NO822067L - IMMUNOCHEMICAL DETERMINATION OF THE ACID PROSTATE PHOSPHATASE - Google Patents

Abstract

1. Claims for the Contracting States BE, CH, DE, FR, GB, IT, LI, NL, SE A process for determining the amount of prostatic acid phosphatase isoenzyme in a biological fluid sample, characterized by a) incubating the sample with (i) a soluble first antibody which selectively binds immunologically to the isoenzyme in the sample without inhibiting the enzymatic activity of this isoenzyme, and (ii) an insolubilized antibody formed by coupling this second antibody to amylose whereby the second antibody selectively binds immunologically to the first antibody without inhibiting the enzymatic activity of the isoenzyme, to obtain a precipitate of a mixture of immunocomplexes which contain isoenzyme-first antibody-second antibody and first antibody-second antibody, and b) isolating the precipitate containing the immunocomplexes and c) measuring the isoenzyme activity in the precipitate. 1. Claims for the Contracting State AT A process for determining the amount of prostatic acid phosphatase isoenzyme in a biological fluid sample, characterized by a) incubating the sample with (i) a soluble first antibody which selectively binds immunologically to the isoenzyme in the sample without inhibiting the enzymatic activity of this isoenzyme, and (ii) an insolubilized antibody formed by coupling this second antibody to amylose, whereby the second antibody selectively binds immunologically to the first antibody without inhibiting the enzymatic activity of the isoenzyme, to obtain a precipitate of a mixture of immunocomplexes which contain isoenzyme-first antibody-second antibody and first antibody-second antibody, and b) isolating the precipitate containing the immunocomplexes and c) measuring the isoenzyme activity in the precipitate.

Description

i<1>

, I D• e acid phosphatases are a heterogeneous group of many isoenzymes,

in which each is specific for a tissue type. As a result of this tissue specificity, the determination of a tissue-specific isoenzyme in the serum is a valuable aid in the identification

■ of abnormalities in this tissue. In several scientific pu^-

: lications have been shown that the level of the isoenzyme's acidic prostatic

j phosphatase (Prostatic Acid Phosphatase; PAP) in serum rises

IN

1 as a result of the presence of a carcinoma of the prostate (R.M. Townsend,

■ Ann. Clin. and Lab. Sei," volume 7, no. 3, pp 254-261, May-I

, June 1977)... i i 'i The measurement or determination of PAP isozyme levels in the serum j

is, however, complicated due to that other isoenzymes of acid j

' phosEatase from tissue not derived from the prostate is present.

In fact, significant levels of isoenzymes j are normally present

' 1 i i acid phosphatases not originating from the prostate present in serum. [Li, C. et al., J. Lab. Clin. With. 82, 446-460 j I

(1973)]. In view of the many sources for the formation<!>of I

increased levels of isoenzymes of acid phosphatase, it is necessary to determine the isoenzyme's PAP, which is specific for this isoenzyme.

Previous attempts to determine the amount of PAP in serum were based on substrate specificity and chemical inhibition. These! ■experiments did not give very good results, as even the specific substrate or the specific inhibitor reacts with isoenzymes other than PAP. According to the state of the art, these attempts caused false positive or negative test results with the accompanying high possibility of incorrect therapy.

Various immunochemical determination methods for PAP were also described. Choe, B.K., et al have in ProC

Soc. Exper. Biol. and Med. 162, 396-400 (1979) described

a method for determining the enzymatic activity

12 5

and the radioactivity of a J-labeled PAP antigen/antibody complex filled with ammonium sulfate. According to this method, PAP is determined by measuring the antigen/antibody complex which. is filled with ammonium sulfate. According to this method, one obtains

1

however, a strong disturbance through acid phosphatases such as is due to the non-specific nature of ammonium sulfate-I

felling.. i | -

To solve the problem of the interference due to other j isoenzymes in determinations for individual, organ-specific I isoenzymes of human alkaline phosphatase, a double anti- j si tJof. fBeinolzy.-mC-h-.eimmm. u 2n 4o 3, lo1.g6i0 sk (19m6e8t) oodg e abv eCshkore evet et ala.v , CSluinss. mCahn eme. t 26å, i l.,j1'

in 1854-1859 (1980). In both of these works it is described that the specific j

antibody for the desired isoenzyme is reacted with. the experimental sample, after which the antibody/isoenzyme complex is reacted in a subsequent step with a second soluble antibody (anti-u-blobulrn). After the centrifugation, the precipitate is examined with regard to residual iso-enzyme activity.. | in

In order for the Sussman and Choe method to proceed accurately, it is necessary that the second soluble antibody reacts in such a way with the antibody/enzyme complex that the isoenzyme to be determined separates completely. This reaction is dependent on factors such as temperature, pH, ionic strength, time, protein concentration etc. Since these factors are difficult to con- ; trollers in a determination method, Sussman and Choe's methods have limited utility in accurately determining the amount of a specific isoenzyme.

