CH648596A5 - REAGENT AND METHOD FOR DETERMINING HUMAN MUSCLE TYPE ALDOLASE. - Google Patents

Description

This invention relates to reagents and a method for the determination of human muscle type aldolase.

Aldolase is usually used to refer to enzymes that catalyze aldol condensation between dihydroxyacetone phosphate and a number of aldehydes.

In the description of this invention, the term “aldolase” encompasses fructose diphosphate aldolase (E.C. 4.1.2.13 fructose-1, 6-phosphate-D-glyceraldehyde-3-phosphate-lyase). The enzyme reversibly cleaves fructose-1,6-di-phosphate into dihydraxyacetone phosphate and D-glyceralde-hyd-3-phosphate. The enzyme therefore holds an important position in the glycolysis system; it is specifically active in muscle tissues. It is therefore a very important enzyme in energy metabolism.

The mammalian aldolase is normally divided into three types: the muscle type or type A, the liver type or type B and the brain type or type C. It is known that the concentration ratios of these isoenzymes vary , depending on the metamorphosis of the living body such as the differentiation of the fetal liver in rats or depending on the involvement of the organism with cancer in humans or rats and similar processes.

Two methods for the determination of human muscle type aldolase are known to date. One is the electrophoresis method. The other is a method that determines the decomposition activities of two substrates. The two substrates are: fructose-1,6-diphosphate (FDP) and fructose-l-phosphate (FIP) for aldolase. This determines the FDP / FIP activity ratio.

However, the methods are problematic.

The problem with electrophoresis is that the migrated spots of human aldolase are so close together that it is very difficult to distinguish them and consequently a precise quantitative determination is not possible.

When determining the FDP / FIP activity ratio method, it is very difficult to determine the FIP aldolase activity. This method also does not always lead to quantitative determinations.

A corresponding method using radio-immunoassay has recently been proposed. Compared to the two methods mentioned above, the new method is more sensitive and also leads to improved quantitative results. However, this new method also has some disadvantages in that the reagent used in it cannot be stored. The radioisotopes used for the labeling decompose too quickly. The implementation of the new method also requires special machines and apparatus and, above all, a suitable space for measuring the activities of the radioisotopes. The handling and disposal of waste also pose major problems.

We have now developed a reagent to determine the presence of human muscle type aldolase in an enzyme immunoassay assay. A method for determining said aldolase with said reagent has also been developed. With the new reagent and the corresponding method, various disadvantages of the above methods are eliminated or at least reduced.

The reagent according to the invention for the determination of human muscle type aldolase is characterized in claim 1; the corresponding method for determining the aldolase mentioned in claim 8.

The individual process steps of claim 8 are illustrated in more detail below.

In step a), the antibody, which is immobilized on a support, is incubated together with the sample. The sample can be a serum or similar liquid. The antigen in the sample, i.e. the human muscle-type aldolase can now react with the immobilized antibody mentioned. After the reaction is completed, the reaction solution is removed. The solid phase is washed with a buffer solution, with distilled water or with similar liquids.

In step b), the solid phase from step a) is incubated together with an enzyme-labeled aldolase antibody of the muscle type. The combination of the enzyme-labeled antibody mentioned and the antigen previously bound to the immobilized antibody is obtained. After this reaction is complete, the reaction solution is again removed and the solid phase is washed with a buffer solution, with distilled water or similar liquids.

In step c) the solid phase from b) is incubated together with an enzyme substrate in order to enable the corresponding enzyme reaction. After a certain time, the reaction is terminated by adding a solution which interrupts the above-mentioned enzyme reaction.

Finally, in step d), the absorption of decomposition products from the substrate is measured in the above enzyme reaction. The measurement of the absorption is carried out by means of an absorption photometer which can be set to a suitable wavelength for the quantitative determination of decomposition products of the substrate.

To determine the abovementioned absorption, 5

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forth a calibration curve from samples with known contents.

FIGS. 1 to 4 show such calibration curves for the content of human muscle type aldolase, which was obtained by means of the enzyme immunoassay method according to Example 5 below.

5 shows a corresponding calibration curve for the following example 6.

6 shows results of determinations of human muscle type aldolase in human serum, which were obtained by means of the enzyme immunoassay method according to the following example 7.

The following description now illustrates the reagent according to the invention and its use.

A) Muscle-type aldolase antibodies

Antiserum is obtained as follows: muscle-type aldolase from a mammal, such as human, rabbit, dog, monkey or cattle, is injected into another animal. The animal to be immunized is preferably selected from the various species of birds. The muscle-type aldolases from the various mammals show no significant immunological differences.

