FR2503184A1 - COMPOSITION FOR DETERMINING PROSTATIC ACID PHOSPHATASE AND METHOD OF USING THE SAME - Google Patents

Abstract

THE PRESENT INVENTION CONCERNS A COMPOSITION WHICH ALLOWS THE DETERMINATION OF PROSTATIC ACID PHOSPHATASE IN A SPECIFIC, QUICK AND SIMPLIFIED MANNER AND WHICH IS COMPOSED OF A SPECIFIC ANTI-PROSTATIC ACID PHOSPHATASE SERUM, A SPECIFIC ANTI-GAMMA-GLOBULIN SERUM. ANIMAL SPECIES USED FOR THE PREPARATION OF ANTI-PHOSPHATASE-PROSTATIC ACID SERUM (SUCH SERUMS MAY BE DILUTED IN AN APPROPRIATE BUFFER SOLUTION), AN APPROPRIATE SUBSTRATE AND AN ENZYMATIC ACTIVITY BLOCKING REAGENT. THE INVENTION ALSO CONCERNS THE PROCESS FOR DETERMINING THE ACTIVITY OF PROSTATIC ACID PHOSPHATASE USING THE COMPOSITION ABOVE.

Description

Composition for the determination of acid phosphatase

  prostate and method of using the same.

  The present invention relates to a new composition designed for the immunochemical determination of prostatic acid phosphatase; it is also relative to

  diagnostic method that uses such a composition.

  Acid phosphatases are known to be

  cellular enzymes found in the body while

  and which are capable of promoting the hydrolysis of the esters

  of phosphoric acid in an acidic environment.

  More particularly, prostatic acid phosphatase

  tick (APP) is a specific species of isoenzyme that is

  by the phosphatase antigens present in the others

  tissues.Scnapparntimin the bloodstream has been associated with the pre-

  of prostate cancer. This type of tumor does not cause symptoms for a long period of time and is often

  located in the back lobe, far from the ureter.

  The clinical stages of cancer are mainly four, namely: 1. A tumor confined to the prostate gland and which can not be detected by palpation;

  2. A tumor still confined within the capsule

  prostate, but which can be detected by proctoscopy;

  3. A tumor that is locally spread with a volun-

  surrounding lymphatic nodules;

  4. A tumor accompanied by metastases.

  However, the activity of acid phosphatase can

  also be exalted by other syndromes, which imposes

  to be able to make a quick diagnosis, to have recourse to

  to an analytical process that is both sensitive and specific.

  It is known in fact that in the case of cancer, regardless of its mode and location, only rapid treatment is likely to provide a good chance of therapeutic success: such treatment, in the case of cancer of the prostate, can be performed only in the early stages of tumor growth, when the levels of acid phosphatase circulating in the blood serum are

very weak.

  The determination of the effectiveness of the APP can be carried out directly by using more or less substrates

  specific, or also indirectly, by using an

  APP bittor such as tartaric acid. In the latter case, the fraction of the enzymatic activity attributable to APP is calculated as the difference between the total activity and the activity

  residual, precisely in the presence of tartaric acid. How-

  However, the use of the latter compound as an inhibitor of the prostatic isoenzyme does not confer a sufficient degree of

  specificity to the dosage because other percentages of phos-

  acid phatase of different origin are also inhibited, such

  that, more particularly, that derived from blood platelets

guines, liver and kidney.

  The use of specific antiserum has recently

  has been suggested for the immunochemical determination of APP,

  thus possible to isolate the prostatic isoenzyme serum

  in which other types of phosphatases are also

  content. Processes such as electrophoretic counter-immunity, and radioimmunoassays are based precisely on a

  such basic immunochemical principle.

  Counterimmunoelectrophoresis is a process that is both relatively simple and specific, but in any case,

  it does not allow quantitative assays.

  Communicative assays allow for quantitative measurement, but there are factors that may limit their potential for practical use, such as the cost of reagents, health hazards from the use of radioisotopes, lnstability of labeled enzymes, and the need to use large amounts of human prostatic acid phosphatase under conditions of purity for

  the preparation of diagnostic devices.