The present invention relates to an immunochemical method for determining the amount of PAP isoenzyme in a quantity of a biological fluid. Increased amounts of PAP isoenzyme in serum indicate a prostate tumor.

The immunological method according to the present invention is a double antibody immunoenzyme method in which one of the antibodies is an insoluble antibody, which is formed by reaction of this antibody with ainylose. The method comprises incubation of the test sample with a first antibody that binds selectively immunologically to the isoenzyme in the sample

, and an insoluble second antibody that binds selectively immunologically to the first antibody.. They . resulting

in immunochemical reactions give insoluble immune complexes 'which

i are easy to isolate. After the isolation of the immune complexes'

are measured in these the above isozyme activities. j 1'"■.■■'!.■!

It was found that<p>roteins such as immunoglobulins (IgG)i, i

il enzymes and lingende can be linked to amylose, a solid carrier.' and gives insoluble proteins such as IgG-amylose, which can be used as immunoabsorbents. It was found that a protein such as e.g. IgG insolubilized by reaction with amylose shows advantages over<p>proteins insolubilized with other solid supports. In particular, the advantage lies in the fact that IgG-amylose can be produced by a very simple reaction. Amylose has a large surface area. for the binding of IgG; and the reaction product obtained, i.e. The IgG amylose can simply be centrifuged at high speeds, but ! remains nevertheless in the performance of an immunochemical determination: for a sufficiently long time in the suspension.

The method according to the present invention has significant advantages over the method according to the state of the art, as the method according to the invention is very specific, fast, extremely accurate and reproducible and enables a specific isolation of the PAP isoenzyme from sera, which contain non- prostatic isoenzymes, and thus enables specific measurement of the amount of the isolated PAP isoenzyme.

