FR2596867A1 - Immunological method of assaying erythropoietin - Google Patents

Abstract

The improved method of assaying erythropoietin according to thei invention consists in reacting a first antibody, specifically reactive with erythropoietin, which is fixed on an insoluble support, with a test sample containing erythropoietin and a second antibody which is specifically reactive with erythropoietin and which is prepared by immunising an animal of a species different from the animal used for preparing the first antibody; in reacting with a defined amount of a third labelled antibody which is specifically reactive with the immunoglobulin of the animal species used for preparing the second antibody; and in then determining the amount of labelled antibody bound to the insoluble support carrying the antibody, or the amount of unbound labelled antibody. Applications: diagnosis of various blood diseases such as anaemia and erythrocytosis, sensitive at 10-20 mU/ml.

Description

 The present invention relates to an immunological method for assaying erythropoietin, more particularly an improved method for the immunological assay of erythropoietin (hereinafter sometimes called "EPO") by a technique known as "sandwich" using a first, a second and third antibody.

EPO is mainly produced in the kidneys and is a hormone with a molecular weight of around 40,000 which stimulates the formation of erythrocytes in the body. It is known that the amount of EPO increases or decreases in body fluids in the case of various anemias and erythrocytoses, and therefore it is very important to measure accurately
The amount of EPO in the body's fluids for the diagnosis of these blood diseases.

 Various methods are known for determining EPO, for example a biological assay technique using small animals (eg mouse or rat) Ccf. "Rinsho Kensa" (Clinicat Test), vol. 22, no. 2, 1978), a technique for measuring the absorption of 59Fe in mouse bone marrow cells (cf. J. Lab. CLin. Med., Vol. 97, no. 2, p. 158-169, 1981) and a technique using mouse fetus cells (cf.

Acta Haematal JAPAN, vol. 44, no. 6, 1981). The biological assay method is the most reliable, but it has an unfavorable lower sensitivity in detection and requires a long time for the measurement and furthermore it is expensive because it uses large amount of animals.

 Recently, an immunological method has been developed, in particular an enzyme immunoassay for the determination of substances which are present in very small amounts in body fluids. However, the enzyme immunoassay is less sensitive for the detection of substances, compared to the radioimmunoassay and, therefore, its applications are limited. In particular, EPO is contained in very small amounts in normal blood, for example 10 to 20 mU / ml (0.1 to 0.3 ng / ml) and, therefore, it has hitherto been considered very difficult to determine by known enzyme immunoassays.

 The Applicant has studied an improved immunoassay for the determination of EPO and found an improved immunoassay for the determination of EPO which consists in reacting an antibody specifically reactive with EPO, fixed on an insoluble support, a sample for testing and an antibody specifically reactive with EPO, labeled with an enzyme, thus forming an antibody-EP0-antibody complex fixed on the insoluble support and then measuring the quantity of the labeled product fixed on the insoluble support (see request for Japanese Patent No. 39687/1980) .However, in this method, the antibody specifically reactive with EPO has a lower titer and therefore may not necessarily be suitable for precise measurement of the very small amount of EPO in the normal blood sample. The low titer of the EPO reactive antibody is considered to be due to the rapid metabolism of EPO in the case of immunization of animals with other than humans with EPO and furthermore, the fact that the animals to which EPO is administered physiologically react with EPO and therefore severe anemia can be induced.

The Applicant has further studied an even improved method of high sensitivity for the immunoassay of
EPO, which can be implemented in a very short time and at low cost in a usual test room and has found that the desired assay can be carried out by a so-called "sandwich" method using a third antibody labeled with an enzyme, which is specifically reactive with Immunoglobulin in animals used for the preparation of the second antibody.

 The object of the invention is to propose an improved method for the immunological assay of erythropofetin. The object of the invention is also to propose an immunoenzymatic assay by a "sandwich" technique for the determination of erythropoietin in body fluids. The object of the invention is further to propose an immunological assay for erythropoietin suitable for the diagnosis of various blood diseases such as anemia and erythrocytosis. These objects and advantages of the invention and others will become clear to the user. 'skilled person reading the following description with reference to the single figure of the accompanying drawing which represents a standard calibration curve of the EPO used according to the invention.

The immunoassay method for erythropoietin according to the invention consists in reacting a first antibody specifically reactive with erythropoietin, which is fixed on an insoluble support, with a test sample containing erythropoietin and a second antibody which is specifically reactive with erythropoietin and which is prepared by immunization of an animal species different from the animal used for the preparation of the first antibody; reacting with a determined amount of a third labeled antibody which is specifically reactive with the animal species immunoglobulin which is used for the preparation of the second antibody; and then to be determined
The amount of the labeled antibody attached to the insoluble support carrying
The antibody or amount of labeled antibody that is not attached.

