FR2527223A1 - ANTI-TREHALASE ANTIBODIES, REAGENT AND METHOD FOR IMMUNO-ENZYMATIC DETERMINATION OF TREHALASE - Google Patents

Abstract

An anti-trehalase antibody is described which is formed as an antigen against purified trehalase by higher animals and by humans. The anti-trehalase antibody is highly suitable for the accurate and simple immunological determination of trehalase in human urine and serum.

Description

Anti-trehalase antibody, reagent and method for the immunoenzymatic determination of trehalase.

 The present invention relates to a novel anti-trehalase antibody.

 Trehalase is an enzyme that hydrolyzes the disaccharide trehalose into two glucose molecules. So far this enzyme has been found in bacteria, mold, yeast, insects, fish, higher plants and higher animals. Among these bio-organisms, trehalase in higher animals is found particularly in the two tissues of the intestines and the kidneys as well as in the blood, urine and excrement.

 It is supposed that in higher animals, trehalase takes part in the mechanism of glucose transfer as physiological role if one judges it from the various discoveries (Kagaku to Seibutsu, vol. 18, nO 12 p.809).

 Taking into account the above findings, this inventor has discovered that the determination of trehalase in human urine is valid for clinical diagnosis, particularly for the diagnosis of diabetes mellitus.

 Until now, as a method for the determination of human urinary trehalase and serum trehalase, only one method is known for determining this enzyme which consists in hydrolyzing it using a trehalose substrate, and in assaying the glucose released. .

 However, in urine, there is a small amount of trehalase and many substances that interfere in determining the activity of trehalase in urine and serum. Thus, it is essentiei to concentrate the urine and to dialyze it to eliminate the substances with low molecular weight, in order to determine trehalase in a body fluid with the method described previously.

 In particular, the urine is dialyzed for a day or more than a day against an appropriate medium (deionized water or buffered saline, etc.) to remove the substances which interfere with the activity determination process.

 Then, the urine is concentrated with a concentration device (Minicon B - 15; Amicon Co.) sample by sample.

Therefore, the first process is extremely complicated.

Given these considerations, this method is not applicable to clinical diagnostics where it is necessary to take a large number of samples in a short time. Generally, to measure a small amount of compounds in a body fluid, an immunodiffusion method and a complement binding assay method which measure antigen from an immunological point of view are used.

 However, these methods are not good as far as sensitivity is concerned. Thus, the current radioimmunoassay method is applied for the determination of compounds in body fluids. However, the radioimmunoassay method pollutes the environment and requires the use of expensive assay equipment and instruments.

 Therefore, this method is unsatisfactory for use in clinical diagnostics.

 An object of the present invention is to provide a new anti-trehalase antibody.

 Another object of the present invention is to provide an antithalhalase antibody which is used as a reagent for assaying human urinary trehalase and serum trehalase by a rapid and simple immunoenzymatic assay method.

 The present invention is accomplished based on the findings that the enzyme-linked immunosorbent assay method is the best way to detect trehalase with respect to sensitivity, safety, equipment and instruments necessary for the assay, and that an anti-trehalase antibody which is produced by purified trehalase which comes from higher animals in the form of antigen, is a new antibody which is essential for the functioning of the immunoassay.

 This antibody has the following immunological properties. The anti-trehalase antibody reacts by an antigen-antibody reaction with intestinal, renal, urinary trehalase and serum from the same species of animals but not with trehalase from different species of animals.

 The aforementioned anti-trehalase antibody can quickly and simply measure trehalase with an antigen antibody reaction between the trehalase antigen and the antibody. That is to say that the anti-trehalase antibody is an essential substance for measuring trehalase in a body fluid and in a tissue extract by the method of immunodiffusion, immunoelectrophoresis, radioimmunological and immuno - enzymatic etc. and also to measure trehalase in tissues by the immunohistochemical technique.