In particular, the method according to the present invention is advantageous because the resulting immune complexes can quickly precipitate out of the reaction mixture and become available for measuring the isoenzyme activity. Furthermore, the reaction mixture that forms the immune complex is independent of factors such as temperature, pH, ionic strength, time, protein concentration - etc. As these factors are missing, they are excluded by the method according to the present invention as possible sources of error, which means that the method according to the present invention enables a more accurate determination of the amount of the specific isoenzyme. ' i ' In the method according to the present invention, it is possible to use as a test sample a sample of a biological liquid such as, e.g. whole blood, plasma, serum, lymph fluid, bile fluid, urine, spinal fluid, saliva, sweat, etc. <! >, as well as stool samples from humans or animals. It is also possible to use liquid preparations from human sources. or animal tissue such as skeletal muscle, heart, kidney, lung, brain, bone marrow, skin etc., but biological fluids are preferred. The preferred biological fluid as a test sample according to the present invention is human serum. - * . i The present invention relates in particular to a method; by determining the amount of PAP isoenzymes in a sample of a biological fluid, which may contain other, non-prostatic isoenzymes or other proteinaceous substances. The method according to the present invention is carried out by isolating the specific PAP isoenzyme of interest, and by measuring the enzymatic activity of the isolated PAP isoenzyme. The isolation of the PAP isoenzyme is carried out by using two different antibodies, a first and a second antibody, which react in a reaction mixture with the sample of the biological fluid, and subsequent separation of the PAP isoenzyme containing the immune complexes from this reaction mixture. According to the present invention, the first antibody can be any antibody which is soluble in the reaction mixture and which. selectively binds to the PAP isoenzyme without significantly inhibiting the enzymatic activity of the PAP isoenzyme. The first antibody can be produced in the usual way by injecting a purified PAP isoenzyme as antigen into an animal and subsequent blood sampling to recover sera containing the antibody. To prepare and purify the PAP isoenzyme and inject the purified PAP isoenzyme as antigen into animals for recovery of anti-PAP serum containing the first antibody, any conventional technique can be used. The anti-PAP serum does not need to be purified and can be used directly in the method according to the invention. The anti-PAP serum, which is obtained by using purified PAP isoenzyme, reacts! not with acid phosphatases from tissues other than the prostate and does not significantly inhibit the enzymatic activity of the PAP-iso<L>i ;.enzyme to which it binds immunologically. This means that the first antibody present in this anti-PApj serum binds specifically and only to the PAP isoenzyme from the test sample and at the same time does not affect the enzymatic activities in ;! t! il the PAP enzyme...' ;The PAP isoenzyme used as an antigen to produce! the first antibody can be recovered from any suitable biological fluid such as prostate fluid, blood, serum or from any biologically suitable fresh prostate tissue. The preferred source of this isoenzyme is animal prostate fluids or tissues. /. particularly preferred is human prostate fluid or tissue. | i ;In order to reduce the presence of non-specific antibodies, it is recommended to purify the PAP isoenzyme to a high degree of purity before it is used for the production of the first antibody. The PAP isoenzyme can be purified by any conventional technique known for such purpose . The preferred purification includes classical, known methods such as affinity chromatography, or electrophoresis in ; The degree of purity of the PAP isoenzyme used to form the first antibody, as well as the specificity of the antibody obtained can be determined by common methods such as immunodiffusion or electrophoresis techniques. The preferred method for determining the purity of the PAP isoenzyme is acrylamin gel electrophoresis. The first antibody can be obtained from any animal species that is. known for this purpose. The animal species are particularly vertebrates such as pigs, cattle, dogs, donkeys, horses, goats, rabbits, rats etc. Among these animals, mammals such as donkeys, sheep and goats are particularly preferred. Particularly preferred are goats. In a preferred aspect of the present invention, the first antibody is produced by immunization of goats, a first animal species, with purified PAP isoenzyme by usual immunological techniques and extraction of goat anti-PAP. The purified PAP isoenzyme is obtained from human prostate tissue. in ; . - 1;: j The first antibody is used in an amount that is sufficient; to bind<p>the donkey's PAP isoenzyme. In order to achieve good results, the antibody in the sample is reduced to an amount that is above the limit which is normally sufficient to bind the PAP-ij isoenzyme. The amount of the required first antibody for each sample can be determined by usual methods. After addition of the first antibody, the test tube is treated with an insoluble second antibody. The time of' ; ' ' the addition of the insoluble second antibody after the addition of the first antibody is not critical, but it is preferred to wait with the addition of the insoluble second antibody for at least 15 minutes after addition; of the first antibody. J ;According to the present invention, an insoluble ; done, second antibody by linking the second antibody through an amine bridge to amylose. The second antibody of this insolubilized second antibody can be any antibody that selectively binds to the first antibody without inhibiting the enzymatic activity of the isoenzyme bound to this first antibody. The second antibody of this insolubilized second antibody can be produced by any conventional technique in an animal species different from that in which the first antibody was produced. The second antibody is produced by immunizing an animal different from that in which the first antibody was produced with gamma-globulin from the blood of the host species used for the production of the first antibody. In this way, the second antibody becomes immunoreactive for the first antibody and forms a complex with it. The amylose used for the formation of the insolubilized second antibody can be a homopolymer of D-glucose. The D-gluco-<p>vranose units in the amylose have mainly (1-4) a-glyco-<.....>t ! lateral binding. Amylose's molecular weight varies depending on the amylose source and ranges from an average molecular weight of approx. 100,000 to approx. 