GB2062226A

GB2062226A - Reagent and method for the determination of human-muscle aldolase

Info

Publication number: GB2062226A
Application number: GB8030730A
Authority: GB
Original assignee: Eisai Co Ltd
Current assignee: Eisai Co Ltd
Priority date: 1979-09-25
Filing date: 1980-09-24
Publication date: 1981-05-20
Also published as: FR2466020B1; IT1141082B; JPS5648894A; IT8024877A0; DE3036184A1; SE8006667L; FR2466020A1; CH648596A5; GB2062226B; BE885376A; NL8005330A; CA1158549A; SE447422B

Abstract

A reagent for the determination of human-muscle aldolase comprises enzyme-labelled muscle aldolase antibody and is used in a process which comprises the following steps: (a) contacting a specimen to be tested with insolubilized muscle aldolase followed by separation of the solid phase; (b) contacting the solid phase with enzyme-labelled muscle aldolase antibody, followed by separation of the solid phase; (c) contacting the solid phase with a substrate for the enzyme; and (d) determining the absorbance of a decomposition product of the substrate.

Description

SPECIFICATION Reagent and method for the determination of the human-muscle aldolase This invention relates to reagents and methods for the determination of the human-muscle aldolase.

Aldolase is a general term for the enzymes which catalize the aldol condensation and cleavage reaction of dihydroxyacetone phosphate and a series of aldehydes.

The aldolase with which this invention is concerned is fructose diphosphate aldolase (E.C. 4.1.2.13. fructose-l, 6-phosphate D-glycelaldehyde-3-phosphate lylase), which reversibly decomposes fructose-l 6-diphosphate into dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. This enzyme occupies the main course of the glycolysis system, and in particular is mainly distributed in muscle, thereby contributing significantly 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 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 humans or rats, and the like.

Two conventional methods are known for determining the human-muscle type aldolase, one of which is the electrophoresis method, and the other is a method which comprises measuring the decomposition activities of two substrates, that is, fructose-l ,6-phosphate (FDP) and fructose-i - phosphate (FIP), for the aldolase [FDP-ALD activity and FIP-ALD activity], thereby determining the activity ratio (FDP/FIP).

These methods however, involve various problems.

In the electrophoresis method, the problem is that the migrated points of the human aldolase isoenzymes are so closely positioned to one another that the discrimination is difficult and the precise quantitative determination cannot be achieved.

For the measuring method of the activity ratio FDP/FIP, it is pointed out that the operation is particularly complicated for the measurement of the FIP-ALD activity, accompanied by no quantitative determination.

Recently, a method by means of radioimmunoassay has been reported. Compared with the aforementioned methods, this method is more excellent in sensibility or sensitivity and also in the quantitative way. However, this method also has some drawbacks since the reagent cannot be stored because of the quick decay of the radioisotope to be used for labeling; special machines and apparatuses and a particular room are required for measuring the radioisotope; handling of the radioisotope during the measurement and the disposal of waste matters thereof are difficult, as the use of the radioisotope is accompanied with a health hazard.

We have developed a reagent to be used for the enzyme-immunoassay of the human muscle aldolase, and a method for the determination of the human-muscle aldolase using said reagent.

According to this method, various drawbacks in the methods of prior art can be dissolved or diminished.

The method provided by this invention is one of enzyme-immunoassay, a so-called "sandwich method". In this method, measurement is effected through the following steps: (a) contacting a specimen with an insolubilized muscle aldolase antibody, followed by separation of the solid phase; (b) contacting the solid phase with a muscle aldolase antibody labelled with enzyme, followed by separation of the solid phase; (c) contacting the solid phase with a substrate for the said enzyme; and (d) measuring absorbance of a decomposition product of said substrate.

The following will illustrate more particularly some preferred aspects of this method.

In the step (a), an antibody insolubilized e.g. by combination with a carrier is incubated together with a specimen, such as serum, etc., so that if present the antigen (human muscle aldolase) in the specimen may react with the insolubilized antibody. After the reaction is over, the reaction solution is removed and the solid phase is washed with buffer solution, distilled water or the like.