 This anti-trehalase antibody is produced by immunizing suitable animals with purified trehalase as an antigen. The previous trehalase purification methods are based on gel filtration, ion exchange chromatography, adsorption chromatography, affinity chromatography and preparative gel electrophoresis. The trehalase obtained by these methods is not homogeneous from the electrophoretic point of view nor extremely pure, and the purification methods are then complicated.

 According to the present invention, the purified trehalase from higher animals which is used as an antigen for the anti-trehalase antibody, is obtained by a method which consists in carrying out more than one chromatography, comprising gel filtration, chromatography with ion exchange or adsorption chromatography for body fluids or tissue extracts from higher animals (except human tissue), performing affinity chromatography or chromatoconcentration for the resulting active trehalase fraction and, if necessary , by performing gel filtration> affinity chromatography or chromatoconcentration for the fraction containing trehalase.

Gel filtration can be carried out using resins such as "Sephacryl S-300", "Sephacryl 5-200", "Sephadex G-200", "Sephadex G-150", "Sephadex G-100", "Ultrogel
ACA-34 "," Sepharose 6B ", hSepharose 4B", "Bio-Gel P-300", "Bio-Gel P-200", etc., and perform adsorption chromatography using resins such as hydroxyapatite, alumina gel and calcium phosphate gel etc., and carry out ion exchange chromatography using resins such as DEAE-celulose, CM-cellulose, ECTEORAcellulose, DEAE- "Sephacel", DEAE- "Sephadex", CM - "Sephadex", QAE- "Sephadex" etc., and perform affinity chromatography using resins such as Phenyl- "Sepharose",
Octyl- "Sepharose", Con A- "Sepharose", Tris- "Sepharose", Trehalose-1, Sepharose ", Lectin-" Sepharose ", Epoxy-" Sepharose ", etc. and chromato-concentration using resins such as PBE-94, PBE-118, etc.

A buffer used in the above mentioned chromatography is suitable and for example phosphate buffer, Tris-HCl buffer, histidine buffer are used
HCl etc.

 In addition, there is no difficulty in this buffer containing more than one reagent such as a nonionic detergent (Tween 80, Triton X-100, etc.), sodium chloride, calcium chloride , manganese chloride and ammonium sulfate etc. The aforementioned buffer can be used as well as z-methyl glucopyranoside, trehalase and Poly buffer etc. as an elution buffer.

 The anti-trehalase antibody is obtained by immunization with an emulsified mixture of adjuvants (complete Freund's adjuvant or incomplete Freund's adjuvant, etc.) and an extremely pure trehalase which is obtained by the above-mentioned methods from human urine. , human serum, kidneys of rabbits, intestines of rats, kidneys of pigs, etc., and a suitable animal which is of different species for antigen sources such as rabbit, guinea pig, goat, dog, cow, horse, etc. is selected and immunized by subcutaneous injection and by injection at the interface between the claws and the pads several times at intervals of one week to two weeks. Then 10 to 14 days after the last injection, the animals are bled and the fraction of the serum is obtained.

 After inactivation of the complement in the serum, the anti-trehalase antibody (anti-trehalase IgG) can be obtained by carrying out precipitation with sulphate and chromatography with ion exchange (DEAE-cellulose, DEAE-Sephacel, etc.). or by gel filtration (Sephadex G-200, Sephacryl S-300, etc.).

 We will now describe the drawings in which FIG. 1 and FIG. 2 show the precipitation line of the double immunodiffusion method (Oucbterlony method) of the anti-trehalase antibody of human urinary trehalase and of the anti-trehalase antibody. rabbit kidney trehalase, respectively, against various purified antigens.

 The present invention is illustrated by the following descriptive and nonlimiting examples in the latter, unless otherwise indicated, the percentages are expressed by weight.

PREPARATION EXAMPLE 1
2 liters of human urine are concentrated up to 100 times by ultrafiltration (registered trademark: Diaflo, membrane PM-10 manufactured by Amicon Cà.) And deoxycholate is added to the concentrated liquid until there is 0.5%, and an aliquot is centrifuged at 1000xG for 10 mirrors.

