CA1158549A - Enzyme-labelled muscle type aldolase antibody - Google Patents
Enzyme-labelled muscle type aldolase antibodyInfo
- Publication number
- CA1158549A CA1158549A CA000360973A CA360973A CA1158549A CA 1158549 A CA1158549 A CA 1158549A CA 000360973 A CA000360973 A CA 000360973A CA 360973 A CA360973 A CA 360973A CA 1158549 A CA1158549 A CA 1158549A
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- CA
- Canada
- Prior art keywords
- muscle type
- type aldolase
- antibody
- aldolase
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
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- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
Novel reagents and process for the correct and simple determination of human-muscle type aldolase. The reagent comprises enzyme-labeled muscle type aldolase antibody.
The process comprises the following steps:
(a) contacting a specimen with insolubilized muscle type aldolase, followed by separation of the solid phase;
(b) contacting the solid phase with enzyme-labeled muscle type aldolase antibody, followed by separation of the solid phase;
(c) contacting the solid phase with substrate of enzyme;
and (d) determining the absorbance of the decomposition products of the substrate.
Novel reagents and process for the correct and simple determination of human-muscle type aldolase. The reagent comprises enzyme-labeled muscle type aldolase antibody.
The process comprises the following steps:
(a) contacting a specimen with insolubilized muscle type aldolase, followed by separation of the solid phase;
(b) contacting the solid phase with enzyme-labeled muscle type aldolase antibody, followed by separation of the solid phase;
(c) contacting the solid phase with substrate of enzyme;
and (d) determining the absorbance of the decomposition products of the substrate.
Description
11~8S~9 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.
The aldolase is a general term of the enzymes which catalize the aldol condensation and cleavage between dihydro-xyacetone phosphate and a series of aldehydes.
The 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, distributes mainly in muscle, thereby contri-buting 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 theconstruction 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 electrophoresi~ method, and the other is a method which comprises measuring the decomposition activities of two sub~trata, that is, fructose-1,6-diphosphate (FDP) and fructose-l-phosphate (FIP~ for the aldolase, [FDP-ALD activity ~.
.
, '' ., ,' ' ~' : .
~iSS5~9 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 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, accompanied by no quantitative determination.
Recently, a method by means of radio-immunoassay has been reported. Comparing with the aforementioned methods, this method is more excellent in the sensibility or sensitivity and also in the quantitative matter. However, this method also has some drawbacks that the reagent can not be stored because of the quick decrement of radioisotope to be used for labeling; special machines and apparatuses and a particu-lar room are required for measuring the radioisotope; handling of the radioisotope in the measurement and the disposal of waste matters thereof are difficult, because 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 type aldolase, and a method for the determination of the human-muscle type aldolase using ~aid reagent. According to this method, various drawbacks in the methods of prior art can be dissolved or diminished.
.
1158~ ~g The method according to this invetnion is one of the enzyme-immunoassay, so-called "sandwich method". In this method, measurement is effected 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 muscle type aldolase antibody labeled with enzyme, followed by separation of the solid phase;
(c) contacting the solid phase with the substrate of said enzyme; and (d) measuring absorbance of the decomposition product of said substrate.
The following will illustrate more particularly this method.
In the step (a), an antibody insolubilized by combi-nation 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 re~ct with said 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 ~tep (b),, the solid phase obtained in the step (a) is incubated together with an enzyme-labeled antibody, resulting in that said enzyme-labeled antibody aombines 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.
', ' 11585 ~9 In the step (c), the solid phase obtained in the step (b) is incubated together with a substrate of enzyme to effect the enzyme reaction. After the predetermined time, the reaction is terminated by~adding a stop solution of the enzyme reaction thereto.
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 the absorbance is effected with an absorption photometer using a suitable wave length for quantitative determination of the decomposition product of the substrate.
The absorbance of the known amount of the standard specimen for control is previously measured with said 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 be determined from said working ~-curve by measuring the absorbance of an unknown amount of specimen with the same measuring system.
Figs. 1-4 in the attached drawings, show working curves of the human-muscle type aldolase obtained by the enzyme-immunoassay in the following Example 5.
Fig. 5 shows a working curve of the human-muscle type aldolase obtained by the enzyme-immunoassay in the following Example 6.