  The Applicant has discovered a process which is the subject of the present invention by which it is possible to perform a quantitative analysis of protease acid phosphatase by a simplified method, with a high sensitivity, a high accuracy and a very satisfactory degree of

  The process according to the invention does not require the use of

  In addition, it provides the advantage of deter-

  undermine prostatic acid phosphatase after it has

  been separated from inactivation factors that may be

  naked in the blood serum to be tested (P. Vihko, Clln Chem.24, 1783 (1979)). In addition, the method according to the invention does not require the adoption of expensive radioactive tracers and

  perishable and does not require further preparation

  of significant amounts of pure enzyme to obtain

  labeled enzyme is necessary for a quantitative assay to be

  radioimmunoassay can be performed.

  In accordance with the present invention, the method

  APP is therefore to add to the sample

  to test a specific antiserum, an anti-gamma serum

  globulin of the same animal species as that used for the preparation of the specific antiserum (these sera may

  diluted in an appropriate buffer solution), centrifugally

  the suspension thus obtained and finally to measure the

  enzymatic content of the residue by addition of a solution of a

appropriate substrate.

  The determination of enzymatic activity in the centrifugation residue can be carried out because the APP enzyme retains its activity even when bound.

to specific antibodies.

  Applicant advantageously uses as

  substrate, the sodium salt of p-nitrophenylphosphoric acid

  that, but satisfactory results can also be obtained when using other salts of this acid, and this

  is also true when using sodium salts, potas-

  sium, ammonium and ammonium derivatives (eg

  hexylammonium, 2-amino-2-ethyl-1,3-propanediol, 2-amino-2-

  methyl-1,3-propanediol), or other cations,

  phenylphosphoric, beta-glycerolphosphoric, alpha-naphthyl-

  phosphoric, phenolphthaleinphosphoric, thymolphthalein-

  phosphoric, alphanaphthyl-AS-BI phosphoric and beta-naphthyl-

phosphoric.

  The method that allows the determination of the activi-

  APP can be implemented advantageously using

  a composition which is also the subject of the present

  vention, consisting of a reagent to perform the control test,

  of a specific serum (or a corresponding antibody fraction)

  dante), an anti-gamma-globulin serum of the same species

  than that used for the preparation of the anti-APP serum (these reagents may be diluted in a suitable buffer solution), and a substrate-buffer system which is designed for the determination of the enzymatic activity. The reaction medium may contain an additive suitable for obtaining a maximum precipitation action on the immunocomplex with a minimum simultaneous effect on other free phosphatases: for example, the medium may contain a polyethylene glycol. The same effect can also be obtained

  maintaining the pH in the range of 4.8 to 5.6.

  As pointed out above, such a composition

  This allows a specific process to be implemented with considerable sensitivity, speed and

  simplicity of execution which are considerably better

  compared to those of the usual immunochemical processes.

  All of these features and other variations that are useful for understanding and implementing the method according to the present invention will be apparent from

  reading the following example in which is also given

  the formulation of the composition, this example being unique

  illustrative and not limiting of the scope of the invention.

EXAMPLE

  0.2 ml of the test sample, which is fresh human blood serum acidified with acetic acid at a pH of about 6, is added to 0.1 ml of anti-APP specific serum, eg from of rabbits, which has been suitably diluted and, separately, to 0.1 ml of normal, appropriately diluted rabbit serum in test tubes (preferably 5 ml test tubes):

  plastic test tubes, provided that they do not pre-

  do not feel adsorption phenomena.

  The dilutions of the serums which are used as reagents are optimized by successive approximations on

individual lots available.

  In the following example, the reagents are: anti-APP specific serum, CP 011 lot, 1: 100 dilution with 0.1 M glycine and 0.5% of bovine seroin albumin solution: pH 7.2 ; normal rabbit serum, lot FB 201, dilution 1:75 with the same solution as above; rabbit and goat anti-gamma globulin serum, lot MB 25, 1:15 dilution with 10 mM phosphate buffer, pH 7.0, 0.15 M NaCl with polyethylene glycol under conditions

optimized corentration.

  The test tube containing the specific anti-APP serum is the test sample, while the test tube

  tests that contains normal rabbit serum is the control.

  After an appropriate incubation time has elapsed

  4 ° C), which can vary from 5 minutes to 24 hours, 30 minutes being the preferred time, 1 ml of rabbit and goat anti-gammoglobulin serum, or of another animal, is added to each test tube. suitably immunized, diluted with 10 mM phosphate buffer, pH 7, 0.15 M NaCl. The polyethylene glycol (6000) in an optimized concentration is also dissolved in the buffer solution to obtain the maximum precipitation effect on the membrane. immunocomplex but

  with minimal influence on other free phosphatases.