The supernatant liquid thus obtained is placed at the top of a column (2.6 x 90 cm) of a filter gel (registered trademark: Sephacryl S - 300 manufactured by Pharmacia, InC.) Which is balanced with 5 mM of a phosphate buffer containing 0.18 of Tween 80 (registered trademark; manufactured by Sigma Chemicals
Co.) and 0.9% sodium chloride, and is eluted with the same buffer. The fraction containing the trehalase thus obtained is concentrated by ultrafiltration (registered trademark: Diaflo, membrane PM - 10 manufactured by Amicon Co.) to 20 ml, and placed at the top of an affinity chromatography column (2.6 x 10 cm) (registered trademark: Phenyl Sepharose CL - 4B manufactured by Pharmacia, Inc.) which is balanced with 5 mM of buffer
Tris-HCl (pH 7.2) containing 0.8 M ammonium sulfate, and eluted with a TriS4rl 5d buffer (Ph 7.2) containing 0.8 M ammonium sulfate but no detergent up to a buffer
5 mM Tris-HCl (Ph 7.2) containing 0.2% Tween 80 but no ammonium sulphate, which is the final buffer (each volume of initial buffer and the final buffer is 150 ml and the total volume is 300 ml), and a fraction containing trehalase is obtained.

 In addition, another fraction containing the trehalase is eluted with a 5 mM Tris-lEl buffer (pH 7.2) containing 0.2% Tween 80.

Each fraction of these trehalase-containing fractions is placed on a column (0.9 x 20 cm) of chroma toconcentration (registered trademark: PBE-94 manufactured by Pharmacia, Inc.) which is equilibrated with 25 mM of histidine buffer. HCl (pH 6.4) containing 0.1 t of Tween 80 and which is eluted with 300 ml of Poly buffer-llCl (pH 4) (registered trademark: Poly buffer 74 manufactured by Pharmacia, Inc). Then the trehalase fraction active is concentrated and placed at the top of a column (1.6 x 70 cm) of a filter gel (registered trademark: Sephacryl
S - 300 manufactured by Pharmacia., Inc.) which is equilibrated with a 5 mM phosphate buffer containing 0.1 t of Tween 80 and 0.95 of sodium chloride, and eluted with the same buffer which makes it possible to obtain the trehalase purified human urinary.

 The recovery and specific activity of purified human urinary trehalase are shown in Table 1, and the enzymatic properties are shown in Table 2.

 Analytical gel electrophoresis, on 7.5t polyacrylamide gel, is carried out against the resulting purified trehalase, the gels being stained with a protein coloring reagent (blue Coomassie, etc.) and shows a homogeneous protein band. .

The trehalase fraction before chromatoconcentration is not homogeneous but is detected by other protein bands with analytical gel electrophoresis
Table 1 Recovery and specific activity of
purified human urinary trehalase
Purified Brute
Total protein (mg 1400 12
Total activity (unit) 348 248
Specific activity (unit / mg 0.25 21
protein)
Table 2 Enzymatic properties of trehalase
purified from human urine
Isoelectric point pH 4.8
Molecular Weight 45,000
Km value (trehalose) 0t91 mM
Ki value (Tris- (hydroxymethyl) aminomethane) 18 mM
Ki value (mercuric chloride) 8 mM
PREPARATION EXAMPLE 2
A male rabbit (weight 2 to 2.5 kg, white, Japanese) is killed by anesthesia (Nembutal, etc.) and the kidneys are removed and washed thoroughly with physiological saline. The kidney cortex is collected and homogenized with the 10 mM phosphate buffer (pH 7.2) containing 5 mM EDTA, 0.95 sodium chloride and 1 mM magnesium chloride. The homogenized product is centrifuged at 300xG for 10 minutes.

The supernatant liquid thus obtained is centrifuged at 110,000 g for 60 minutes and the precipitate is suspended in the aforementioned phosphate buffer, then papalne is added to the suspension until a final concentration of 0 is obtained. 01t, and the aliquot is incubated at 37 "C for 30 minutes and then centrifuged at 110,000xG for 60 minutes.