In the step (b), the solid phase obtained in the step (a) is incubated together with enzymelabeled antibody provided by the invention, resulting in that the enzyme-labeled antibody 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 or the like.

In the step (c), the solid phase obtained in the step (b) is incubated together with a substrate for the enzyme to effect an enzyme reaction. After a predetermined time, the reaction is terminated by adding a stop solution for the enzyme reaction.

In the step (d), the absorbance of the decomposition product of the substrate in the reaction solution of the step (c) is measured. The measurement of absorbance is effected with an absorption photometer using a suitable wave length for quantitative determination of the composition product of the substrate.

The absorbance of known amounts of a standard specimen for control can be previously measured with the measuring system so as to set up a working curve of the amount of antigen versus the absorbance. The amount of antigen in the specimen can then be determined from the working curve by measuring the absorbance of the unknown amount of specimen with the same measuring system.

The following description illustrates some preferred aspects of the reagent of the invention for determination to be used with the present method. Reference is made to the accompanying drawings, in which: Figures 1 to 4 show working curves for the human-muscle aldolase obtained by the enzymeimmunoassay in the following Example 5.

Figure 5 shows a working curve for the humanmuscle aldolase obtained by the enzymeimmunoassay in the following Example 6.

Figure 6 shows the results of determination of the human-muscle aldolase in the human serums obtained by the enzyme-immunoassay in the following Example 7.

A) The Muscle Type Aldolase Antibody Antiserum is obtained by administering muscle aldolase of a mammal such as human, rabbit, dog, monkey, bovine, etc. into another animal.

The recipient is preferably a bird, i.e. from a different species, because the muscle aldolases do not always show significant immunological distinction between the mammals. Illustrative of the preferred birds include, for example, fowl, turkey, duck and the like.

It is preferable then to add to the resulting antiserum inactivated serum from the animal which is the source of the muscle aldolase, thereby removing by absorption the other impure antibodies.

Purification of the antiserum may be effected by means of affinity chromatography using a support combined with the mammal-muscle aldolase, the mammal being selected from human, rabbit, monkey, bovine, etc., for example. Support materials to be used for this purpose include agarose, cross-linked dextran, etc. The combined support can be prepared by activating a support with bromocyan or the like reagent, adding the activated support to a solution of the muscle aldolase, and stirring the mixed solution at a low temperature. The purified muscle aldolase antibody is obtained by adding the antiserum to a column filled with the combined support, and effecting affinity chromatography. Elution of the muscle aldolase antibody from the column may be carried out with a weak alkaline solution.

In this affinity chromatography, the sensitivity varies in the measuring method of the present invention, depending on the combination of the kind of the muscle aldolase combined with the support and the kind of the muscle aldolase used for the preparation of the antiserum.

In the case of the human muscle aldolase antiserum, a preferably working curve is obtained as shown in Figure 1 by using the human-muscle aldolase antibody which is prepared by purification with the support combined with the human muscle aldolase. In the case of rabbit muscle aldolase antiserum, the more preferable working curves are obtained by purification with the use of the support combined with the human muscle aldolase or the support combined with the bovine muscle aldolase, than with the support combined with the rabbit muscle aldolase, as shown in Figures 2, 3 and 4.

B) The Enzyme-Labelled Muscle Type Aldolase Antibody As suitable enzymes to be used for the labelling, there may be mentioned the enzymes which are generally used for enzymeimmunoassay, for instance, alkaliphosphatase, peroxidase, p-D-galactosidase, glucoamylase, glucose oxidase, and the like.

The labelling may be achieved by any of the conventional methods, such as for example the Glutaraldehyde method, Nakane method, Maleimide method, Mixed acid Anhydride method, Carbodiimide method, etc. In the glutaraldehyde method, the labelling is achieved by adding enzyme to the antibody, and adding glutaraldehyde so that the concentration may reach 0.2 to 0.8%, and finally effecting the reaction at room temperature. The reaction ratio of the enzyme to the antibody is preferably 1:1 by concentration, although it may be 1:2 to 2:1.