Fig. 6 shows the results of determination of the human-muscle type aldolase in the human serums obtained by the enzyme-lmmunoassay in the following Example 7.
The following description illustrates the reagent for determination to the used with the present method.
, ~
g A) The muscle type aldolase antibody Antiserum is obtained by immunizing the muscle type aldolase of a mammal such as human, rabbi~, dog, moneky, bovine, etc. to the other animal.
The immune animal is preferably selected from the different species of birds, because the muscle type aldolases do not show significant immunological distinction between the mammals.
Illustrative of the prefered birds include, for example, fowl, turkey, duck and the like.
It is preferable to add the inactivated serum of the animal which is the source of the muscle type aldolase, to the resulting anti-serum, thereby removing by absorption the other impure antibody. :~
The purification of these antiserum may be effected by means of affinity chromatography using support combined with the mammal-muscle type aldolase, the mammal being selected from human, rabbit, monkey, bovine, etc., for example.
Support materials to be used for~this purpose are agarose, bridged dextran, etc. The combined support can be prepared by activating a support with bromocyane or the like, adding the activated support to the solution of the muscle type aldolase, and stirring the mixed solution at a low temperature.
The purified muscle type aldolase antibody is provided by adding the antiserum to a column filled with said combined Qupport, and effecting the affinity chromatography. Elution of the mu~cle type aldolase antibody from the column may be carried out with a weak alkaline solution.
-` ~1S85~
In this affinity chromatography, the sensitivity varies in the measuring method of the present invention, depending on the combination of a kind of the muscle type aldolase combined with the support and a kind of the muscle type aldolase used for the preparation of the anti~serum.
In the case of the human muscle type aldolase antiserum, a preferable working curve is obtained as shown in Fig. 1 by using the human-muscle type aldolase antibody which is prepared by purifying with the support combined with the human muscle type aldolase. In the cases of the rabbit muscle type aldolase antiserum, more preferable working curves are ob-tained by purifying with the use of the support combined with the human muscle type aldolase and the support combined with the bovine muscle type aldolase, than the support combined with the rabbit muscle type aldolase as shown in Figs. 2, 3 and 4. - --B) The enzyme-labeled muscle type aldolase antibodY
As the enzymes to be used for the labeling, there may be mentioned the enzymes which are generally used for the enzyme-immunoassay, for example,~alkaliphosphatase, peroxidase, ~-D-galactosidase, glucoamylase, glucose oxydase, and the like.
The labeling may be achieved by any conventional methods, such, Por example, as Glutalaldehyde method, Nakane method, Maleimide method, Mixed acid unhydrides method, Carbodiimid method, etc. In Clutalaldehyde method, the labeling is achieved by adding enzyme to the antibody, and adding glutal-aldehyde so that the concentration may reach to 0.2 - 0.8%, and finally effecting the reaction at a 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.
' :' , ' " : ' :
:
, ~ .
:
, llS85'~g The enzyme-labeled antibody is generally used as a reagent by diluting it with a buffer solution and the like.
It is also desirable to add an inactivated rabbit serum in an amount of above 20% to said diluted solution. As shown in Fig. 4, the addition of the inactivated rabbit serum provides more preferable working curve than those provided by a buffer solution alone or by the buffer solution containing the bovine serum albumin.
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, silicone rubber, metal, ~tc. 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.
D) The others As a substrate, there are used substrates of enzymes which have been used for the enzyme-labeled antibody. When the enzyme used is alkaliphosphatàse, there are used as substrate~ p-nitrophenyl phosphate, ~-glycerol phosphate, phenyl phosphate, ~-naphthyl phosphate, phenolphthalein phosphate, or the like.
As the stop solution for the enzyme reactions, there can be used known solutions to the respective enzymes. In the ~i585~1t9 case of the alkaliphosphatase, the suitable stop solution is lN sodium hydroxide solution.
The above descriptions have been made to the sandwich method, but the human muscle type aldolase according to this invention can be also determined by the immunoenzymometrîc assay. This method comprises reacting the enzyme-labeled antibody with a specimen ~antigen), followed by separation of the enzyme-labeled antibody-antigen reactant (B) from unreacted enzyme-labeled antibody (F), and determining the~
enzyme activity of either said (B) or (F) with the substrate.