Preferred birds include, for example, the domestic chicken, turkey, ducks and the like.

It is advantageous to add the inactivated serum of the animal from which the muscle aldolase is derived to the resulting antiserum, the other impure antibodies being removed by absorption.

The purification of this antisera can be carried out by means of affinity chromatography on supports which are combined with aldolase from mammalian muscles.

Such aldolases preferably originate from human muscles, rabbit muscles, monkey muscles, cattle muscles or the like. The carrier material is usually agarose, cross-linked dextran, etc. The loaded carrier can be prepared by first activating it with cyanogen bromide or similar substances and then adding it to the muscle-type aldolase solution. The solution is then stirred at a low temperature. The purified muscle-type aldolase antibody is obtained by passing the antiserum over a column filled with the above-mentioned carrier. Elution of the muscle-type aldolase antibody from the column can be carried out using a weakly alkaline solution.

In the affinity chromatography described, the sensitivity of the measurement method varies according to the combination between the carrier and the aldolase used. The type of aldolase used to prepare the antiserum also has an impact on the accuracy of the measurement method.

In the case of a human-muscle type aldolase antiserum, a calibration curve as shown in Fig. 1 is obtained. Aldolase antibody of the human muscle type is used, which is cleaned on a carrier which is loaded with aldolase of the human muscle type. In the cases of rabbit muscle-type aldolase antiserum, corresponding calibration curves are obtained if carriers are loaded with human muscle-type, bovine muscle-type and rabbit muscle-type aldolase for cleaning. The corresponding calibration curves are shown in FIGS. 2, 3 and 4.

B) Enzyme-labeled muscle-type aldolase antibody

To label the antibody, those enzymes can be used which are generally used in enzyme,

immunoassay method can be used. Examples of such enzymes are: alkali phosphatase, peroxidase, β-D-galactosidase, glucoamylase, glucose oxidase, and the like.

The marking can be carried out using conventional methods. For example, the glutaraldehyde method, the nakane method, the maleimide method, the mixed method using acids and anhydrides, the carbodiimide method, etc. are known. In the glutaraldehyde method, the label becomes an antibody by adding the enzyme and simultaneous addition of glutaraldehyde. The concentration of the latter compound can be 0.2 to 0.8%. The reaction is then completed at room temperature. The reaction ratios of the enzyme to the antibody are preferably 1: 1, but they can vary in the ratios 1: 2 to 2: 1.

The enzyme-labeled antibody is usually diluted with a buffer solution before being used as a reagent. Other liquids can be used instead of the buffer solution. It is also advantageous to add approximately 20% inactivated rabbit serum to the diluted solution mentioned. As shown in Fig. 4, the addition of inactivated rabbit serum leads to a preferred calibration curve compared to a pure buffer solution of the antibody or with a buffer solution of the antibody which additionally contains bovine serum albumin.

C) Immobilized muscle-type aldolase antibody

An insoluble solid is used as the carrier,

which is loaded with antibody. Illustrative examples of such solids include: polystyrene, cellulose, agarose, glass, cross-linked dextran, silicone rubber, metals, etc. The materials can be in the following forms: tubing, microtiter plates, powders, beads, plates, foils, etc.

In the case of polystyrene microtiter plates, the antibody can be combined with the surface of the plate. For this purpose, the antibody is diluted in a suitable buffer solution and the diluted solution is left on the plate.

D) Other substances

Substrates of those enzymes which were used for labeling the antibodies are used as substrates. If the enzyme is alkali phosphatase, p-nitrophenyl phosphate, β-glycerol phosphate, phenyl phosphate, β-naphthyl phosphate, phenolphthalein phosphate or the like are used as substrates.

The known agents can be used as a solution to interrupt the enzyme reaction. In the case of alkali phosphates, an IN sodium hydroxide solution is a suitable solution for interrupting the enzyme reaction.

The above descriptions have been made regarding the sandwich method. However, the reagent according to the invention for determining human muscle type aldolase can also be determined using immunoenzymetric methods. This method comprises the reaction of enzyme-labeled antibody with a sample representing the antigen, followed by separation of the anti-body antigen reactant B from the unreacted enzyme-labeled antibody (F). The enzyme activity of both (B or F) on the substrate is then determined. The gel filtration method can be used to separate (B) and (F), but the absorption method using immobilized antigen also gives acceptable results. The enzyme-labeled is also used in the determination of human muscle type aldolase by immunoenzymetry

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Human muscle type aldolase antibodies as described above are used as the reagent.

In the following experiments and examples will further illustrate the invention.