The enzyme-labelled antibody is generally employed as a reagent by diluting it with a buffer solution or the like. It is also desirable to add inactivated rabbit serum in an amount of above 20% to the diluted solution. As shown in Figure 4, the addition of the inactivated rabbit serum provides a more preferable working curve than that provided by a buffer solution alone or by the buffer solution containing the bovine serum albumin.

C) The Insolubilized Muscle Aldolase Antibody The carrier employed as in insoluble solid which is combinative with antibody, Illustrative of such solids include, for example, polystyrene, cellulose, agarose, glass, cross-linked dextran, silicone rubber, metal, etc. Suitable forms include a tube, mocrotiter plate, powder, sphere, disc, plate, foil, etc.

In the case of a polystyrene microtiter plate, the antibody may be combined with the wall surface of the plate. 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.

D) The Others As substrate, there can be used enzyme substrates which have been used for the enzymelabelled antibodies. When the enzyme used is alkaliphosphatase, suitable substrates comprise for instance p-nitrophenyl phosphate, p-glycerol phosphate, phenyl phosphate, p-nathphyl phosphate, phenolphthalein phosphate or the like.

As the stop solution for the enzyme reaction, there can be used known stop solutions for use with the respective enzymes. In the case of the alkaliphosphatase, a suitable stop solution is 1 N sodium hydroxide solution.

The above description has been given in terms of reagents for the sandwich method, but according to this invention human muscle aldolase can be also determined by an immunoenzymometric assay. This method comprises reacting the enzyme-labelled antibody with a specimen (antigen), followed by separation of the enzyme-labelled antibody-antigen reagent (B) from unreacted enzyme-labelled antibody (F), and determining the enzyme activity of either said (B) or (F) with the substrate. The method used for the separation of (B) and (F) may be the gel filtration method, the absorption method by means of insolubilized antigen, etc. In determination of the human-muscle aldolase by means of the immunoenzymometric assay, the enzyme-labelled human-muscle aldolase antibody as described above is used as the reagent.

The following experiments and examples will further illustrate this invention.

Experiment 1 Human-Muscle Type Aldolase Anti-Serum The human-muscle type aldolase (1 mg) was dissolved in 0.5 ml of physiological salt water, and to the solution was admixed the equal volume of Freund complete adjuvant. The product was administered by injection to the muscle of a fowl (White Leghorn). The human-muscle type aldolase was administered respectively in the same procedure as described above, two weeks, and also three weeks thereafter. One week after the last administration, the blood was collected from the fowl to obtain the human-muscle type aldolase anti-serum.

The normal-human serum (NHS) inactivated at 560C for 2 hours was added to this anti-serum, so that it may contain the content of 10%. The mixture was incubated at 370C for one hour, and 'allowed to stand overnight at 40C. The product was centrifuged at 3000 rpm for 20 minutes. The supernatant was collected to obtain the humanmuscle type aldolase anti-serum containing absorbed NHS.

Experiment 2 Rabbit-Muscle Type Aldolase Anti-Serum The rabbit-muscle type aldolase (2 mg) was dissolved in 1 ml of physiological salt water.

Thereafter, the same procedure as described in Experiment 1 was repeated to obtain the rabbitmuscle type aldolase anti-serum.

To this anti-serum, was added normal-rabbit serum (NRS) inactivated at 560C for 3 hours, so as to contain the content of 10%. Thereafter, the same procedure as described in Experiment 1 was repeated to obtain the rabbit-muscle type aldolase anti-serum containing absorbed NRS.

Experiment 3 Human-Muscle Type Aldolase Antibody Sepharose 4BTM (Pharmacia Fine Chemicals AB; Agarose) (10 g), and the human-muscle type aldolase (10 mg) were allowed to stand at 40C overnight in 1 5 ml of 0.1 M sodium carbonate buffer solution (pH 9.0).