As the method for the separation of (B) and (F), there may be mentioned the gel filtration method, the absorption method by means of insolubilized antigen, etc. Also in determination of the human-muscle type aldolase by means of the immunoenzy-mometric assay, the enzyme-labeled human-muscle type aldolase antibody as described above is used as the reagent.
Follwoing 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 admini-~tered respectively in the same procedure as described above, two week-~, 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.
.
'~
.
1158S'~9 The normal-human serum (NHS) inactivated at 56C for
OF THE HUMAN-MUSCLE TYPE ALDOLASE
This invention relates to reagents and a method for the determination of the human-muscle type aldolase.
The aldolase is a general term of the enzymes which catalize the aldol condensation and cleavage between dihydro-xyacetone phosphate and a series of aldehydes.
The 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, distributes mainly in muscle, thereby contri-buting 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 theconstruction 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 electrophoresi~ method, and the other is a method which comprises measuring the decomposition activities of two sub~trata, that is, fructose-1,6-diphosphate (FDP) and fructose-l-phosphate (FIP~ for the aldolase, [FDP-ALD activity ~.
.
, '' ., ,' ' ~' : .
~iSS5~9 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 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, accompanied by no quantitative determination.
Recently, a method by means of radio-immunoassay has been reported. Comparing with the aforementioned methods, this method is more excellent in the sensibility or sensitivity and also in the quantitative matter. However, this method also has some drawbacks that the reagent can not be stored because of the quick decrement of radioisotope to be used for labeling; special machines and apparatuses and a particu-lar room are required for measuring the radioisotope; handling of the radioisotope in the measurement and the disposal of waste matters thereof are difficult, because 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 type aldolase, and a method for the determination of the human-muscle type aldolase using ~aid reagent. According to this method, various drawbacks in the methods of prior art can be dissolved or diminished.
.
1158~ ~g The method according to this invetnion is one of the enzyme-immunoassay, so-called "sandwich method". In this method, measurement is effected 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 muscle type aldolase antibody labeled with enzyme, followed by separation of the solid phase;
(c) contacting the solid phase with the substrate of said enzyme; and (d) measuring absorbance of the decomposition product of said substrate.
The following will illustrate more particularly this method.
In the step (a), an antibody insolubilized by combi-nation 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 re~ct with said 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 ~tep (b),, the solid phase obtained in the step (a) is incubated together with an enzyme-labeled antibody, resulting in that said enzyme-labeled antibody aombines 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.
', ' 11585 ~9 In the step (c), the solid phase obtained in the step (b) is incubated together with a substrate of enzyme to effect the enzyme reaction. After the predetermined time, the reaction is terminated by~adding a stop solution of the enzyme reaction thereto.
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 the absorbance is effected with an absorption photometer using a suitable wave length for quantitative determination of the decomposition product of the substrate.
The absorbance of the known amount of the standard specimen for control is previously measured with said 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 be determined from said working ~-curve by measuring the absorbance of an unknown amount of specimen with the same measuring system.
Figs. 1-4 in the attached drawings, show working curves of the human-muscle type aldolase obtained by the enzyme-immunoassay in the following Example 5.
Fig. 5 shows a working curve of the human-muscle type aldolase obtained by the enzyme-immunoassay in the following Example 6.
Fig. 6 shows the results of determination of the human-muscle type aldolase in the human serums obtained by the enzyme-lmmunoassay in the following Example 7.
The following description illustrates the reagent for determination to the used with the present method.
, ~
g A) The muscle type aldolase antibody Antiserum is obtained by immunizing the muscle type aldolase of a mammal such as human, rabbi~, dog, moneky, bovine, etc. to the other animal.
The immune animal is preferably selected from the different species of birds, because the muscle type aldolases do not show significant immunological distinction between the mammals.
Illustrative of the prefered birds include, for example, fowl, turkey, duck and the like.
It is preferable to add the inactivated serum of the animal which is the source of the muscle type aldolase, to the resulting anti-serum, thereby removing by absorption the other impure antibody. :~
The purification of these antiserum may be effected by means of affinity chromatography using support combined with the mammal-muscle type aldolase, the mammal being selected from human, rabbit, monkey, bovine, etc., for example.
