NL8005330A - REAGENT AND METHOD FOR DETERMINING ALDOLASE OF THE HUMAN MUSCLE TYPE. - Google Patents

Description

1 '* N.O. 29,471

Reagent and method for the determination of aldolase of the human muscle type.

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

Aldolase is a general expression for the enzymes that catalyze the aldol condensation and cleavage between dihydroxyacetone phosphate and a range of aldehydes.

The aldolase of the present invention is intended for a fructose diphosphate aldolase (E.C. 4. 1. 2.

13. fructose-1,6-phosphate D-glycelaldehyde-3-phosphate lyase), which decomposes reversible fructose-1,6-diphosphate into dihydroxyacetone phosphate and D-glycelaldehyde-3-phosphate. This enzyme is involved in the major course of the glycolysis system and in particular mainly spreads in the muscle, thereby contributing significantly to the energy metabolism.

Mammalian aldolase includes three types of isoenzymes, i.e. the muscle type (A type), the liver type (B type) and the brain type (C type). It is known that the composition ratio of these isoenzymes varies according to the meta-morphosis of the living body, such as differentiation of the fetal liver in the rat, cancer formation of the organism in humans, the rat and the like.

Two methods are known for the determination of the human muscle type aldolase, one of which is the electrophoresis method of 23 and the other is a method of measuring the decomposition activity of two substrates, i.e. fructose-1.6- diphosphate (FDP) and fructose-1-phosphate (FIP) for the aldolase, [FDP-ALD activity and FIP-ALD activity], determining the activity ratio (FDP / FIP).

However, these methods pose several problems.

The problem with the electrophoresis method is that the migrated points of the human aldolase isoenzymes are so close together that the distinction is difficult and the precise quantitative determination cannot be achieved.

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In the method of measuring the EDP / PIP activity ratio, it is emphasized that the implementation is particularly complicated for the measurement of the FIP-ALD activity, accompanied by no quantitative determination.

5 Recently, a method has been reported by radioimmunoassay. Compared to the aforementioned methods, this method is better in the sensitivity of the assay and the sensitivity of the instrument, and also in a quantitative sense. However, this method also has some drawbacks in that the reagent cannot be stored due to the rapid decrease in the radioisotope to be used for marking; special machines and devices and a special room are required for the measurement of the radioisotope; handling the radioisotope in measurement 15 and removing waste products therefrom are difficult because the use of the radioisotope is associated with a health hazard.

A reagent has now been developed which can 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 this reagent. According to this method, various disadvantages can be solved or reduced according to the known methods.

The method of the invention is an enzyme immunoassay called "sandwich method". In this method, the measurement is performed by the following steps: (a) a sample is contacted with an insoluble made muscle type aldolase antibody followed by solid phase secretion (b) the solid phase is contacted with an enzyme-labeled muscle type aldolase antibody followed by solid phase secretion; (C) the solid phase is contacted with the substrate of the enzyme and (d) the absorbance of the decomposition product of the enzyme is measured.

More specifically, the following will illustrate this method 40.

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In step (a), an antibody, insolubilized by combination with a carrier, is incubated together with a sample, such as serum, etc., so that the antigen (human muscle type aldolase) in the sample can react with the insolubilized antibody . After the reaction is complete, the reaction solution is removed and the solid phase is washed with a buffer solution, distilled water or the like.

In step (b), the solid phase obtained in step 10 (a) is incubated together with an enzyme-labeled antibody, with the result that the enzyme-labeled antibody combines with the antigen which has previously been combined with the insolubilized antibody. After the reaction is complete, the reaction solution is removed and the solid phase is washed with a buffer solution, distilled water or the like.

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

In step (d), the absorbance of the decomposition product of the substrate in the reaction solution of step (c) is measured. The absorbance is measured with an absorption photometer using an appropriate wavelength for the quantitative determination. of the decomposition product of the substrate.