The resulting Sepharose 4B gel combined with the human-muscle type aldolase was packed into a column, and washed with 0.05 M trishydrochloric acid buffer solution (pH 8.0) containing 0.5 N sodium chloride. To this column were added 3 ml of the NHS-absorbed humanmuscle type aldolase anti-serum obtained in the Experiment 1, and the anti-serum was flowed out with 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 0.5 N sodium chloride.

Thereafter, 0.1 N 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 humanmuscle type aldolase antibody.

Experiment 4 Rabbit-Muscle Type Aldolase Antibody The procedure described in Experiment 3 was repeated except that 3 ml of the NRS-absorbed rabbit-muscle type aldolase anti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the rabbit-muscle type aldolase. The rabbit-muscle type aldolase antiserum (3 mg) was obtained.

Experiment 5 Rabbit-Muscle Type Aldolase Antibody The same procedure as described in Experiment 3 was repeated except that 3 ml of the NRS-absorbed rabbit-muscle type aldolase antiserum described in Experiment 2 were added to the Sepharose 4B gel combined with the humanmuscle type aldolase. The rabbit-muscle type aldolase antibody (3 mg) was obtained.

Experiment 6 Rabbit-Muscle Type Aldolase Antibody The same procedure as described in Experiment 3 was repeated except that 3 ml of the NRS-absorbed rabbit-muscle type aldolase anti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the bovine-muscle type aldolase. There were thus obtained the rabbit-muscle type aldolase antibody (3 mg).

Experiment 7 lnsolubilized Human-Muscle Type Aldolase Antibody Into each of the holes provided on a polystylene microtiter plate, 200 jt41 of 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 25 yI/ml of the human-muscle type aldolase antibody described in Experiment 3 was added, and then allowed to stand overnight at 40C. 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.

Experiment 8 lnsolubillzed Rabbit-Muscle Type A Idolase Antibody Into each of the holes provided on a polystylene microtiter plate, 200 ,ul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 25 yg/ml of the rabbit-muscle type aldolase antibody described in Experiment 4 was added, and then allowed to stand overnight at 40C. The solution was removed from the plate. The plate was washed with distilled water to obtain the microtiter plate combined with the rabbit-muscle type aldolase antibody.

Experiment 9 Insolubilized Rabbit-Muscle Type Aldolase Antibody Into each of the holes provided in a polystylene microtiter, 200 ,ul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 25 yg/ml of the rabbit-muscle type aldolase antibody described in Experiment 5 was added, and then allowed to stand overnight at 40C. The solution was removed from the plate. The plate was washed with-distilled water to obtain the microtiter plate combined with the rabbit-muscle type aldolase antibody.

Experiment 10 Insolubilized Rabbit-Muscle Type Aldolase Antibody Using the rabbit-muscle type aldolase antibody which was obtained in Experiment 6, there was obtained the microtiter plate combined with the rabbit-muscle type aldolase antibody, in a similar manner as described in the preceding Experiment 9.

EXAMPLE 1 Alkaliphosphatase-Labeled Human-Muscle Type Aldolase Antibody Into 0.3 ml of water containing 1 mg of alkaliphosphatase (relative activity 1000 unit/mg), were added 0.2 ml of 0.05 M phosphoric acid buffer solution (pH 7.0) containing 1 mg of the human-muscle type aldolase antibody described in Experiment 3 and 50 yl of 2.5% glutalaldehyde solution. The mixture was allowed to stand for 30 minutes at the room temperature, followed by subjection to dialysis with 0.05 M trishydrochloric acid buffer solution (pH 8.0) overnight. The alkaliphosphatase-labeled humanmuscle type aldolase antibody was obtained.

EXAMPLE 2 Alkaliphosphatase-Labeled Rabbit-Muscle Type Aldolase Antibody The same procedure as described in Example 1 was repeated, except that 1 mg of the rabbit muscle type aldolase antibody described in Experiment 4 was used with 1 mg of alkaliphosphatase. The alkaliphosphatase-labeled rabbit-muscle type aldolase antibody was obtained.