Support materials to be used for~this purpose are agarose, bridged dextran, etc. The combined support can be prepared by activating a support with bromocyane or the like, adding the activated support to the solution of the muscle type aldolase, and stirring the mixed solution at a low temperature.
The purified muscle type aldolase antibody is provided by adding the antiserum to a column filled with said combined Qupport, and effecting the affinity chromatography. Elution of the mu~cle type aldolase antibody from the column may be carried out with a weak alkaline solution.
-` ~1S85~
In this affinity chromatography, the sensitivity varies in the measuring method of the present invention, depending on the combination of a kind of the muscle type aldolase combined with the support and a kind of the muscle type aldolase used for the preparation of the anti~serum.
In the case of the human muscle type aldolase antiserum, a preferable working curve is obtained as shown in Fig. 1 by using the human-muscle type aldolase antibody which is prepared by purifying with the support combined with the human muscle type aldolase. In the cases of the rabbit muscle type aldolase antiserum, more preferable working curves are ob-tained by purifying with the use of the support combined with the human muscle type aldolase and the support combined with the bovine muscle type aldolase, than the support combined with the rabbit muscle type aldolase as shown in Figs. 2, 3 and 4. - --B) The enzyme-labeled muscle type aldolase antibodY
As the enzymes to be used for the labeling, there may be mentioned the enzymes which are generally used for the enzyme-immunoassay, for example,~alkaliphosphatase, peroxidase, ~-D-galactosidase, glucoamylase, glucose oxydase, and the like.
The labeling may be achieved by any conventional methods, such, Por example, as Glutalaldehyde method, Nakane method, Maleimide method, Mixed acid unhydrides method, Carbodiimid method, etc. In Clutalaldehyde method, the labeling is achieved by adding enzyme to the antibody, and adding glutal-aldehyde so that the concentration may reach to 0.2 - 0.8%, and finally effecting the reaction at a 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.
' :' , ' " : ' :
:
, ~ .
:
, llS85'~g The enzyme-labeled antibody is generally used as a reagent by diluting it with a buffer solution and the like.
It is also desirable to add an inactivated rabbit serum in an amount of above 20% to said diluted solution. As shown in Fig. 4, the addition of the inactivated rabbit serum provides more preferable working curve than those provided by a buffer solution alone or by the buffer solution containing the bovine serum albumin.
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, silicone rubber, metal, ~tc. 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.
D) The others As a substrate, there are used substrates of enzymes which have been used for the enzyme-labeled antibody. When the enzyme used is alkaliphosphatàse, there are used as substrate~ p-nitrophenyl phosphate, ~-glycerol phosphate, phenyl phosphate, ~-naphthyl phosphate, phenolphthalein phosphate, or the like.
As the stop solution for the enzyme reactions, there can be used known solutions to the respective enzymes. In the ~i585~1t9 case of the alkaliphosphatase, the suitable stop solution is lN sodium hydroxide solution.
The above descriptions have been made to the sandwich method, but the human muscle type aldolase according to this invention can be also determined by the immunoenzymometrîc assay. This method comprises reacting the enzyme-labeled antibody with a specimen ~antigen), followed by separation of the enzyme-labeled antibody-antigen reactant (B) from unreacted enzyme-labeled antibody (F), and determining the~
enzyme activity of either said (B) or (F) with the substrate.
As the method for the separation of (B) and (F), there may be mentioned the gel filtration method, the absorption method by means of insolubilized antigen, etc. Also in determination of the human-muscle type aldolase by means of the immunoenzy-mometric assay, the enzyme-labeled human-muscle type aldolase antibody as described above is used as the reagent.
Follwoing 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 admini-~tered respectively in the same procedure as described above, two week-~, 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.
.
'~
.
1158S'~9 The normal-human serum (NHS) inactivated at 56C for
2 hours was added to this anti-serum, so that it may contain the content of 10%. The mixture was incubated at 37C for one hour, and allowed to stand overnight at 4C. The product was centrifuged at 3000 rpm for 20 minutes. The supernatant was collected to obtain the human-muscel type aldolase anti-serum containing absorbed NHS.