The absorbance of the known amount of the standard control sample is pre-measured with this measurement system to determine a working curve of the amount of antigen against the absorbance. The amount of antigen in the sample can be determined from the working curve by measuring the absorbance of an unknown amount of sample using the same measuring system.

1-4 of the accompanying drawings show working curves of the human muscle aldolase obtained by the enzyme immunoassay of Example Y.

40 lie. 5 shows a working curve of the 80 05 3 aldolase human muscle type obtained according to the enzyme immunoassay of Example VI.

Fig. 6 shows the results of the determination of the human muscle type aldolase in the human sera obtained by the enzyme immunoassay of Example VII.

The following description illustrates the reagent for the determination to be used in the present method.

A) The antibody of muscle type aldolase

Antiserum is obtained by immunizing the muscle-type aldolase of a mammal such as human, rabbit, dog, monkey, bovine, etc. in the other animal.

The immune animal is preferably selected from different types of birds, because the muscle type aldolase does not show a clear immunological distinction between the mammals.

Preferred examples of birds are fowl, turkey, duck and the like.

It is preferable to add the inactivated serum of the animal, which is the source of the muscle 20 aldolase, to the obtained anti-serum, thereby removing the other impure antibody by absorption.

The purification of this antisera can be performed by affinity chromatography using a carrier combined with the muscle type aldolase of a mammal, the mammal being selected from human, rabbit, monkey, bovine, etc., for example. Carrier materials which can be used for this purpose are agarose, coupled dextran, etc. The combined carrier can be prepared by activating a carrier with cyanogen bromide or the like, adding the activated carrier to the aldolase solution of stir the muscle type and mixed solution at a low temperature. The purified muscle type aldolase antibody is provided 55 by adding the anti-serum to a column filled with this combined support and performing the affinity chromatography. Elution of the muscle type aldolase antibody from the column can be performed with a weakly alkaline solution.

In this affinity chromatography, the sensitivity in the measurement method of the present invention varies depending on the combination of the type of muscle type aldolase combined with the vehicle and the type of muscle type aldolase used to prepare the anti- serum.

In anti-serum of human muscle type aldolase, a preferred working curve is obtained as indicated in Figure 1 using the human muscle type aldolase antibody prepared by purification with the carrier in combination with the aldolase of the human muscle type. In rabbit muscle type aldolase anti-sera, more preferred working curves are obtained by purification using the vehicle combined with the human muscle type aldolase and the vehicle combined with bovine muscle type aldolase than the vehicle combined with rabbit muscle type aldolase as indicated in Figures 2, 3 and 4.

B) The enzyme-labeled aldolase antibody of the muscle type

As enzymes to be used for labeling may be mentioned the enzymes generally used for the enzyme immunoassay, for example, alkali phosphatase, peroxidase, β-D-galactosidase, glucoamylase, glucose-oxidase and the like.

Labeling can be accomplished by any of the usual methods, such as, for example, the glial aldehyde method, the Nakane method, the maleimide method, the mixed acid anhydrides method, the carbodiimide method, etc. In the gint aldehyde method labeling is accomplished by adding enzyme to the antibody and adding glutaraldehyde so that the concentration can reach 0.2-0.8% and finally carrying out the reaction at room temperature. The reaction ratio of the enzyme to the antibody 35 is preferably 1: 1 by concentration, although it may be 1: 2 to 2: 1.

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 8005330 of about 20% to the dilute solution. As indicated in Figure 4, the addition of the inactivated rabbit serum gives a more preferred working curve than that obtained by a buffer solution alone or by the buffer solution containing bovine serum albumin.

C) The insolubilized antibody of the aldolase of the kidney type

Insoluble solids which can be combined with an antibody can be used as a carrier. Hot examples of such solids include, for example, polystyrene, cellulose, agarose, glass, coupled dextran, silicone rubber, metal, etc. Forms may include tube, microtiter plate, powder, sphere, disk, plate, foil, etc.