 According to the method of the invention, even if the second antibody specifically reactive with EPO has a lower titer, the titer can be increased by using the third labeled antibody which is specifically reactive with the immunoglobulin of the species of animal used for the preparation of the second antibody, which allows an efficient immunological determination of EPO.

The third antibody can be easily prepared by immunization of an animal species, other than the animal species used for the preparation of the second antibody, with the immunoglobulin of this latter animal species, and the third antibody thus obtained has a much higher titer than that of the second antibody.

The first and second antibodies used according to
The invention are prepared by immunization of different animal species, other than humans, for example rabbit, cow, goat, sheep, guinea pig, etc., with a purified EPO which is obtained from L urine from patients with panmyelophtisia (aplastic anemia) who produce a relatively large amount of EPO. Either of the first and second antibodies may be a monoclonal antibody. In a preferred embodiment of the invention, the first antibody is an antibody prepared by immunization of an animal species such as Le rabbit, goat or cow with purified EPO, the second antibody is a monoclonal antibody from the mouse and the third antibody is an antibody prepared by immunization of an animal other than the mouse with a mouse immunoglobulin.

 The purification of EPO can be carried out by a combination of known purification techniques such as affinity chromatography, gel filtration, etc. In order to obtain an antibody with high specificity, EPO is purified to as high a level as possible. The purified EPO is administered subcutaneously to animals such as rabbits and cows and is thus immunized to prepare the first and second antibodies. The third antibody, specifically reactive with the immunoglobulin of the animal species used for the preparation of the second antibody, is prepared by immunization of an animal other than the animal used for the preparation of the second antibody with an immunoglobulin purified from this. last animal.

The monoclonal antibody specifically reactive with EPO is prepared in a known manner, for example by immunization of a BALB / C mouse with purified EPO, isolation of the spleen cells of the immunized mouse, fusion of the cells with myeloma cells using polyethylene glycol and repeated cloning of the fused cells in the HAT medium.

 The first antibody as prepared above is fixed on an insoluble support. The insoluble support consists of a polystyrene tube, polystyrene beads, silicone fragments, microplates, etc., preferably a polystyrene tube or polystyrene beads. The binding of the first antibody to the insoluble support is carried out by combination of known chemical or physical techniques. For example, the physical method consists in dissolving the first antibody in an appropriate buffer solution, adding the insoluble support to it, allow the mixture to stand between OOC and room temperature for several hours overnight, preferably at 4-20 "C for 4-16 h, to wash the antibody attached to the resulting insoluble support with physiological saline, etc. to give the antibody attached to the desired insoluble support (hereinafter referred to simply as "antibody fixed to support"). The antibody fixed to support can be stored stably for at least one year by adding a stabilizer such as serum - beef albumin.

 The third antibody labeled with a labeling substance (hereinafter referred to simply as "labeled antibody") is prepared in the following manner.

 The third antibody is an antibody against the immunoglobulin of an animal species used for the preparation of the second antibody specifically reactive with EPO. For example, when the second antibody is a monoclonal antibody from the mouse, the third antibody is an antibody against mouse immunoglobulin and, when the second antibody is an antibody prepared using a rabbit, the third antibody is an antibody against rabbit immunoglobulin. The labeling substances used in this invention include enzymes, isotopes, fluorescent substances, metallic substances, etc. Preferred labeling substances are enzymes such as peroxidase, alkaline phosphatase, B-galactosidase, glucose oxidase, glucose-6 acid -phosphoric dehydrogenase, glucosed dehydrogenase, etc., among which particularly preferred are peroxidase, glucose oxidase and glucose-6-phosphoric acid dehydrogenase. The labeling of the third antibody by the enzymes can be carried out by known techniques, for example by oxidation of the sugar chain of the enzyme by periodic acid and fixation of the aldehyde group produced on the amino group of the third antibody, or by introduction of a maleimido group into the enzyme and then fixation of the group with the thiol group of the third antibody.

Using the antibody attached to a support and
The labeled antibody prepared above, the determination of EPO can be carried out as follows.

 First, the antibody fixed on a support is reacted with a test sample containing EPO and the second antibody specifically reactive with EPO at 0-500C for one hour to one night, preferably at 15 -37 "C for 2-4 h.