Experiment ?
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 l was-repeated to obtain the rabbit-muscle type aldolase anti-serum.
To this anti-serum, was added normal-rabbit serum (NRS) inactivated at 56C 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 B Human-muscle type aldolase antibody Sepharose 4B- ~(Pharmacia Fine Chemicals AB; Agarose) (lOg), and the human-muscle type aldolase (10 mg) were allowed to stand at 4C overnight in 15 ml of O.lM sodium carbonate buffer solution (pH 9.0).
~'a ~I~a de~n4 r ~
The resulting Sepharose 4B~gel combined with the human-muscle type aldolase was packed into a column, and washed with 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 0.5 N sodium chloride. To this column were added
Experiment ?
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 l was-repeated to obtain the rabbit-muscle type aldolase anti-serum.
To this anti-serum, was added normal-rabbit serum (NRS) inactivated at 56C 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 B Human-muscle type aldolase antibody Sepharose 4B- ~(Pharmacia Fine Chemicals AB; Agarose) (lOg), and the human-muscle type aldolase (10 mg) were allowed to stand at 4C overnight in 15 ml of O.lM sodium carbonate buffer solution (pH 9.0).
~'a ~I~a de~n4 r ~
The resulting Sepharose 4B~gel combined with the human-muscle type aldolase was packed into a column, and washed with 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 0.5 N sodium chloride. To this column were added
3 ml of the NHS-absorbed human-muscle type aldolase anti-serum *3 obtained in the Experiment 1, and the anti-serum was flowed out with 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) 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.o) to obtain 3 mg of the human-muscle 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 ar,ti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the rabbit-muscle type aldolase. The rabbit-muscle type aldolase anti-cerum (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 anti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the human-muscle 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 excep~ 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 Insolubilized human-muscle type aldolase antibody Into each of the holes provided on a polystylene microtiter plate, 200Jul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25Jug/ml of the human-muscle type aldolase antibody described in Experiment 3 was added, and then allowed to stand overnight at 4C. The solu-tion 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 Insolubilized rabbit-muscle type aldolase antibody Into each oP the holes provided on a polystylene microtiter plate, 2OOJU1 of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25~ug/ml of the rabbit-muscle type aldolase antibody described in Experiment 4 was added, and then allowed to stand overnight at 4C. The solution was removed from the plate. The plate was washed with di~tilled water to obtain the microtiter plate combined with the rabbit-muscle type aldolase antibody.
Experiment 9 Insolubilized rabbit-muscle type aldolase antibody Into each oP the holes provided in a polystylene microtiter, 200Jul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25 ~g/ml oP the rabbit-muscle type aldolase antibody described in Experiment 5 was added, i~585 ~9 and 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 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 micro-titer 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 Experi-ment 3 and 50Jul of 2.5% glutalaldehyde solution. The mixture was allowed to stand for 30 minutes at the room temperature, followed by subjection to di~alysis with 0.05 M tris-hydro-chloric acid buffer solution (pH 8.0) overnight. The alkaliphosphatase-labeled human-muscle type aldolase anti-body wa~ obtained.
Example 2 Alkaliphosphatase-labeled rabbit-muscle type aldolase antibody The same procedure as described in Examp`le 1 was re-peated, except that 1 mg of the rabbit-muscle type aldolase ' - :... .
~S8S~
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 re-peated, except that the rabbit-muscle type aldolase antibody described in Experiment 5 was used with 1 mg of alkaliphos- -~
phatase. 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 re-peated, except that the rabbit-muscle type aldolase antibody described in Experiment 6 was used with 1 mg of alkaliphos-phatase. There was thus obtained the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody.
ExamPle 5 Enzyme-immunoassay A standard antigen solution of 500 ng/ml - 7.6 mg/ml was prepared by diluting the human-muscle type aldolase serially two-fold with the normal rabbit serum inactivated at 56C for 2 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 37C. The reaction ~olution was removed. The-plate was washed 4 times with distilled water. On the other hand, the alkaliphosphatase-'. : . .