In the case of the polystyrene microtiter plate, the antibody can combine with the wall surface of the plate, when the antibody is suitably diluted with a buffer solution or the like, after which the diluted solution is added to the plate and finally is kept.

D) The others

Substrates of enzymes used for the enzyme-labeled antibody are used as the substrate. When the enzyme used is alkali phosphatase, as substrates p-nitrophenyl phosphate, β-glycerol phosphate, phenyl phosphate, β-naphthyl phosphate, phenolphthalein phosphate or the like are used.

As the stop solution for the enzyme reactions, known solutions for the respective enzymes can be used. With the alkali phosphatase, a suitable stop solution is a 1 F sodium hydroxide solution.

The foregoing descriptions have been made with respect to the sandwich method, but the human muscle type aldolase of the present invention can be determined by the immunoenzymometric assay. This method involves reaction of the enzyme-labeled antibody with a sample (antigen), followed by separation of the enzyme-labeled antibody-antigen reagent (B) from unreacted enzyme-labeled antibody (F) 40 and determination of enzyme activity of either (B) or 80 05 330 7 (F) with the substrate As methods for the separation of (B) and (E), mention may be made of the gel filtration method, the absorption method by insolubilized antigen, etc. Also at the determination of the human muscle type aldolase by the immunoenzyme metric assay, the enzyme-labeled human trace type aldolase antibody, as described above, is used as a reagent.

The following experiments and examples will further illustrate the present invention.

Experiment 1

Anti-serum of human muscle type aldolase 1 mg Human muscle type aldolase was dissolved in 0.5 ml of physiological saline and to the solution the equal volume of complete additive according to Breund was added with mixing. The product was added by injection to the muscle of a chicken (White Leghorn). The human muscle type aldolase was administered by the same method as described above for two weeks and also three weeks after, respectively. One week after the last administration, blood was collected from the chicken to obtain human muscle type aldolase antiserum.

The normal human serum (HHS) inactivated at 56 ° C for 2 hours was added to this anti-serum so that it can contain 10%. The mixture was incubated at 37 ° C for one hour and stored at 4- ° C overnight. The product was centrifuged at 5000 rpm for 20 minutes. The supernatant was collected to obtain anti-serum of human muscle aldolase containing absorbed HHS. Experiment 2

Anti-serum of rabbit muscle type aldolase 2 mg Rabbit muscle type aldolase was dissolved in 1 ml physiological saline. Then, the same method as described in experiment 1 was repeated to obtain the anti-serum of rabbit muscle type aldolase.

To this anti-serum normal rabbit serum (HES) 40 inactivated at 56 ° C for 3 hours was added, so that 8005330 8 contained 10%. Then, the same method as described in Experiment 1 was repeated to obtain rabbit serum aldolase anti-serum containing absorbed HES.

5 Experiment 5

Antibody of aldolase of the human muscle type

Sepharose 4B - (Pharmacia Fine Chemicals AB; Agarose) (10 g) and 10 mg human muscle type aldolase were stored overnight at 4 ° C in 15 ml of a 0.1 molar sodium carbonate buffer solution having a pH of 9 * 0.

The resulting Sepharose 4-B gel combined with the human muscle type aldolase was filled in a column and washed with a 0.05 molar tris hydrochloric acid buffer solution of pH 8.0 containing 0.5 N sodium chloride. summed up. To this column, 5 ml of human muscle aldolase anti-serum containing NHS absorbed, obtained in Experiment 1, was added and the anti-serum was sputtered with a 0.05 molar tris hydrochloric acid buffer solution with a pi of 8. 0, containing 0.5 N sodium chloride 20. Then, a 0.1 N aqueous sodium carbonate solution was added to the column to elute the human muscle type aldolase antibody. The antibody eluate fraction was subjected to dialysis with a 0.05 molar tris hydrochloric acid buffer solution with a pH of 8.0 to obtain 3 mg of human muscle type aldolase antibody.

