Note: Descriptions are shown in the official language in which they were submitted.
~14125'11
REAGENT AND METHOD FOR THE DETERMINATION
OF THE HUMAN-MUSCLE TYPE ALDOLASE
This invention relates to reagents and a method for the
determination of the human-muscle type aldolase.
Aldolase is a general term of the enzymes which
catalyze aldol condensation and cleavage between dihydro-
xyacetone phosphate and a variety of aldehydes.
Aldolase according to this invention is meant for a
fructose diphosphate aldolase (E.C. 4. 1. 2. 13. fructose-l,
6-phosphate D-glycelaldehyde-3-phosphate lyase) which
reversibly decomposes fructose-1,6-diphosphate into dihydro-
xyacetone phosphate and D-glycelaldehyde-3-phosphate. This
enzyme occupies the main course of glycolysis system, and
particularly, distrib~ites mainly in muscle, thereby contrib-
uting importantly to the energy metabolism.
Mammal aldolase comprises three types of isoenzymes,
that is, the muscle type (A type), the liver type (B type)
and the brain type (C type). It is known that the construc-
tion ratio of these isoenzymes varies in accordance with the
metamorphosis of the living body, such as differentiation of
the fetal liver in rat, canceration of organism in human,
rat, and the like.
There have been conventionally known two methods for
determining the human-muscle type aldolase, one of which is
the electrophoretic method, and the other is a method which
comprises measuring the decomposition activities of two
substrates, that is, fractose-1,6-diphosphate (FDP) and
fructose-l-phosphate (FIP) for aldolase, [FDP-ALD activity
, ~
and FIP-ALD activity], thereby determining the activity
ratio (FDP/FIP).
These methods, however, involve various problems.
In the electrophoretic method, the problem is that the
migrated points of the human aldolase isoenzymes are so
closely positioned one another that the discrimination is
difficult and the precise quantitative determination can not
be achieved.
In the measuring method of the activity ratio FDP/FIP,
it is pointed out that the operation is particularly compli-
cated for the measurement of the FIP-ALD activity, accompa-
nied by no quantitatiVe determination.
Recently, there has been reported a method for determin-
ing aldolase by means of radioimmunoassay through competition
(double antibody method). Comparing with the aforementioned
methods, this method is more excellent in the sensitivity
and also in the quantification. However, this method also
has some disadvantage that there is required a large amount
of the second antibody for the separation of B and F, as
well as handling procedure itself of the separation lS very
complicated, resulting in the requ~ements of a long period
of time and much labor cost. There is further observed an
appearance of non-specific reaction.
The present invention relates to a radioisotope-labeled
muscle type aldolase antibody and a method for the determi-
nation of the human-muscle type aldolase by means of radio-
immunoassay through so-called "Sandwich method" using the
above-mentioned aldolase antibody as a reagent. According
to this method, various defects in the methods of prior art
can be dissolved or diminished. More particularly, the
method of the present invention is remarkable in both the
sensitivity for the determination an~d the quantitative
matter. Also in the comparison with a double-antibody
method, the method of the present invention is characterized
in the points that the second antibody is not required; the
separation procedure for B and F is simple and convenient;
and non-specific reaction is outstandingly low.
Accordins to so-called "Sandwich method" of the present
invention, determination of human-muscle type aldolase is
carried out through the following steps:
(a) contacting a specimen with an insolubilized muscle
type aldolase antibody, followed by separation of the
solid phase;
(b) contacting the solid phase with a radioisotope-labeled
muscle type aldolase antibody, followed by separation
of the solid phase; and
(c~ determining the radioactivity of the solid phase.
The following will illustrate more particularly this
method.
In the step (a), an antibody insolubilized by combina-
tion with a carrier is incubated together with a specimen,
such as serum, etc., so that the antigen (human muscle type
aldolase) in the specimen may react with said insolubilized
antibody. After the reaction is over, the reaction solution
is removed and the solid phase is washed with buffer solu-
tion, distilled water,physiological saline water or the like.