585~g labeled musele type aldolase antibody was diluted to 150-fold with 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 30% of the normal rabbit serum inactivated at 56C for 2 hours. Said diluted solution (0.1 ml) was added to said plate, and then allowed to stand for 60 minutes at 37C. The reaction solution was removed. The plate was washed 4 times with distilled water. A solution of 5 mg/ml of p-nitrophenyl phosphate in o.i 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 37C for 60 minutes, followed by addition of 0.1 ml of 1 N sodium hydro-xide 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 sy~tem, 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 alkaliphosphatase-labeled human-muscle type aldolase antigen in Example 1 was used as an alkaliphosphatase-labeled muscle type aldolase antigen, res-pectively.
Fig. 2 shows the working curve in the case where the mlcrotlter plate comblned with the rabbit-muscle type aldolase antibody in Experiment 8 was used as~a microtiter combined with muscle type aldolase antibody, and the alkali-. .
: .
~.
, ~
~, ,, 1~58~g -phosphatase-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 lO 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. 1-4, the horizontal axes indicate the con-centration (ng/ml) of the human-muscle type aldolase, and the vertical axes indicate the absorbance on 405 m~u ( OD405 mJU ) 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-mùscle type aldolase antibody (Fig. l) is used, and followed by the order of the insolubilized 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 thè cases wherein the rabbit-muscle type aldolase antibody is used, the more ilS8~
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 rabbit-muscle type aldolase is used.
Example 6 Enzyme-immunoassay Effects of the type of diluent on the enzyme-labeled 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 human-musGle 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.o) containing 30% concentration of the normal rabbit serum inactivated at 56C for 2 hours.
(B) 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 1% concentration of the bovine serum albumin inactivated at 56C for 30 minutes.
(C) 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) as control.
~ i, ~ :, ......... ..
, ~S85~9 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-$olution. Each working curve obtained is shown in Fig. 4. The horizontal axis in Fig. 4 indicates the concentration of the human-muscle type aldolase (ng/ml) and the vertical axis indicates the absorbance on 405 mJu (D40s mu) 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-immunoassay 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 useq 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 ~erum of the cancer patients in a higher concèntration than those in the other benign ill patients and those in the healthy human.
,
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.o) to obtain 3 mg of the human-muscle 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 ar,ti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the rabbit-muscle type aldolase. The rabbit-muscle type aldolase anti-cerum (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 anti-serum described in Experiment 2 were added to the Sepharose 4B gel combined with the human-muscle 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 excep~ 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 Insolubilized human-muscle type aldolase antibody Into each of the holes provided on a polystylene microtiter plate, 200Jul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25Jug/ml of the human-muscle type aldolase antibody described in Experiment 3 was added, and then allowed to stand overnight at 4C. The solu-tion 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 Insolubilized rabbit-muscle type aldolase antibody Into each oP the holes provided on a polystylene microtiter plate, 2OOJU1 of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25~ug/ml of the rabbit-muscle type aldolase antibody described in Experiment 4 was added, and then allowed to stand overnight at 4C. The solution was removed from the plate. The plate was washed with di~tilled water to obtain the microtiter plate combined with the rabbit-muscle type aldolase antibody.
Experiment 9 Insolubilized rabbit-muscle type aldolase antibody Into each oP the holes provided in a polystylene microtiter, 200Jul of 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 25 ~g/ml oP the rabbit-muscle type aldolase antibody described in Experiment 5 was added, i~585 ~9 and 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 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 micro-titer 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 Experi-ment 3 and 50Jul of 2.5% glutalaldehyde solution. The mixture was allowed to stand for 30 minutes at the room temperature, followed by subjection to di~alysis with 0.05 M tris-hydro-chloric acid buffer solution (pH 8.0) overnight. The alkaliphosphatase-labeled human-muscle type aldolase anti-body wa~ obtained.
Example 2 Alkaliphosphatase-labeled rabbit-muscle type aldolase antibody The same procedure as described in Examp`le 1 was re-peated, except that 1 mg of the rabbit-muscle type aldolase ' - :... .
~S8S~
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 re-peated, except that the rabbit-muscle type aldolase antibody described in Experiment 5 was used with 1 mg of alkaliphos- -~
phatase. 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 re-peated, except that the rabbit-muscle type aldolase antibody described in Experiment 6 was used with 1 mg of alkaliphos-phatase. There was thus obtained the alkaliphosphatase-labeled rabbit-muscle type aldolase antibody.