Experiment 4

Antibody of aldolase of the koni.ine muscle type

The method described in expeAnent 3 was repeated, except that 3 ml of rabbit muscle type aldolase anti-serum absorbed with NES described in Experiment 2 were added to the Sepharose 4B gel combined with rabbit muscle type aldolase . 3 mg Rabbit serum type aldolase anti-serum were obtained.

Experiment 5

Antibody of aldolase of the koni.ine muscle type

The same procedure as described in Experiment 3 was repeated, except that 3 ml of rabbit aldolase antiserum absorbed with NES, described at ex-80 05 330 9 periment 2, were added to the Sepharose 4B gel combined with aldolase from the human muscle type. 3 mg Rabbit muscle type aldolase antibody were obtained.

5 Experiment 6

Antibody of aldolase of the konilne muscle type

The same method as described in Experiment 3 was repeated, except that 3 ml of rabbit muscle type aldolase antiserum absorbed with NES, described in experiment 10, was added to the Sepharose 4B gel combined with bovine muscle type aldolase. 3 mg rabbit muscle type aldolase antibody was obtained.

Experiment 7 Insolubilized human aldolase antibody of the muscle type

To each of the wells provided on a polystyrene microtiter plate, 200 µl of a 0.05 molar tris hydrochloric acid buffer solution with a pH of 8.0 containing 25 µg / ml of human muscle type aldolase antibody were described in experiment 3 contained, added and then stored at 4 ° C overnight. 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

Solubilized antibody from rabbit muscle type aldolase

To each of the wells provided on a polystyrene 30 microtiter plate, 200 µl of a 0.05 molar tris hydrochloric acid buffer solution with a pH of 8.0 containing 25 µg / ml rabbit muscle type aldolase antibody described in Experiment 4 was added. and then stored at 4 ° C overnight. 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 antibody of aldolase of the ko-40 nine muscle type ft Λ Λ C T T Λ 10

200 µl of a 0.05 molar tris hydrochloric acid buffer solution with a pE of 8.0 containing 25 µg / ml rabbit muscle type aldolase antibody were described to each of the wells provided in a polystyrene microtiter plate as described in experiment 5 »was added and then stored at 4 ° C overnight. 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.

10 Experiment 10

Insolubilized antibody of aldolase of the koinine muscle type

Using rabbit muscle type aldolase antibody obtained in experiment 6, the microtiter plate was combined with rabbit muscle aldolase antibody obtained in a similar manner as described in the previous experiment 9.

Example I

Alkali phosphatase-labeled human aldolase antibody

To 0.5 ml of water, containing 1 mg of alkali phosphatase (relative activity 1000 units / mg), 0.2 ml of a 0.05 molar phosphoric acid buffer solution (pH 7> 0) was added, which contained Ί mg of aldolase antibody of the human muscle types described in experiment 5 contained and added 50 µl of a 2.5 percent glutalaldehyde solution. The mixture was kept at ambient temperature for 50 minutes, followed by dialysis overnight with a 0.05 molar tris hydrochloric acid buffer solution having a pH of 8.0. The human muscle type aldolase antibody labeled with alkali phosphatase was obtained.

Example II

Alkali phosphatase labeled antibody of aldolase of the konin muscle type

The same method as described in Example 1 was repeated except that 1 mg rabbit muscle type aldolase antibody described in Experiment 4 was used 40 with 1 mg alkali phosphatase. The alkaline phosphatase-labeled 80 05 330 11 rabbit muscle type aldolase antibody was obtained.

Example III

Alkali phosphatase-labeled aldolase antibody of 5 betiulin muscle type

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

Example IV

Alkali phosphatase-labeled rabbit aldolase antibody, muscle type 15 The same procedure as described in Example I was repeated, except that the rabbit muscle type aldolase antibody described in Experiment 6 was used with 1 mg of alkali phosphatase. Thus, the alkaline phosphatase-labeled rabbit muscle-type aldolase antibody was obtained.