The test sample containing EPO consists of serum, blood plasma, urine, etc. The first antibody to be fixed on an insoluble support and the second antibody must be prepared using different animal species from each other. If the first antibody and the second antibody are prepared using animals of the same species, the third labeled antibody, against an immunoglobulin of the previous animal, will bind in a non-specific manner regardless of the amount of EPO present in the sample therefore it is impossible to measure the amount of EPO.

 By the above reaction, the supported antibody is bound with the second antibody in proportion to the amount of EPO. The third labeled antibody is reacted with the resulting reaction product, at 0-500 ° C. for one hour to one night, preferably at 15-37 ° C. for 2-4 h, the third labeled antibody being fixed on the support carrying the antibody proportional to the amount of EPO.

 Finally, the quantity of the labeled antibody fixed on the support carrying the antibody is measured and the quantity of EPO contained in the test sample is calculated from the calibration curve which was previously plotted using a standard sample of EPO. When the labeling substance is an enzyme, the enzyme activity is measured and when the labeling substance is an isotope, the radioactivity is counted.

 According to the present invention, the titer of the antibody is increased by the third antibody and therefore it can be used for the assay of EPO with high sensitivity even in an amount as low as 10 to 20 mU / ml (0 , 1 to 0.3 ng / ml) in normal blood to which known techniques could not be applied. Thus, the method according to the invention is useful for the determination of erythropoietin in body fluids and is therefore suitable for the diagnosis of various blood diseases such as anemia and erythrocytosis, in which the level of erythropoietin is unusually increased or decreased.

 The following examples illustrate the invention without, however, limiting its scope.

Example 1 (a) Preparation of the antibody fixed on a support
Preparation of the polyclonal antibody: The polyclonal antibody against erythropoSetin is prepared by immunizing a rabbit three times with purified EPO (from a patient suffering from anemia) in the following manner and the ant is treated; - resulting serum in the usual way to prepare the purified IgG.

First immunization: 400 μg / ml of purified EPO are dissolved in a phosphate-buffered saline solution (STP) and the solution is mixed with the same amount of adjuvant.
Freund to give an emulsion. 1.5 ml of the emulsion is injected subcutaneously into a rabbit weighing approximately 2 kg at 10 to 15 points along the spine.

 Second immunization: Two weeks after the first immunization, 1 ml of the same emulsion as above is injected at 10 to 15 points in the same manner as in the first immunization.

 Third immunization: Another two weeks later, an emulsion is prepared using a solution at? 00 pg / ml of an antibody in the STP in the same manner as described in the first immunization and 1 ml of the 10-point emulsion.

 The day before the third immunization, a small amount of blood is collected from the animal and the anti-EPO activity of the serum is determined. One week after the third immunization (final immunization), the blood collection is completed and the collected blood is subjected to the separation of the serum to give an anti-EPO serum. An anti-EPO IgG is prepared from the anti-EPO serum by a known technique, that is to say by subjecting it to precipitation with ammonium sulphate at 50 X and to fractionation by chromatography on DEAE-cellulose. , by rejecting substances not adsorbed on DEAE-cellulose.

 Then add a solution of 20 ml of the first antibody in 0.1 M NaHCO 3 to 100 polystyrene beads of 6.4 mm in diameter and the mixture is left to stand at 4 "C for 16 h and washed with saline physiological to give an antibody attached to polystyrene beads.

(b) Preparation of the anti-IaG of mouse labeled with peroxidase
To 0.2 mg of peroxidase (manufactured by Toyo Boseki KK), 0.2 ml of Nais, 0.06 M is added and the mixture is reacted at room temperature for 20 min, then it is dialyzed against an acetate buffer sodium (pH 4) overnight. After dialysis, the mixture is adjusted to pH 9.5 and 5 mg of rabbit anti-mouse IgG antibody (made by DAKO CoD) is added thereto and the mixture is reacted at room temperature for 2 h. the reaction mixture with sodium borohydride. The entire reaction mixture is subjected to gel filtration.
Sephadex G-20d and the active fractions are collected to give the mouse anti-IgG labeled with peroxidase.

(c) Determination of EPO
The second antibody used is a mouse anti-EPO monoclonal antibody (as described in the first publication of Japanese patent No. 155395/1984).

 Antibody attached to a polystyrene bead prepared in (a) above, 50 µl of a test sample containing EPO and 200 µl of 0.01 M phosphate buffer are added to a polystyrene tube (12 x 75 mm) and the mixture is reacted at 37 "C for 2 h and washed with physiological saline solution.