Experiment 1 Human muscle type aldolase antiserum

One mg of human muscle type aldolase is dissolved in 0.5 ml of an aqueous physiological saline. The same volume of Freund's additional solution was added to the solution. The product was administered by injection into the muscle of a domestic chicken (White Leghorn). The same injection took place two or three weeks later. One week after the last injection, the animal's blood was collected and the human muscle type aldolase antiserum was obtained therefrom. Normal human serum (NHS), which had been previously inactivated at 56 ° C. for two hours, was added to the antiserum obtained. In the end, the solution contained 10% of the human serum mentioned. The mixture was then incubated at 37 ° C for 1 hour and left at 4 ° C overnight. The product was centrifuged at 3000 rpm for 20 minutes. The clear liquid above was separated and the human muscle-type alcoholase antiserum containing the normal human serum absorbed was obtained therefrom.

Experiment 2 Rabbit muscle type aldolase antiserum

Rabbit muscle type 2 mg aldolase was dissolved in 1 ml of aqueous physiological saline. The procedure was then the same as in Experiment 1. The aldolase antiserum of the rabbit muscle type was obtained in this way. So much normal rabbit serum (NRS) was added to this antiserum that the solution finally contained 10% of it. The above-mentioned normal rabbit serum was previously inactivated at 56 ° C. for 3 hours. Then, the procedure was again the same as in Experiment 1, and the rabbit muscle-type aldolase antiserum containing the NRS was finally obtained.

Experiment 3 Human muscle type aldolase antibodies

10 g of Sepharose 4B ^ from Pharmacia Fine Chemicals AB, consisting of agarose and 10 mg of human muscle type aldolase, were left overnight at 4 ° C. in 15 ml of a 0.1 M sodium carbonate buffer solution at pH 9.

The Sepharose 4B gel thus obtained, which was now loaded with human muscle type aldolase, was packed in a column. The package was washed with a 0.05 M trishydrochloride buffer solution with 0.5 N sodium chloride at pH 8.0. 3 ml of the aldolase antiserum with absorbed NHS of the human muscle type from Experiment 1 were then added to the column. The antiserum was washed out with a 0.05 M trihydrochloride buffer solution with 0.5 N sodium chloride at a pH of 8.0. Then, a 0.1 N aqueous solution of sodium carbinate was added to the column, whereby the human muscle type aldolase antibody was eluted. The fraction with the antibody was subjected to dialysis. The counter solution was the above 0.05 M Tris hydrochloride buffer solution at pH 8.0. Finally, 3 mg of human muscle type aldolase antibodies were obtained.

Experiment 4 Rabbit muscle-type aldolase antibodies

The procedure from Experiment 3 was repeated, except that 3 ml of the aldolase antiserum with absorbed rabbit muscle type NRS from Experiment 2 was added to the column. The Sepharose had previously been loaded with rabbit muscle-type aldolase. 3 mg of rabbit muscle type aldolase antibody were obtained.

Experiment 5 Rabbit muscle-type aldolase antibodies

The same procedure as in Experiment 3 was repeated with the exception that 3 ml of aldolase antiserum with absorbed rabbit muscle type NRS from Experiment 2 was added to the column coated with Sepharose 4B gel. The Sepharose had previously been loaded with human muscle type aldolase. 3 mg of rabbit muscle type aldolase antibody were obtained.

Experiment 6 Rabbit muscle type aldolase antibodies

The procedure from Experiment 3 was repeated, except that 3 ml of aldolase antiserum with absorbed rabbit muscle type NRS from Experiment 2 was added to the column coated with Sepharose 4B gel. The Sepharose 4B had previously been loaded with bovine muscle type aldolase. Finally, 3 mg of rabbit muscle type aldolase antibodies were obtained.

Experiment 7

Immobilized human muscle type aldolase antibody

200 μl of a 0.05 M Tris hydrochloride buffer solution of pH 8.0 were added to each well on a polystyrene microtiter plate. Each portion contained 25 µg / ml human muscle type aldolase antibody from Experiment 3. The solutions were left overnight at 4 ° C. The solutions were then removed from the plate. The plate was washed with distilled water and washed thus obtained a microtiter plate loaded with human muscle type aldolase antibody.

Experiment 8

Immobilized rabbit muscle aldolase antibody

Type

200 μl of a 0.05 M Tris hydrochloride buffer solution at pH 8.0 were added to each well of a polystyrene microtiter plate. Each position contained 25 µg per ml of rabbit muscle-type aldolase antibody from Experiment 4. The plate with the solutions was left at 4 ° C overnight. Then the solutions were removed from the plate. The plate was washed with distilled water to obtain a microtiter plate loaded with rabbit muscle-type aldolase antibody.