EXAMPLE 3 Alkaliphosphatase-Labeled Rabbit-Muscle Type Aldolase Antibody The same procedure as described in Example 1 was repeated, except that the rabbit-muscle type aldolase antibody described in Experiment 5 was used with 1 mg of alkaliphosphatase. The alkaliphosphatase-labeled rabbit-muscle type aldolase antibody was obtained.

EXAMPLE 4 Alkaliphosphatase-Labeled Rabbit-Muscle Type Aldolase Antibody The same procedure as described in Example 1 was repeated, except that the rabbit-muscle type aldolase antibody described in Experiment 6 was used with 1 mg of alkaliphosphatase. There was thus obtained the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody.

EXAMPLE 5 Enzyme-lmmunoassay A standard antigen solution of 500 mg/ml--7.6 mg/ml was prepared by diluting the human-muscle type aldolase serially two-fold with the normal rabbit serum inactivated at 560C for2 hours. Said standard solution (0.1 ml) was added to the microtiter plate combined with the muscle type aldolase antibody, then stood for 60 minutes at 370C. The reaction solution was removed. The plate was washed 4 times with distilled water. On the other hand, the alkaliphosphatase-labeled muscle type aldolase antibody was diluted to 1 50-fold with 0.05 M trishydrochloric acid buffer solution (pH 8.0) contining 30% of the normal rabbit serum inactivated at 560C for 2 hours. Said diluted solution (0.1 ml) was added to said plate, and then allowed to stand for 60 minutes at 370C. The reaction sdlution was removed.The plate was washed 4 times with distilled water. A solution of 5 mg/ml of p-nitrophenyl phosphate in 0.1 M sodium carbonate buffer solution (pH 9.0) containing 0.001 M magnesium chloride was prepared separately. This solution (0.1 ml) was added to said plate and allowed to react at 370C for 60 minutes, followed by addition of 0.1 ml of 1 N sodium hydroxide to terminate the reaction.

The reaction solution was diluted to 10-fold with distilled water. Absorbance on 405 m,u was measured with a spectrophotometer to set up the working curve between the absorbance and the concentration of the human-muscle type aldolase.

Fig. 1 shows the working curve in the case of the above mentioned assay system, wherein the microtiter plate combined with the human-muscle type aldolase antibody in Experiment 7 was used as a microtiter plate combined with muscle type aldolase antibody, and the alkaliphosphataselabeled human-muscle type aldolase antigen in Example 1 was used as an alkaliphosphataselabeled muscle type aldolase antigen, respectively.

Fig. 2 shows the working curve in the case where the microtiter plate combined with the rabbit-muscle type aldolase antibody in Experiment 8 was used as a microtiter combined with muscle type aldolase antibody, and the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody in Example 2 was used as an alkaliphosphatase-labeled muscle type aldolase antibody, respectively.

Fig. 3 shows the working curve in the case where the microtiter plate combined with the rabbit-muscle type aldolase antibody in Experiment 9 was used as a microtiter plate combined with muscle type aldolase antibody, and the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody in Example 3 was used as an alkaliphosphatase-labeled muscle type aldolase antibody, respectively.

Fig. 4 shows the working curve in the case where the microtiter plate combined with the rabbit-muscle type aldolase antibody in Experiment 10 was used as a microtiter plate combined with muscle type aldolase antibody, and the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody in Example 4 was used as an alkaliphosphatase-labeled muscle type aldolase antibody, respectively.

In Figs. 4, the horizontal axes indicate the concentration (ng/ml) of the human-muscle type aldolase, and the vertical axes indicate the absorbance on 405 mFl(OD405 my) As shown in Figs. 1-4, the most preferable working curve is obtained in the insolubilized antibody and the enzyme-labeled antibody wherein the human-muscle type aldolase antibody (Fig. 1) is used, and followed by the order of the insolubiiized antibody and the enzyme-labeled antibody wherein the rabbit-muscle type aldolase antibody (Figs. 4, 3 and 2) is used.It is noted that, when comparing the cases wherein the rabbitmuscle type aldolase antibody is used, the more preferable working curves are respectively obtained in the case (Fig. 4) wherein the support combined with the bovine-muscle type aldolase is used, as well as well as the case (Fig. 3) wherein the support combined with the human-muscle type aldolase is used, for the purification of the antibody by means of affinity chromatography, than the curve obtained in another case (Fig. 2) wherein the support combined with the rabbitmuscle type aldolase is used.