In the step (b), the solid phase obtained in the step
(a) is incubated together with a radioisotope-labeled
1291
antibody, resulting in that said radioiso-tope-labeled anti-
body combines with the antigen previously combined with the
insolubilized antibody. After the reaction is over, the
reaction solution is removed, and the solid phase is washed
with a buffer solution, distilled water, physiological
saline water or the like.
In the step (c), the radioactivity of the solid phase
obtained in the step (b) is determined using a well-type
scintilation counter, etc.
The radioactivity of the standard antigen is previously
measured with said determination system, so as to set up a
standard curve of the amount of antigen versus the radio-
activity. The radioactivity of a specimen is measured with
the same determination system, and then the amount of
antigen in the specimen is determined from said standard
curve.
Fig. 1 in the attached drawing shows a standard curve
of the human-muscle type aldolase obtained by the radio-
immunoassay in the following Exampie 2.
The following description illustrates the reagent for
determination to be used with the present method and the
muscle type aldolase antibody to be used with the production
of the reagent.
A) The muscle type aldolase antibody
Antiserum is obtained by immunizing the muscle type
aldolase of a mammal such as human, rabbit, dog, monkey,
etc. to the other animal.
The immune animal is preferably selected from the
species of birds, because the muscle type aldolases do not
~141~
show significant immunological distinction between the
mammals. Illustrative of the preferred birds include, for
example, fowl, turkey, duck and the like.
In the purification of the resulting antiserum, there
can be used a method which comprises adding the inactivated
serum of the animal which is the source of the muscle type
aldolase, and removing by absorption the other impure anti-
body; an affinity chromatography using support combined the
muscle type aldolase; and the like.
B) Radioisotope-labeled muscle type aldolase antibody
The labeling of muscle type aldolase antibody by means
of radioisotope can be achieved by any conventional methods.
For example, the labeling is carried out by chloramine-
T method using 125I, 13~I, etc. as the radioisotope, and the
like method.
C) The insolubilized muscle type aldolase antibody
As a carrier, there are used an insoluble solid which
is combinative with an antibody. Illustrative of such solid
include, for example, polystyrene, cellulose, agarose, glass,
bridged dextran, metal, etc. There may be mentioned a form
such as tube, microtiter plate, powder, sphere, disc, plate,
foil, etc.
In the case of the polystyrene microtiter plate, the
antibody may combine with the wall surface of the plate,
when the antibody is properly diluted with a buffer solution
or the like, the diluted solution is then added to the plate,
and the whole is finally allowed to stand.
The above descriptions have been made to the sandwich
method, but the human muscle type aldolase can be also
determined by the immunoradiometric assay using radioisotope-
labeled muscle type aldolase antibody which is the reagent of
the present invention. This method comprises reacting the
radioisotope-labeled antibody with a specimen (antigen),
followed by separation of the radioisotope-labeled antibody-
antigen reactant (B) from unreacted radioisotope-labeled anti-
body (F), and determining the radioactivity of either said
(B) or (F). As the method for the separation of (B) and (F),
there may be mentioned the gel filtration method, the absorp-
tion method by means of insolubilized antigen, etc.
Following experiments and examples will further illus-
trate this invention.
Experiment 1
Human muscle type aldolase antibody
The human-muscle type aldolase (1 mg) was dissolved in
physiological salt water (0.5 ml), and to the solution was
admixed the equal volume of Freund complete adjuvant. The
product was immunized by subcutaneous injection to a fowl
(~hite Leghorn) at the intervals of one week, so that the
injections may amount to five times. One week after the
last immunization, the blood was collected from the fowl to
obtain the anti-serum.
Sepharose 4BTM (Pharmacia Fine Chemicals AB; Agarose)
(10 g) which was activated with bromo-cyanide, and the
human-muscle type aldolase (10 mg) were allowed to stand at
4C overnight in 15 ml of 0.1M sodium carbonate buffer solu-
tion (pH 9.0).