The precipitate thus obtained is suspended in the 10 mM phosphate buffer (pH 7.2) then Triton
X-100 (registered trademark manufactured by Sigma Chemicals Co.) and deoxycholate are added to the suspension to have a final concentration of 0.5% of each product, and the suspension is centrifuged at 110,000 G for 60 minutes.

The clear supernatant liquid obtained is placed on the top of a column (2.6 x 30 cm) of ion exchange chromatography (registered trademark: DEAE - Sephacel manufactured by Pharmacia Inc.) which is balanced with a buffer
5 mM Tris - HCl containing 0.2% Triton X - 100 (pH 7.2), and is eluted with a linear gradient of sodium chloride ranging from 0 to 0.5 M in the same buffer. The trehalase-containing fraction is concentrated by ultrafiltration (Diaflo,
PM - 10 Amicon Co.) up to 15 ml and ammonium sulphate is added to have a final concentration of 0.8M.

Then, the enzyme solution is placed on a column (2.6 × 20 cm) of chromatography; zia by affinity (registered trademark: Phenyl
Sepharose CL-4B manufactured by phannacia, Inc.) which is balanced with ulltamlzon Tl'IS-liCl 5 mx! (pH 7.9) containing 0.8M of azmonium sulphate, and eluted with a linear gradient of 0.8M of ammonium sulphate without detergent up to (', 2 of Triton X-100 without sulphate of ammonium in 5 mM Tris - HCl buffer (pH 7.2), and another fraction containing trehalase and eluted with buffer
5 mM Tris-HCl containing 0.25 Triton X-100. The last fraction containing trehalase is placed on top of a column (2.6 x 20 cm) of adsorption chromatography (hydroxyapatite: manufactured by Seikagaku Kogyosha Co. ) which is balanced with a 5 mM Tris-HCl buffer (pH 7.2) containing 0.1% of Triton X - 100 and eluted with a linear gradient of 0 to 0.2 M phosphate buffer containing 0.1% of Triton
X -100.

The first fraction of affinity chromatography (registered trademark: Phényl - Sepharose CL - 4B manufactured by
Pharmacia Inc.) and the active adsorption chromatography fraction (hydroxyapatite: manufactured by Seikagaku Kogyosha
Co.) are placed on the top of an affinity chromatography column (1.6 x 15 cm) (registered trademark: Con A
Sepharose manufactured by Pharmacia, Inc.) which is equilibrated with a Tris F.C1 - 5 ruz buffer containing 0.15 of Triton X-100, 1 mM of calcium chloride, 1 mM of manganese chloride and 0.25 M of sodium chloride, and is eluted with the same buffer, and another fraction containing trehalase is eluted with the same buffer containing 20 mM -methylglucopyranoside.

Each of the fractions containing the trehalase thus obtained (4 fractions) is placed on the top of a column (0.9 x 25 cm) of chromatoconcentration (registered trademark
PBE-94: manufactured by Pharmacia, Inc.) which is equilibrated with 25 mM histidine buffer (PH 6.4) containing 0.1% of
Triton X -100, and eluted with a Poly buffer - HCl (pH 4) (registered trademark: Poly buffer 74 manufactured by Pharmacia Inc.).

Finally the Poly buffer in the purified trehalase solution is purified with a gel filtration column (1.6 x 70 cm) (registered trademark: Sephacryl S - 200 manufactured by Pharmacia Inc.) which is balanced with a 5m Tris buffer -
HCl containing 0.18 of Triton X - 100 and 0.9 t of sodium chloride, and elected with the same buffer which makes it possible to obtain the purified trehalase from the rabbit kidney.

 The recovery and specific activity of purified rabbit renal trehalase and the enzymatic properties of purified trehalase are shown in Table 3 and in Table 4 respectively.

TABLE 3 Recovery and specific activity of trehalase
purified rabbit kidney.