EXAMPLE 6 Enzyme-lmmunoassay Effects of the type of diluent on the enzymelabeled antibodies were studied by means of the immunoassay system according to Example 5.

There were used the alkaliphosphatase-labeled human-muscle type aldolase antibody described in Example 1 as an enzyme-labeled antibody, and the microtiter plate combined with the humanmuscle type aldolase described in Experiment 7 as an insolubilized antigen.

Following diluents were used in this Example: (A) 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 30% concentration of the normal rabbit serum inactivated at 560C for 2 hours.

(B) 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 1% concentration of the bovine serum albumin inactivated at 560C for 30 minutes.

(C) 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) as control.

The enzyme-labeled antibodies diluted with each of such diluents to 150-fold respectively were used. The working curve was set up by the same procedure as described in Example 5 using the standard antigen solution. Each working curve obtained is shown in Fig. 4. The horizontal axis in Fig. 4 indicates the concentration of the humanmuscle type aldolase (ng/ml) and the vertical axis indicates the absorbance on 405 m (OD405m ) The symbols A, B and C indicate the respective working curves obtained in each case where the diluents (A), (B), and (C) are used respectively. As shown in the drawing, the most preferable working curve is working curve A which is obtained in the case where the diluent containing the inactivated rabbit-serum is used.

EXAMPLE 7 Enzyme-lmmunoassay Determination was achieved for the concentrations of the human-muscle type aldolase in the serums of various cancer patients, of benign ill patients and of healthy humans by means of the immunoassay system as described in Example 5.

The enzyme-labeled antibody used in this Example was the alkaliphosphatase-labeled human-muscle type aldolase antibody described in Example 1, and the insolubilized antibody used was the microtiter plate combined with the human-muscle type aldolase described in Experiment 7. The serum of specimen was directly used without diluting.

Results are shown in Fig. 6. As shown in the drawings, the human-muscle type aldolase tends to be contained in the serum of the cancer patients in a higher concentration than those in the other benign ill patients and those in the healthy human.

Claims

1. A reagent for the determination of humanmuscle aldolase which comprises enzyme-labeled muscle aldolase antibody.

2. A reagent according to claim 1, wherein the enzyme is alkaliphosphatase.

3. The reagent according to claim 1, wherein the muscle aldolase antibody is human muscle aldolase antibody.

4. A reagent according to claim 1, wherein the muscle aldolase antibody is rabbit-muscle aldolase antibody.

5. A reagent according to claim 4, wherein the rabbit-muscle aldolase antibody is one which was purified by means of affinity chromatography using a support supporting human-muscle aldolase.

6. A reagent according to claim 4, wherein the rabbit-muscle aldolase antibody is one which was purified by means of affinity chromatography using a support supporting bovine-muscle aldolase.

7. A reagent according to claim 1, wherein the enzyme-labeled muscle aldolase antibody has been diluted with a buffer solution containing inactivated rabbit serum.

8. A process for the determination of humanmuscle aldolase which comprises the following steps: (a) contacting a specimen with an insolubilized muscle aldolase antibody, followed by separation of the solid phase; (b) contacting the solid phase with enzymelabeled muscle aldqlase antibody, followed by separation of the solid phase; (c) contacting the solid phase with a substrate for the enzyme; and (d) determining the absorbance of a decomposition products of the substrate.

9. A process according to claim 8, in which rabbit-muscle aldolase antibody purified by means of affinity chromatograph using a support supporting bovine-muscle aldolase is employed as the muscle aldolase antibody in the insolubilized muscle aldolase antibody and the enzyme-labeled muscle aldolase antibody.