The resulting Sepharose 4B gel combined with the human-
muscle type aldolase was packed into a column, and washed
L1291
with 0.05 M tris-hydrochloric acid buffer solution (pH 8.0)
containing 0.5 N sodium chloride. To this column was added
the above-mentioned human-muscle type aldolase anti-serum,
and the anti-serum was flowed out with 0.05 M tris-hydro-
chloric acid buffer solution (pH 8.0) containing 0.5 N
sodium chloride. Thereafter, 0.1 M aqueous sodium carbonate
solution was added to the column to elute the human-muscle
type aldolase antibody. The fraction of antibody eluate
was subjected to dialysis with 0.05 M tris-hydrochloric acid
buffer solution (pH 8.0) to obtain 3 mg of the human-muscle
type aldolase antibody.
Experiment 2
_nsolubilized human-muscle type aldolase antibody
Into each of the holes provided on a polystylene micro-
titer plate, was added every 200 ~1 of solution of the human-
muscle type aldolase antibody which was adjusted to OD280 m~
=0.10, and the whole was then allowed to stand overnight
at 4C. The solution was removed from the plate. The plate
was washed with distilled water to obtain the microtiter
plate combined with the human-muscle type aldolase antibody.
EXAMPLE 1
5I-labeled human muscle type aldolase antibody
Na I of 250 ~ curie (10 ~1), 0.5 M phosphate buffer
(pH 7.4)(12.5 ~1), human muscle type aldolase antibody (1.85
mg/ml)(10 ~1) and Chloramine-T (3 mg/ml)(12.5 ~1) were
reacted one another for 20 seconds in a small test tube
coated with silicone. After the reaction was over, sodium
metabisulfite (6 mg/ml)(25 ~1) were added to the mixture,
so as to cease the reaction. To the resulting mixture were
added potassium iodide (50 mg/ml)(12.5 ~1) and 2~ egg albumin
(250 ~1) in 0.05 M phosphate buffer. The whole was then
subjected to gel-filtration by means of Sephadex G-25TM
(Pharmacia Fine Chemicals AB; bridged-dextran), so that the
free 125I may be removed. There was thus obtained l25I-
labeled human muscle type aldolase antibody, the specific
activity thereof being 11.6 ~ curie/~g.
EXAMPLE 2
Radioimmunoassay (Sandwich method)
Into each of the holes which were provided on microtiter
plate combined with human muscle type aldolase antibody, 100
~ of the standard solution of the human muscle type aldolase
were respectively added. The whole was reacted at 37C for
2 hours. After the reaction was over, the supernatant layer
were removed, and the remainder was washed thrice with
physiological saline solution. To the washed holes was then
respectively added 100 ~1 of 125I-labeled human muscle type
aldolase antibody (about 200,000 cpm). The whole was
reacted at room temperature for 16 hours. After the reaction
was over, the supernatant layer was removed. The remainder
was washed thrice with physiological saline solution.
After the contained water was thoroughly exhausted, the
plate was cut, and the respective hole portions were sepa-
rated one another. The individual aliquots were respective-
ly placed in small test tubes. Radioactivity was counted
in a well-type scintilation counter.
As shown in Figure 1, there was obtained the standard
curve wherein about 8 - 1000 ng/ml were plotted as measur-
able ranges. In the Figure, the horizontal axis (logarithm)
indicates the concentration (ng/ml) of the human muscle type
aldolase, and the vertical axis indicates the radioactivity
(cpm).
~ luman muscle type aldolases in various human serums
were then determined through the above assay system. There
were thus observed the data that the human muscle type
aldolase of 25 normal subject amount to 165 + 40 ng/ml, all
of which were less than 210 ng/ml. On the contrary, the
human muscle type aldolase of 20 cancer patients showed
higher values than 210 ng/ml, excludir.g two examples. The
20 cancer patients comprise 7 hepatomae, 8 gastric cancers,
3 large intestine cancers, and 2 pancreatic cancers.
