above, to measure the absorbance of the column filtrate.
W~en calculated from the measured ~alue by reference to the
calibration cur~e in item (3) above, the concentration of
a-fetoprotein in the sample was found to be 151 ng/ml.
the similar mode in the
For comparison, the same sample was subjected to/RIA, so
10 that the concentration of a-fetoprotein in the sample was
estimated to be 192 n$/ml.
Example 5 Assay of calcitoninr
(1) Preparation of an antibody
A commercial preparation of calcitonin having~been
isolated from an extract of swine thyroid glands was used
as the antigen. A commercial preparation of serum
containing an antibody having been produced by injecting
the antigen into a rabbit wa~ treated in the same manner as
20in item (1) of Example 1, to thereby isolate the antibody
therefrom.
(2) Preparation of a labeled antigen and an antiantibody-
on-carrier
The procedure in item ~1) of Example 2 was followed
25to prepare a labeled antigen. On the other hand, an
antiantibody-on-carrier was prepared by conducting the same
procedure as in item (2) of Example 2.
(3) Preparation of antigen solutions having different
concentrations
An aqueous solution of the antigen ~as diluted -to
prepare a series of antigen solutions containing to 150
MRC mU/ml of the antigen ("MRC U" refers to ~he unit amount
of calcitonin in the standard bioassay method).
(4) Construction of a calibration curve
To O.1-ml portions of a solution containing the
labeled antigen prepared in item (2) above were added 0.5
ml each of solutions containing smaller amounts of the
~Z1~3'~2
16
~nt~xX~ ~ item (1) as well as 0.1 ml each of the antiqen solutions
prepared in item (3). After completion of the reaction,
each of the reaction mixtures ~as pas~ed ~ ough a colt~n.
~hereafter, the column w~s treated foll~w~g the ~ pr~e
as in item (3) of Example 2 to measure the absorbance of
filtrate. The meastlred ~alues were plotted against the
amounts of antigen initially used to construct a calibration
curve as shown in Fig. 3.
(5) Determination of calcitonin in a swine thyroid extract
A swine thyroid extract sample having an unknown
ooncentration of c~citon~ was treated follcw~ the ~ proc~e
as in item (3) above to measure the absorbance of filtrate.
When calculated fro~ the measured value by reference to the
calibration curve shown in Fig. 3, the concentration ,of
1~ calcitonin in the sample was found to be 75 MRC mU/ml.
Example 6 Assay of thyroxine.
(1) Preparation of a labeled antigen and an antibody
A commercial preparation of thyroxine having been
20 isolated from an extract of swine thyroid glands was used
as the antigen. The same procedure as in item (1) of
Example 2 was conducted to prepare a labeled antigen. On
the other hand, a commercial preparation of serum containing
an antibody corresponding to the antigen was treated in
25 the same manner as in item (1) of Example 1 to isolate the
antibody therefrom.
(2) Preparation of an antibody-on-carrier
The same procedure as in item (2) of Example 1 was
conducted to prepare an antibody-on-carrier.
30 (3) Preparation of a~tigen solutions having different
concentrations
An aqueous solution of the antigen was diluted to
prepare a series of antigen soltltions having different
concentrations.
(4) Construction of a calibration curve
To i-ml portions of a solution containing the labeled
antigen prepared in item (1) were added the antigen solutions
.,~
~5;~ZZ
prepared in item (3). Each of the resulting mixtures was
passed throush a column packed with a smaller amo~nt of the
antibody-on-carrier, and the column ~as treated in the same
procedure as in item (3) of Example 1 to measure the
5 absorbance of the column filtrate. The measured values were
plotted against the amounts of antigen initially used to
construct a calibration curve.
(5) Determination of thyroxine in a swine thyroid extract
A swine thyroid extract samPle with an unknown
~0 concentration of thyroxine was treated f~ w mg the same procedure
as in item (3) above to measure the absorbance of the column
filtrate. When calculated from the measured value by
reference to the calibration curve constructed in (4) above,
the concentration of thyroxine in the sample was found to be
the similar node m the
15 10.5 ~g/dl. ~or comparison, the same sample was subjected to/
RIA, so that the concentration of thyroxine therein was
estimated to be 11.0 ~ug/dl.