ExamPle 5 Enzyme-immunoassay A standard antigen solution of 500 ng/ml - 7.6 mg/ml was prepared by diluting the human-muscle type aldolase serially two-fold with the normal rabbit serum inactivated at 56C for 2 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 37C. The reaction ~olution was removed. The-plate was washed 4 times with distilled water. On the other hand, the alkaliphosphatase-'. : . .
585~g labeled musele type aldolase antibody was diluted to 150-fold with 0.05 M tris-hydrochloric acid buffer solution (pH 8.0) containing 30% of the normal rabbit serum inactivated at 56C for 2 hours. Said diluted solution (0.1 ml) was added to said plate, and then allowed to stand for 60 minutes at 37C. The reaction solution was removed. The plate was washed 4 times with distilled water. A solution of 5 mg/ml of p-nitrophenyl phosphate in o.i 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 37C for 60 minutes, followed by addition of 0.1 ml of 1 N sodium hydro-xide 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 sy~tem, 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 alkaliphosphatase-labeled human-muscle type aldolase antigen in Example 1 was used as an alkaliphosphatase-labeled muscle type aldolase antigen, res-pectively.
Fig. 2 shows the working curve in the case where the mlcrotlter plate comblned with the rabbit-muscle type aldolase antibody in Experiment 8 was used as~a microtiter combined with muscle type aldolase antibody, and the alkali-. .
: .
~.
, ~
~, ,, 1~58~g -phosphatase-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 lO 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. 1-4, the horizontal axes indicate the con-centration (ng/ml) of the human-muscle type aldolase, and the vertical axes indicate the absorbance on 405 m~u ( OD405 mJU ) 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-mùscle type aldolase antibody (Fig. l) is used, and followed by the order of the insolubilized 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 thè cases wherein the rabbit-muscle type aldolase antibody is used, the more ilS8~
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 rabbit-muscle type aldolase is used.
Example 6 Enzyme-immunoassay Effects of the type of diluent on the enzyme-labeled 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 human-musGle 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.o) containing 30% concentration of the normal rabbit serum inactivated at 56C for 2 hours.
(B) 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) containing 1% concentration of the bovine serum albumin inactivated at 56C for 30 minutes.
(C) 0.05 M tris-hydrochloric acid buffer solution (pH 8.o) as control.
~ i, ~ :, ......... ..
, ~S85~9 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-$olution. Each working curve obtained is shown in Fig. 4. The horizontal axis in Fig. 4 indicates the concentration of the human-muscle type aldolase (ng/ml) and the vertical axis indicates the absorbance on 405 mJu (D40s mu) 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-immunoassay 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 useq 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 ~erum of the cancer patients in a higher concèntration than those in the other benign ill patients and those in the healthy human.
,
Claims (9)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A reagent for the determination of human-muscle type aldolase which comprises an enzyme-labelled antibody capable of binding with human-muscle type adolase already bound to a muscle type aldolase antibody, whereby the human-muscle type aldolase is sandwiched between said enzyme-labelled antibody and said muscle type aldolase antibody.
2. The reagent according to claim 1, wherein said enzyme is alkaliphosphatase.
3. The reagent according to claim 1, wherein said muscle type aldolase is human-muscle type aldolase.
4. The reagent according to claim 1, wherein said muscle type aldolase is rabbit-muscle type aldolase.
5. The reagent according to claim 4, comprising using rabbit-muscle type aldolase antibody which was purified by means of affinity chromatography using a support combined with the human-muscle type aldolase.
6. The reagent according to claim 4, comprising using rabbit-muscle type aldolase antibody which was purified by means of affinity chromatography using a support combined with the vobine muscle type aldolase.
7. The reagent according to claim 1, wherein said enzyme-labeled antibody to be associated to a muscle type aldolase has been diluted with a buffer solution containing in-activated rabbit serum.