Example V Enzvm immunoassay

A standard anti-dissolution solution of 500 ng / ml - 7.6 mg / ml was prepared by successive two-fold dilution of the human muscle type aldolase with the normal rabbit serum, which was activated for 2 hours at 56 ° C. Said standard solution (0.1 ml) was added to the microtiter plate combined with the muscle type aldolase antibody and then 60 ml nutritional value and stored at 37 ° 0. The reaction solution was removed. The plate was washed four times with distilled water. On the other hand, the alkali phosphatase-labeled aldolase muscle type antibody was diluted 150-fold with a 0.05 molar tris hydrochloric acid buffer solution with a pH of 8.0 containing 30% normal rabbit serum, inactivated for 2 hours at 56 ° C . This diluted solution (0.1 ml) was added to the plate and then stored at 37 ° 0 for 60 minutes. The reaction solution was removed. The plate was washed four times with distilled water.

40 A solution of 5 mg / ml p-nitrophenylphosphate in a 0.1% carbon monoxide sodium carbonate buffer solution of pH 910 containing 0.001 molar magnesium chloride was prepared separately. This solution (0.1 ml) was added to the plate and reacted at 37 ° C for 60 minutes, followed by addition of 0.1 ml of 1 N sodium hydroxide to complete the reaction. The reaction solution was diluted 10-fold with distilled water. The absorbance at 405 nyu was measured with a spectrophotometer to determine the working curve between the absorbance and the concentration of the human muscle type aldolase.

lie. 1 shows the working curve in the case of the aforementioned assay system, wherein the microtiter plate combined with the human muscle type aldolase antibody was used in Experiment 7 as a microtiter plate combined with the muscle type aldolase antibody and the alkali phosphatase labeled antigen from human muscle type aldolase in Example I, respectively, was used as an alkali-phosphatase labeled antigen from muscle type aldolase.

Fig. 2 shows the working curve in the case where the microtiter plate combined with the rabbit muscle type aldolase antibody was used in experiment 8 as a microtiter combined with muscle type aldolase antibody and the alkaline phosphatase-labeled rabbit muscle type aldolase antibody. for example II, respectively, was used as an alkali phosphatase-labeled muscle aldolase antibody.

FIG. 3 shows the working curve in the case where the microtiter plate combined with the rabbit muscle type aldolase antibody was used in experiment 9 as a microtiter plate combined with muscle type aldolase antibody and the alkaliphos-fatase labeled aldolase antibody. the rabbit muscle type in Example III, respectively, was used as an alkaline phosphatase labeled antibody of aldolase of the muscle type.

Fig. 4 shows the working curve in the case where 40 the microtiter plate combined with the rabbit muscle type 80 05 330 13 aldolase antibody was used in experiment 10 as a microtiter plate combined with muscle type aldolase antibody and the alkaline phosphoryl. fatase labeled rabbit muscle type aldolase antibody in Example IV, respectively, was used as an alkali phosphatase labeled muscle type aldolase antibody.

In Figs. 1-4, the horizontal axes indicate the concentration (ng / ml) of human muscle type aldolase and the vertical axes indicate the absorbance at 405 nyu (° D405 iyp *).

As can be seen from Figs. 1-4, the most preferred working curve is made in the insolubilized antibody and the. enzyme-labeled antibody, using human muscle type aldolase antibody (Fig. 1), and followed by the sequence of the insolubilized antibody and enzyme-labeled antibody, wherein rabbit muscle type aldolase antibody (Fig. 4, 3 and 2) is used. It is noted that, compared to the cases where rabbit muscle type aldolase antibody is used, the more preferred working curves are respectively obtained in the case (Fig. 4), wherein the carrier is combined with aldolase of the bovine muscle type is used, as is the case (Fig. 3) where the carrier is used in combination with human muscle type aldolase, for purification of the antibody by affinity chromatography, other than the curve obtained in a different case (Fig. 2), wherein the carrier is used in combination with rabbit muscle type aldolase,

Example VI Enzyme Immunobile Eel

The influence of the type of diluent on the enzyme-labeled antibodies was studied by the immunoassay system of Example V.