Purified Brute
Total protein (mg) 3,250 4.1
Total activity (unit) 5 390 603
Specific activity (unit / mg of protein) 1.38 142
TABLE 4 Enzymatic properties of renal trehalase from
purified rabbit Enzymatic properties
Molecular weight 180,000 optimum pH pH 5.9
Km value (trehalose) 2.54 mM
Ki value (mercuric chloride) 43.1 M
"(phlorizin) 1.65 mM
(Tris (hydroxymethyl) 8.20 mM
aminomethane)
PREPARATION EXAMPLE 3
1 liter of human urine is precipitated by saturation with ammonium sulphate (saturation at 90 t), and and suspended in the 10 mM phosphate buffer (pH 7.2) in which deoxycholate has been added until a final concentration of 0.5%, the clear supernatant liquid being obtained by centrifugation. The supernatant liquid thus obtained is placed on top of a column (2.6 x 30 cm) of chromatography with ion exchange (registered trademark: DEAE - cellulose manufactured by Bio Rad Laboratories) which is balanced with a 5 mAt phosphate buffer (pH 7.2) containing 0.2 t of Triton X - 100, and eluted with a linear gradient of sodium chloride ranging from 0 to 0, 5 M in the same buffer, the fraction containing the trehalase thus obtained is placed on the top of a column (2.0 x 10 cm) of affinity chromatography (Trehalose - Sepharose) which is balanced with a buffer
Tris 1 (11 -5S1 (pH 7.2) containing 0.1% Triton X - 100 then is eluted with the same buffer containing 0.1 M of Trehalose.

The fraction containing the trehalase is placed on the top of a gel filtration column (2.6 x 70 cm) (registered trademark: Ultrogel ACA - 34 manufactured by Bio-Rad
Laboratories) which is equilibrated with a Tris FCl-5 M1 buffer containing 0.1 t of Triton X - 100 and 0.9% sodium chloride, then eluted with the same buffer.

 The purified human urinary trehalase thus obtained is homogenized by electrophoresis with analytical gel electrophoresis as in Preparation example 1.

EXAMPLE 1
The purified trehalase solution (0.5 mg / ml) prepared from human urine is mixed with a complete Freund's adjuvant at equal volume to obtain an emul
water type or / in oil (W / O) Two milliliters of the emulsion are injected at the interface of the claws and the net pads of the rabbit's feet, each injection being 100 μl and the rest of this emulsion is injected subcutaneously into the backs of rabbits.

 The injection of the emulsion is repeated three times at intervals of two weeks after the first injection.

Ten days after the last injection the rabbits are fasted overnight and then bled from the ear muscle and carotid.

 The blood thus obtained is treated at 370C for 120 minutes and centrifuged at 3000 revolutions / minute for 10 minutes, which gives a clear supernatant liquid (antiserum).

 An equal volume of 0.98 sodium chloride solution is added to the antiserum in which the complement is inactivated by heat treatment at 56 "C for 30 minutes. Then1 after reduction of solubility with ammonium sulfate (0 ~ "33% saturation) the serum is centrifuged at 11,000 rpm for 30 minutes. The precipitate obtained is dissolved in 20 mM phosphate buffer (pH 8) and dialyzed overnight against the same buffer. The ammonium sulphate fraction thus obtained is placed on the top of a column (1.6 x 10 cm) of chromatography with ion exchange (registered trademark: DEAE-cellulose manufactured by Brown Co.) which is balanced with a 20 mM phosphate buffer and eluted with the same buffer.

 The anti-trehalase antibody (anti-trehalase IgG) is obtained in the form of the fraction which has passed through the column of DEAE-cellulose.

 The IgG anti-trehalase thus obtained is found to be homogeneous judging by the precipitation line on the immunoassay, that is to say by the double method of immunodiffusion (Ouchterlony method) compared to the various purified trehalases described below.

EXAMPLE 2
A purified trehalase solution (0.5 mg / ml) from the rabbit kidney is mixed with an equal volume of complete Freund's adjuvant to make an emulsion.

Two ml of the emulsion are injected at a rate of 0.2 ml by subcutaneous injection into each of the guinea pigs.