  Under the conditions thus adopted, the concentration of polyethylene glycol in the buffer diluent

  range from 0.5% to 4%, with 1.5% being the preferred percentage

  re. The same effect can be obtained by diluting the rabbit anti-gamma globulin serum with an acid pH buffer, for example with a 50 mM citrate buffer, having a pH ranging from

  4.8 to 5.6, the preferred value being 5.3.

  After an appropriate incubation at 4 C (5 minutes

  at 24 hours, preferably 30 minutes), centrifugation, for example 1500 g for 15 minutes, is carried out to separate the immunocomplex from the supernatant liquid, the latter

being rejected.

  The precipitate is placed in a bath of thermal water

  (37 C) and is stirred in a substrate solution

in a suitable buffer.

  Any substrate with conditions

  that it can be hydrolysed by acid phosphatases,

  because the prostatic isoenzyme has been separated immi

  is lying. After a time considered necessary for sufficient evolution from the substrate substances that can

  determined by a subsequent chemical reaction

  If these or other chromogenic substances can occur, the reaction is stopped by an alkaline solution.

  For example, the determination of the activity

  using the sodium salt of p-nitrophenylphosphate is

described below.

  The test tubes which contain the precipitate obtained by centrifugation of the test sample and the control are added with 0.5 ml each of p-nitrophenylphosphate sodium salt dissolved in a buffer.

  50 mM citrate, pH 5.0 + 0.1 at a concentration of 6 mM.

  After incubation for 30 minutes (exact time) at 37 ° C, 1 ml of solution is added to stop the reaction, which is a solution of NaOH containing 26.3 mM Na 3 PO 4. The difference in optical density at 405 nm between the test sample and the control is read. This difference, multiplied by 13.44, gives the enzymatic activity attributable to APP in mU / ml of the

serum tested.

Claims (8)

  1. Composition designed for the determination of the activity   Prostatic acid phosphatase (APP), characterized in that it comprises an anti-APP specific serum, an anti-gamma-globulin specific serum of the animal species used for the preparation of the anti-APP serum, a suitable substrate, a reagent to stop the enzymatic activity,   a buffer system and optionally a polyethylene glycol.   2. Composition designed for the determination of phosphorus   prostatic acid phatase according to claim 1, characterized   in that the substrate is a salt of sodium, potassium, ammonium, cyclohexylammonium, 2-amino-2-ethyl-1,3-propanedlol,   2-amino-2-methyl-1,3-propenediol, p-nitrophenyl-   phosphoric, phenylphosphoric, P-glycerolphosphoric,   alpha-naphthylphosphoric, beta-naphthylphosphoric, phenol-   phosphoric phthalein, thymphthaphthalphosphoric acid, naphthyl-AS- BI phosphoric.   3. Composition designed for determining the activity   Prostatic acid phosphatase according to claim 1 or 2, characterized in that the substrate is constituted   by the sodium salt of p-nitrophenylphosphoric acid.   4. Method for determining the activity of phospha-   Prostatic acid tase characterized by the fact that it consists of:   a) add to the sample to be tested a specific anti-serum   and an anti-gamma-globulin serum of the animal species used for the preparation of the specific anti-serum, b) centrifuging the suspension thus obtained, c) adding to the residue a solution of a suitable substrate, d) measure the enzymatic activity.   5. Method for determining the activity of phospha-   Prostatic acid base according to Claim 4, characterized in that the substrate is a salt of sodium, potassium, ammonium, cyclohexylammonium, 2-amino-2-ethyl-1,3-propanediol,   2-amino-2-methyl-1,3-propanedio, p-nitrophenyl-   phosphoric, phenylphosphoric, beta-glycerolphosphoric,   alpha-naphthylphosphoric, beta-naphthylphosphoric, phenol-   phosphinephosphoric, thymolphthaleinphosphoric, alpha- Phosphoric naphthyl-AS-BI.   6. Method for determining the activity of phospha-   Prostatic acid base according to Claim 5, characterized in that the substrate consists of the sodium salt   p-nitrophenylphosphoric acid.   7. Process for determining the activity of phospha-   Prostatic acid plate according to claim 4, characterized in that the operation a) is carried out in the presence of polyethylene glycol.   8. Method for determining the tactivity of phospha-   Prostatic acid tase according to claim 4, characterized in that step a) is carried out at pH inclusive between 4.8 and 5.6.