The alkali phosphatase-labeled human muscle type aldolase antibody described in Example 1 was used as an enzyme-labeled antibody 40 and the microtiter plate combined with aldolase of the an n <2. 3 7.0 Human muscle type described "Bio Experiment 7 was used as an insolubilized antigen.

The following diluents were used in this example: 5 (A) a 0.05 molar tris hydrochloric acid buffer solution with a pH of 8.0 containing a concentration of 30% of the normal rabbit serum inactivated at 56 ° C for 2 hours, (B) a 0.05 molar tris hydrochloric acid buffer solution with a pH 10 of 8.0 containing a concentration of 1% of the bovine serum albumin inactivated at 56 ° C for 30 minutes, (0) a 0.05 molar tris hydrochloric acid buffer solution with a pi of 8.0 as a control.

The enzyme-labeled antibodies diluted with each of these solutions to 150 fold were used, respectively. The working curve was set up according to the same method as described in Example V using the standard antigen solution. Each working curve 20 obtained is shown in Fig. 5. The horizontal axis in Figure 5 shows the concentration of aldolase of the human muscle type (ng / ml) and the vertical axis shows the absorbance at 405 µyu (OD ^ q ^ h). The symbols A, B and C indicate the respective working curves obtained in each case, using the diluents (A), (B) and (C) respectively. As indicated in the drawing, the most preferred working curve is the working curve A obtained in the case using the diluent containing the inactivated rabbit serum.

Example VII Etc / m assay test

The assay was accomplished for the concentrations of human muscle type aldolase in sera from various cancer patients, benign patients and healthy humans by the immunoassay system described in Example V.

The enzyme-labeled antibody used in this example was the alkaline phosphatase-labeled human muscle type aldolase antibody described in Example 1, and the insolubilized antibody used was the microtiter plate combined with aldolase of the human muscle type described in experiment 7 · The serum sample was used directly without dilution.

Results are shown in Fig. 6. As indicated in the figures, the human muscle type aldolase tends to be present in the serum of cancer patients at a higher concentration than that in the 10 other benign patients and those in the healthy man.

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Claims (9)

1. Reagent for the determination of human muscle type aldolase, characterized by the presence of enzyme-labeled muscle type aldolase 5 antibody. Reagent according to claim 1, characterized in that the enzyme is alkali phosphatase. Reagent according to claim 1 or 2, characterized in that the aldolase antibody of the muscle type is aldolase antibody of the human muscle type. 4. Reagent according to claim 1 or 2, characterized in that the aldolase antibody of the muscle type is aldolase antibody of the rabbit muscle type. Reagent according to claim 4, characterized in that the rabbit muscle type aldolase antibody is an antibody purified by affinity chromatography using a vehicle combined with the human muscle type aldolase. 6. Reagent according to claim 4, characterized in that the rabbit muscle type aldolase antibody is an antibody purified by affinity chromatography using a carrier combined with bovine muscle type aldolase. Reagent according to claim 1, characterized in that the enzyme-labeled muscle type aldolase antibody is diluted with a buffer solution containing inactivated rabbit serum. 8. Method for the determination of human muscle type aldolase, characterized in that the following steps are used: (a) contacting a sample with an insoluble muscle type aldolase antibody followed by secretion of the solid phase * - (b) contacting the solid phase with enzyme-labeled muscle aldolase antibody followed by secretion of the solid phase; 8005330 17 (c) contacting the solid phase with enzyme substrate and (d) determining the absorbance of the decomposition products of the substrate. 5 9. A method according to claim 8, characterized in that use is made of rabbit muscle type aldolase purified by affinity chromatography using a carrier combined with bovine muscle type aldolase, as muscle type aldolase antibody. in an insolubilized muscle type aldolase antibody and an enzyme-labeled muscle type aldolase antibody. 4th * * * * * * - * 80 05 330