 The above-mentioned emulsion is injected another ten days after the first injection. Ten days after the injection the guinea pigs are bled from the ventral aorta. The blood is treated according to the same method as in Example 1 until the stage where the ammonium sulfate fraction is obtained.

The ammonium sulfate fraction thus obtained is placed on the top of a gel filtration column (2.6 x 90 cm) (registered trademark: Sephacryl S - 300 manufactured by Pharmacia, Inc.) which has been balanced with a 10 mM phosphate buffer (pH 7.2) containing 0.9 t of sodium chloride and eluted with the same buffer, which gives the anti-trehalase antibody (anti-trehalase IgG).

 The anti-trehalase antibody thus obtained is recognized as being homogeneous with the double immunodiffusion method (Ouchterlony method).

 The method of Ouchterlony is applied to the anti-trehalase antibody of human urinary trehalase, which was obtained by the method described in Example 1, a purified human urinary trehalase which is a starting material for an antibody, a trehalase purified rabbit urine and purified rabbit kidney trehalase to obtain a precipitation line respectively and the results obtained are shown in Figure 1.

 The Ouchterlony method is performed by digging holes in a 1% agar gel plate as shown in the figure, then the antigen and antibody are poured into these holes and kept at 40C for 48 hours in a chamber wet. During this incubation both the antigen and the antibody which are proteins diffuse in the agar and react with each other in a suitable place.

If the antigen and the antibody react with each other, (if the antigen and the antibody engage the same antigenic power, they will react with each other) the antigen-antibody complex (immune complex) will appear as a line of white precipitation on the agar plate.

 Consequently, the appearance of precipitation indicates the production of an antigen-antibody complex.

 In particular, as shown in FIG. 1, the anti-trehalase antibody of human urinary trehalase, produces the precipitation line vis-à-vis purified human urinary trehalase, purified human renal trehalase but not vis-à-vis -vis of purified rabbit urinary trehalase and purified rabbit renal trehalase. This indicates that human urinary trehalase has the same antigenicity as human renal trehalase, but has different antigenicity to trehalase from rabbit urine and rabbit kidney.

 FIG. 2 shows the characterization of the rabbit renal trehalase antithalhalase, which was carried out as in FIG. 1.

 In particular, FIG. 2 shows that the rabbit renal trehalase antibody has the same antigenic power as rabbit urinary trehalase and as rabbit renal trehalase, but it has a different antigenic capacity vis-à-vis human urinary trehalase .

 The embodiments of the present invention in which the exclusive properties or advantages are characterized in the appended claims.

Claims (7)

 1. Anti-trehalase antibody which is produced against trehalase-purified from higher animals as an antigen.  2. anti-trehalase antibody according to claim 1, characterized in that an anti-trehalase antibody produced against the purified superior animaix trehalase is prepared by a process which consists in placing fluids of the body or tissue extracts - (except human tissue) from superior animals on at least one gei filtration, adsorption chromatography and ion exchange chromatography column, then place the fraction containing the trehalase obtained on an affinity chromatography and / or chromatoconcentration column, and then, if necessary> placing the fraction containing the trehalase thus obtained on at least one gel filtration column, adsorption chromatography, ion exchange chromatography and affinity chromatography.  3. Anti-trehalase antibody according to claims 1 or 2, characterized in that the superior animal is human.  4. Reagent for the determination of human urinary trehalase or serum trehalase, characterized in that the anti-trehalase antibody as defined in any one of claims 1 to 3 is used.  5. A method of immuno-enzymatic determination of trehalase, characterized in that the anti-trehalase antibody as defined in any one of claims 1 to 3 is used.  6. Method for measuring trehalase in the body fluid, in a tissue extract by the methods of immunodiffusion, immunoelectrophoresis, radiooimmunological, immunotenzymatic, characterized by the fact that the anti-trehalase antibody is used as defined in any one of claims 1 to 3.  7. A method of measuring trehalase in tissues by the immunohistochemical technique, characterized in that the anti-trehalase antibody as defined in any one of claims 1 to 3 is used.