8. A process for the determination of human-muscle type aldolase which comprises the following steps of: (a) providing a solid carrier, contacting the carrier with an anti-body to be associated to a muscle type aldolase to deposit the antibody on the carrier, contacting a specimen with the anti-body, followed by separation of the resulting solid phase; (b) contacting the solid phase with enzyme-labeled antibody to be associated to a muscle type aldolase, followed by separation of the resulting solid phase; (c) contacting the solid phase with substrate of enzyme; and (d) determining the absorbance of the decomposition products of the substrate.
9. The process according to claim 8, comprising using the antibody to be associated to a rabbit-muscle type aldolase, which was purified by means of affinity chromatography using a support combined with the bovine muscle type aldolase, as antibody to be associated to muscle type aldolase in a deposit-ed antibody to be associated to muscle type aldolase and an enzyme-labeled antibody to be associated to muscle type .lase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12204779A JPS5648894A (en) | 1979-09-25 | 1979-09-25 | Reagent for measuring human muscular aldolase and its determination |
| JP122,047/79 | 1979-09-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1158549A true CA1158549A (en) | 1983-12-13 |
Family
ID=14826285
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000360973A Expired CA1158549A (en) | 1979-09-25 | 1980-09-24 | Enzyme-labelled muscle type aldolase antibody |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS5648894A (en) |
| BE (1) | BE885376A (en) |
| CA (1) | CA1158549A (en) |
| CH (1) | CH648596A5 (en) |
| DE (1) | DE3036184A1 (en) |
| FR (1) | FR2466020A1 (en) |
| GB (1) | GB2062226B (en) |
| IT (1) | IT1141082B (en) |
| NL (1) | NL8005330A (en) |
| SE (1) | SE447422B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56125667A (en) * | 1980-03-07 | 1981-10-02 | Eisai Co Ltd | Reagent for measurement and measuring method for human muscle type aldolase |
| JPS57208459A (en) * | 1981-06-19 | 1982-12-21 | Eisai Co Ltd | Measuring method using enzyme-labelled antibody and reagent |
| DE3145936A1 (en) * | 1981-11-20 | 1983-06-01 | Behringwerke Ag, 3550 Marburg | Method for the enzyme immunological determination of lipase |
| JP6754116B2 (en) * | 2016-04-26 | 2020-09-09 | 学校法人近畿大学 | Colorectal cancer marker |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2128670B2 (en) * | 1971-06-09 | 1977-06-30 | Merck Patent Gmbh, 6100 Darmstadt | IMMUNOLOGICAL ISOENZYME DETERMINATION METHOD |
| JPS54147097A (en) * | 1978-05-11 | 1979-11-16 | Eisai Co Ltd | Reagent for detecting muscleetype aldolase |
-
1979
- 1979-09-25 JP JP12204779A patent/JPS5648894A/en active Pending
-
1980
- 1980-09-23 CH CH7209/80A patent/CH648596A5/en not_active IP Right Cessation
- 1980-09-24 IT IT24877/80A patent/IT1141082B/en active
- 1980-09-24 NL NL8005330A patent/NL8005330A/en not_active Application Discontinuation
- 1980-09-24 CA CA000360973A patent/CA1158549A/en not_active Expired
- 1980-09-24 GB GB8030730A patent/GB2062226B/en not_active Expired
- 1980-09-24 FR FR8020472A patent/FR2466020A1/en active Granted
- 1980-09-24 SE SE8006667A patent/SE447422B/en not_active IP Right Cessation
- 1980-09-24 BE BE0/202218A patent/BE885376A/en not_active IP Right Cessation
- 1980-09-25 DE DE19803036184 patent/DE3036184A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| FR2466020A1 (en) | 1981-03-27 |
| BE885376A (en) | 1981-03-24 |
| JPS5648894A (en) | 1981-05-02 |
| SE8006667L (en) | 1981-03-26 |
| IT1141082B (en) | 1986-10-01 |
| GB2062226B (en) | 1983-11-30 |
| NL8005330A (en) | 1981-03-27 |
| DE3036184A1 (en) | 1981-04-16 |
| IT8024877A0 (en) | 1980-09-24 |
| SE447422B (en) | 1986-11-10 |
| FR2466020B1 (en) | 1983-07-08 |
| GB2062226A (en) | 1981-05-20 |
| CH648596A5 (en) | 1985-03-29 |
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