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Experiment 9

Immobilized rabbit muscle aldolase antibody

Type

200 μl of 0.05 M Tris hydrochloride buffer solution at pH 8.0 were added to each well of a polystyrene microtiter plate. Each position contained 25 µl / ml of rabbit muscle-type aldolase antibody from Experiment 5. The plate with the solutions was left overnight at 4 ° C. The solutions were then removed. The plate was then washed with distilled water and there was thus obtained a microtiter plate loaded with rabbit muscle type aldolase antibody.

Experiment 10

Immobilized rabbit muscle aldolase antibody

Type

Using the rabbit muscle-type aldolase antibody from Experiment 6, a microtiter plate loaded with rabbit muscle-type aldolase antibody was obtained by doing the same as in Experiment 9 described above.

The following examples illustrate the invention.

example 1

Human muscle type aldolase antibody labeled with alkali phosphatase

To 0.3 ml of water containing 1 mg of alkali phosphatase with a relative activity of 1000 units / mg, 0.2 ml of a 0.05 M phosphoric acid buffer solution of pH 7.0 was added. The buffer solution additionally contained 1 mg aldolase antibody of the human muscle type from experiment 3 and 50 μl of a 2.5% glutaraldehyde solution. The mixture was left at room temperature for 30 minutes. Then it was hydrolyzed overnight against a 0.05 M Tris hydrochloride acid buffer solution of pH 8.0. The alkali phosphatase-labeled aldolase antibody of the human muscle type was thus obtained.

Example 2

Rabbit muscle-type aldolase antibody labeled with alkali phosphatase

The same procedure as in Example 1 was repeated except that 1 mg of rabbit muscle type aldolase antibody from Experiment 4 was used together with 1 mg of alkali phosphatase. The rabbit muscle-type aldolase antibody labeled with alkali phosphatase was obtained.

Example 3

Rabbit muscle-type aldolase antibody labeled with alkali phosphatase

The same procedure as in Example 1 was repeated, except that 1 mg of rabbit muscle-type aldolase antibody from Experiment 5 was used together with 1 mg of alkali phosphatase. The rabbit muscle-type aldolase antibody labeled with alkali phosphatase was obtained.

Example 4

Rabbit muscle-type aldolase antibody labeled with alkali phosphatase

The same procedure as in Example 1 was repeated with the exception that 1 mg of aldolase antibody from

Rabbit muscle type from Experiment 6 was used together with 1 mg of alkali phosphatase. The rabbit muscle-type aldolase antibody labeled with alkali phosphatase was obtained.

Example 5 Enzyme Immunoassay

A standard antigen solution of 500 ng / ml - 7.6 mg / ml was prepared by serial dilution of human muscle type aldolase with normal rabbit serum which had been inactivated at 56 ° C for 2 hours. 0.1 ml of this standard solution was added to the microtiter plate which was loaded with muscle-type aldolase antibody. The solution remained on the plate at 37 ° C for 60 minutes. Then the reaction solution was removed. The plate was washed four times with distilled water. In addition, the muscle-type aldolase antibody labeled with alkali phosphatase was diluted 150-fold. A 0.05 M Tris-hydrochloric acid buffer solution of pH 8.0 with 30% normal rabbit serum which had been inactivated at 56 ° C. for 2 hours was used as the dilution solution. 0.1 ml of this solution was added to the above plate and remained there at 37 ° C for 60 minutes. Then this reaction solution was also removed. The plate was again washed four times with distilled water. A solution of 5 mg / ml of p-nitrophenyl phosphate in 0.1 M sodium carbonate buffer solution at pH 9.0, which additionally contained 0.001 M magnesium chloride, was provided separately. 0.1 ml of this solution was then added to the plate and remained there at 37 ° C for 60 minutes. Then 0.1 ml of a 0.1 N sodium hydroxide solution was added to stop the enzyme reaction. The reaction solution was diluted ten times with distilled water. The absorption of this solution at 405 µm was measured with a spectrophotometer. This allowed the calibration curve, i.e. the relationship between the absorption of this solution and the different concentrations of human muscle type aldolase are established.

1 shows the calibration curve obtained above. The microtiter plate with the human muscle type aldolase antibody from Experiment 7 was used. As the enzyme-labeled aldolase antibody, the alkali phosphatase-labeled aldolase antibody of the Huma muscle type from Example 1 was used.

FIG. 2 shows the corresponding curve in the case of the microtiter plate which was loaded with the rabbit muscle-type aldolase antibody from experiment 8. The enzyme-labeled aldolase antibody of the muscle type used for this purpose was the alkali phosphatase-labeled aldolase antibody of the rabbit muscle type from Example 2.