250 μl of the anti-EPO monoclonal antibody diluted 1000 times are added thereto and the mixture is reacted at 37 ° C. for 2 h, then washed with physiological saline solution.

 Separately, the mouse anti-IgG labeled with peroxidase prepared in (b) above is diluted 700 times in a 0.5% serum albumin buffer from beef. 250 μl of the diluted labeled antibody thus obtained are added to the above polystyrene tube and the mixture is reacted at 37 ° C. for 2 h and then washed with physiological saline solution.

500 μl of a citrate buffer containing 3 mgiml of o-phenylenediamine and 0.02% of H 2 O 2 are then added and the mixture is reacted at room temperature for 1 h. The reaction is stopped by adding 2 ml of 1N H2SO4. The absorbance of the reaction mixture is measured at 492 nm.

 The resulting standard EPO calibration curve is shown in the attached figure. The EPO detection sensitivity of 10 mU / ml is calculated from the figure.

Example 2 (a) Preparation of the anticorns fixed on a support
To 100 polystyrene beads 6.4 mm in diameter, 20 ml of a solution of an anti mouse monoclonal antibody are added.
EPO (described in the 1st publication of Japanese patent 155395/1984) in 0.1 M NaHC03 and the mixture is left to stand at 4 "C for 16 h and then washed with physiological saline to give the antibody fixed on beads of desired polystyrene.

(b) PreDaration of the peroxidase-labeled laDin anti-laG
Example 1 (b) is repeated, except that a sheep anti-rabbit IgG antibody made by Cappel Co. is used instead of the mouse anti-IgG (manufactured by DAKO CoD to prepare the anti - Rabbit IgG labeled with the desired peroxidase.

(c) Dosaae of EPo
The second antibody used is a rabbit antibody prepared as in Example 1 (a).

 The antibody fixed on a polystyrene bead prepared as in (a) above, 50 μl of a test sample containing EPO and 200 μl of 0.01 M phosphate buffer are added to a tube of polystyrene (12 x 75 mm) and the mixture is reacted at 370C for 2 h and washed with physiological saline solution. 250 μl of the second antibody (diluted 500 times with 0.01 M phosphate buffer) are added thereto and the mixture is reacted at 370C for 2 h and then washed with physiological saline solution. The peroxidase-labeled rabbit anti-IgG prepared in (b) above is diluted 500 times with a perphosphate buffer containing 0.5% of bovine serum albumin. 250 μl of the diluted labeled antibody thus obtained are added to the above polystyrene tube and the mixture is reacted at 370C for 2 h and then washed with physiological saline solution. 500 μl of citrate buffer containing 3 mg / ml of o-phenylenediamine and 0.02% of H 2 O 2 are then added and the mixture is reacted at room temperature for 1 h. The reaction is stopped by adding 2 ml of H2SO4 IN. The absorbance of the reaction mixture is measured at 492 nm. EPO can be detected by this technique at a content of 25 mU / ml.

Example 3 (a) Preparation of anti-IaG of mice stained with qlucoseoxvdase
10 mg glucose oxidase (manufactured by Toyo Boseki
KK), 2 mg of N-C4-succinimidoxycarbonylcyclohexyl) -maleimide are added in portions at 5 min interval and the reaction mixture is subjected to gel filtration using "Sephadex G-25" (registered trademark ) and the enzyme fractions are collected and then concentrated. 10 mg of an IgG fraction obtained by reduction of rabbit anti-mouse IgG antibody produced by DAKO Co. are added thereto and the mixture is reacted at 4 ° C. overnight. reaction to gel filtration of "Sephadex G-200" (registered trademark) and the active fractions are collected to give the mouse anti-IgG labeled with glucose oxidase.

(b) EPO Dose
The antibody fixed on a polystyrene bead prepared in Example 1 (a), 50 μl of a test sample containing EPO and 200 μl of 0.01 M phosphate buffer are added to a tube of polystyrene (12 x 75 mm) and the mixture is reacted at 37 "C for 2 h and washed with physiological saline solution.