3 shows the corresponding curve for the case in which the microtiter plate was loaded with the aldolase antibody of the rabbit muscle type from experiment 9. The enzyme-labeled aldolase antibody of the muscle type used here was the aldolase antibody of the rabbit muscle type from Example 3 labeled with alkali metal phosphates.

FIG. 4 shows the calibration curve for the case in which the microtiter plate was combined with rabbit muscle-type aldolase antibody from experiment 10. The enzyme-labeled aldolase antibody of the muscle type was the alkali phosphatase-labeled aldolase antibody of the rabbit muscle type from Example 4.

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1 to 4, the horizontal axis shows the concentration in ng / ml of human muscle type aldolase, and the vertical axis shows the absorption at 405 mji.

As can be seen from Figures 1 to 4, preferred calibration curves are obtained by using aldolase antibodies of the human muscle type. Of the cases with rabbit muscle-type aldolase antibodies (FIGS. 2, 3 and 4), FIG. 4 shows the most unambiguous calibration curve. This is the case where the carrier is loaded with bovine muscle type aldolase. 3, i.e. the case where the wearer is loaded with human muscle type aldolase leads to acceptable results. In the case of Fig. 2, i.e. when loading the carrier with rabbit muscle type aldolase, the relationship between concentration and absorption is not very clear.

Example 6 Enzyme Immunoassay

The effects of the type of diluent on the enzyme-labeled antibodies were examined by the Immunoas-say system according to Example 5.

The enzyme-labeled antibody used was the alkali phosphate-labeled aldolase antibody of the human muscle type from Example 1. As the immobilized antigen, the aldolase of the human muscle type from Experiment 7 was bound on the microtiter plate.

The following diluents were used in this example:

A) 0.05 M Tris hydrochloric acid buffer solution (pH 8.0) with 30% normal rabbit serum which had been inactivated at 56 ° C for 2 hours;

B) 0.05 M Tris hydrochloric acid buffer solution at pH 8.0 with a% bovine serum albumin which had been inactivated at 56 ° C for 30 minutes;

C) 0.05 M Tris hydrochloric acid buffer solution at pH 8.0

as comparison.

The enzyme-labeled antibody was diluted 150-fold with each of the above diluents. Then 5 the corresponding calibration curves were drawn up according to the information from Example 5. All calibration curves obtained are shown in FIG. 5. The horizontal axis shows the concentration of human muscle type aldolase in ng / ml and the vertical axis shows the absorption at 405 io mn. The symbols A, B and C indicate the different diluents. As the A position shows, the preferred calibration curves are obtained with the diluent (A), i.e. in the case of the diluent containing inactivated rabbit serum.

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Example 7 Enzyme Immunoassay:

The concentration of aldolase of the human muscle type was determined in the serum of various cancer patients. As a comparison, the same determination was carried out on patients without cancer and on healthy people. The determination method was the immunoassay system from Example 5.

25 The enzyme-labeled antibody used was the aldolase antibody of the human muscle type from example 1 labeled with alkali phosphatase. The immobilized antibody was the aldolase of the human muscle type from experiment 7 on the microtiter plate. 30 The sera to be examined were used directly, i.e. without dilution.

The results are summarized in FIG. 6. As the figure shows, the concentration of aldolase of the human muscle type is generally greater or at least more dispersed in cancer patients than in those who do not suffer from cancer and in healthy people.

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Claims (7)

648596 2nd PATENT CLAIMS 1. Reagent for the determination of human muscle type aldolase, characterized by a content of enzyme-labeled muscle type aldolase antibody. 2. Reagent according to claim 1, in which said enzyme is alkali phosphatase. 3. Reagent according to claim 1, in which said aldolase antibody is of the human muscle type. 4. Reagent according to claim 1, in which said aldolase antibody is of the rabbit muscle type. 5. Reagent according to claim 1, in which said muscle-type aldolase antibody is present in a buffer solution containing inactivated rabbit serum. 6. A method for determining aldolase of the human-muscle type using a reagent according to claim 1, characterized by the following steps: a) bringing the sample to be examined into contact with an immobilized aldolase antibody of the muscle type and subsequent separation of the solid phase, b) contacting the solid phase from a) with a reagent according to claim 1 with subsequent separation of the solid phase, c) contacting the solid phase from b) with an enzyme substrate and c) determining the absorption of decomposition products from the above substrate. 7. The method according to claim 6, comprising the use of rabbit muscle-type aldolase antibody which has been purified by affinity chromatography on a support loaded with bovine muscle-type aldolase as immobilized muscle-type aldolase antibody Shape.