As a second antibody, 250 μl of an anti-EPO monoclonal antibody (described in the first publication of Japanese patent 155395/1984) diluted 1000 times in 0.01 M phosphate buffer is added thereto and the mixture is reacted at 37 "C for 2 h and then washed with physiological saline solution. Separately, the glucose anti-mouse IgG anti-IgG prepared in (a) above is diluted 400 times with 0 perphosphate buffer. 0.5 X beef serum albumin 250 µl of the diluted labeled antibody thus obtained are added to the above polystyrene tube and the mixture is reacted at 370C for 2 h and then washed with physiological saline Separately, 250 μl of a substrate containing 0.1% p-hydroxyphenylacetic acid, 0.002 X of peroxidase and 2% glucose are added thereto and the mixture is reacted at 30 ° C. for 1 h. The reaction is stopped by adding glycine buffer
NaOH. The fluorescence of the reaction mixture is measured at an excitation wavelength of 320 nm and at a fluorescence wavelength of 450 nm. According to this process, EPO can be detected at 120 mU / ml.

Example 4 (a) Preparation of anti-1G of mouse marinated with slucose-6-ohosDhoriaue-dehvdrosenase acid (G6PDH)
To 5 mg of G6PDH (manufactured by Toyo Boseki KK), 2 mg of N- (4-succinimidoxycarbonyl-cyclohexyl) -maleimide are added and the mixture is reacted at 30 "C for 1 h. The reaction mixture is subjected to Filtration on gel of "Sephadex G-25" (registered trademark) and the enzyme fractions are collected and then concentrated. 5 mg of unelgG prepared by reduction of a rabbit anti-mouse IgG antibody (manufactured) by DAKO Co.) and the mixture is reacted at 4 "C overnight. The reaction mixture is subjected to gel filtration of" Ultrogel AcA44 "(registered trademark, manufactured by LKB, Stockholm, Sweden) and collects the active fractions to give the anti-mouse IgG labeled with G6PDH.

(b) Determination of EPO
50 μl of the first antibody prepared in Example 1 (a) are added to wells of a polystyrene microplate (manufactured by Coaster Corp.) and the mixture is reacted at 40C overnight and washed with saline physiological.

50 µl of a test sample containing EPO is added to each well and the mixture is reacted at 37 "C for 2 h and then washed with physiological saline. An antibody is used as the second antibody anti-EPO mouse monoclonal (described in the first publication of Japanese patent 155395/1984) and it is diluted 1000 times with a 0.01 M phosphate buffer. 50 μl of the antibody diluted in the wells are added. above and the mixture is reacted at 37 "C for 2 h and washed with physiological saline. Separately, the mouse anti-IgG labeled with G6PDH prepared in (a) above is diluted 750 times with a phosphate buffer containing 0.5 X of bovine serum albumin. 50 μl of the antibody are added. marked diluted thus obtained in the wells and the mixture is reacted at 37 "C for 2 h and then washed with physiological saline solution.

Next, 200 μl of a substrate containing 30 mM glucose-6-phosphoric acid and 2 mM nicotinamide-adenine-dinucleotide (NAD) are added to each well, and the mixture is reacted at 370C for 30 min. The reaction mixture is added to a substrate for spectral analysis which contains Luciferase (manufactured by Toyo Boseki KK), NAD reduced-flavinemononucleotide (NADH-FMN) oxide reductase (manufactured by Toyo
Boseki KK), flavin-mononucleotide (FMN) and decanal and reduced NAD (NADH) is measured by the emission of luminescence using a TD-4000 measuring device (manufactured by Laboneience
Co.). Consequently, EPO can be assayed at a content of 5 mU / ml.

 It is understood that the invention is not limited to the preferred embodiments described above by way of illustration and that a person skilled in the art can make various modifications and changes thereto without however departing from the scope and the spirit of the invention.

Claims (4)

1. Method for the immunological assay of erythropoietin, characterized in that it consists in reacting a first antibody, specifically reactive with erythropoietin, which is fixed on an insoluble support, with a test sample containing I ' erythropoietin and a second antibody, which is specifically reactive with erythropoietin and which is prepared by immunization of an animal of a species different from the animal used for the preparation of the first antibody; reacting with a determined amount of a third labeled antibody, which is specifically reactive with the animal's immunoglobulin of the species used for the preparation of the second antibody; and then determining the amount of labeled antibody attached to the insoluble support carrying the antibody or the amount of unlabeled labeled antibody. 2. Method according to claim 1, characterized in that the insoluble support is chosen from a polystyrene tube, polystyrene beads, silicone fragments and microplates. 3. Method according to claim 1, characterized in that the third labeled antibody is labeled with a substance chosen from enzymes, isotopes, fluorescent substances and metallic substances. 4. Method according to claim 3, characterized in that the labeling substance is chosen from peroxidase, alkaline phosphatase, ss-galactosidase, glucose oxidase, glucose-6-phosphoric acid-dehydrogenase and glucose- dehydrogenase.