1,100,000. ;according to the fifth possibility for the present invention, they can! t i' for the formation of the reaction product of the antibody and amylose, an amylose from any source and of any molecular weight is used. If desired, the amvlos can now be cleaned in the Vanliq way before 1 ! ;: " ' " i , the reaction with the antibody. The degree of purity, on the other hand, is not critical, but it is preferred that the amylose has a purity between ' 50 and 100%... !: ;The method of binding the second antibody or a second protein through an amine bridge to the amylose to form .it. insolubilized second antibody can be any conventional technique for coupling poly saccharides to the protein. According to a preferred aspect of the present invention, the second antibody or a second protein is covalently attached to activated amylose using a conventional Schlff base reaction (Guthrie, R.D. Adv. Carbohydrate Chem. 16:101, 196.1). According to the preferred method, amylose containing several D-glucopyranose units is activated by means of an oxidizing agent such as sodium periodate (HaCTO^), which oxidizes α-1,4-linked glucose units and gives amylosedialdehyde. The resulting amylosedialdehyde then reacts with the primary amino group of the added second antibody or protein and forms a Schiff base or an aldimine. The Schiff base is treated with a reducing agent and provides a primary amino bridge between the protein and the amylose. Among the usual reducing agents which are suitable for this purpose, a reducing agent such as sodium borohydride (NaBH ) is normally used. The procedure for producing the second antibody/amylose product is illustrated in the following reaction diagram. In this scheme, n means several D-glucopyranose units. ; in ; ; To form the insolubilized antibody, the antibody is reacted with amylose in the above-mentioned manner in order to link the antibody through an amine bridge to the amylose. When manufacturing-. of the insoluble soil antibody', the amount of amylose in the antibody is not critical. However, it is normally used; in approx. 1 - 200 parts by weight of amylose per weight fraction antibody. Man! prefer to use approx. 8-20 parts by weight of amylose per weight fraction antibody. i ' ;i i ;i ! According to the present invention, the immune complexes that are formed by the addition of the second antibody are separated from the sample solution or mixture using standard techniques, and the enzymatic action of PAP is measured in the precipitate obtained. , According to the present invention, the enzymatic activity is therefore measured in the precipitation. Common methods for separating the precipitate from the sample solution include filtration and chromatographic methods. The most preferred method for separating the precipitate from the<p>robe solution is centrifugation with subsequent decantation of the supernatant with measurement of the precipitates. This enables measurement of the amount of PAP in the sample. The measurement or determination of the activity of the PAP isoenzyme isolated from the test sample can be done by any conventional method. These methods include colorimetric methods, such as e.g. described in "Methoden der enzymatischen Analyse" published by H.U. Bergmeyer, 3rd edition 1974, vol. 1, p 145; and also fluorimetric methods. The invention also relates to a diagnostic test kit system for quantitative determination of the presence of the PAP isoenzyme in a biological fluid. The components of the test kit system are: (a) a first container containing an antibody which is; capable of selectively binding to the PAP isoenzyme,; (b) a second container containing a second antibody which, through coupling to the amylose through an amine binding is insoluble and which is capable of selectively immunologically reacting with the first antibody in the first container and (c) a substrate reagent with which the enzymatic activity of the PAP isoenzyme can be measured. i i i When all the components of the diagnostic test kit system according to the present invention are used, the person skilled in the art is able to determine the enzymatic activity of the PAP enzyme i in a free sample of a biological fluid containing non-1-nrostatic isoenzymes. The test kit is suitable for clinics, hospitals, laboratories and the individual doctor, who must determine the amount of PAP isozyme in a sample of a biological fluid. The following examples further illustrate the invention, but should not limit the scope and idea of the invention. ;<! >;EXAMPLE 1<1>;Purification of.PAP ;Del_A ;i ;~ ' i .100 g of frozen human prostate tissue is placed at 4 C in a 1 in'homogenization solution containing 0.05 M ethylenediamine- i tetraacetic acid ( ÉDTA) and 0.01% (w/w) tween 80. The frozen tissue is allowed to thaw in the homogenization solution for about 1 and a half hours. After thawing, the tissue is homogenized in a mixer until no more lumps are present in the resulting1 solution. During the homogenization which is not carried out for longer than 20 seconds, the mixture is in an ice bath. The resulting homogenized material is then stirred overnight at 4°C. Material is then centrifuged for one hour at 1 - 4°C and 16,300 Xg. The obtained supernatant of approximately 240 ml is dialyzed three times against 4 liters of 0.005 M sodium phosphate buffer with pH 6.3 containing 0.9% sodium chloride. During dialysis, the original volume is increased from approximately 240 ml to approximately 260 ml. ;The dialyzed material obtained is then placed in a 1000 ml lyophilization bottle which is suitable for lyophilization by quick freezing over dry ice and acetone and then lyophilized overnight. The lyophilized material obtained is then reconstituted with 20.0 ml of 0.005 M sodium phosphate buffer with pH 6.3 containing 0.9% sodium chloride, during which the reconstituted material used for the subsequent part B is obtained. ;Part_B;An LKB column is packed with 100 ml of a pre-swollen con-A sepharose 4B gel in the aforementioned buffer, after which the gel is allowed to settle without passing through the column. The packed column obtained is then washed with 400 ml of the above-mentioned buffer. ' '.! The reconstituted material from the above-mentioned part A is applied to the LKB column thus prepared. Mari then lets the material sit on the column for three hours.. Then sends. 800 ml of the aforementioned buffer is passed through the column at a flow rate of 40 ml per hour. Then run 500 ml of a 0.0 - 0.3 M mannose gradient, which was obtained > separately in 0.005 M sodium phosphate buffer of pH 6.3 with 0.9% sodium chloride and filtration through activated carbon, at a flow rate of 40 ml per hour through the column, as ! fractions of 10 ml are collected (total of 50 fractions). 500 ml of 1.0 M mannose are then passed through the column and a total of 50 fractions are collected in the same way. ' All fractions are tested for protein and PAP activity. Fractions containing a PAP activity with a specific activity of 2000 Sigma units/ml protein are pooled, dialyzed against 0.05 M sodium phosphate buffer pH 6.3 with 0.9% sodium chloride and concentrated using an amicon filter system to a ! 4.0 ml of concentrated dialysate for the following part C. Part_C A 7.5% acrylamide gel is prepared using a Buchler system. The following buffer was likewise prepared for the Blichler system: 0.03 5 M 3-alanine in 0.014 M acetic acid for the upper buffer of the system and 0.14 M 3-alanine in 0.056 M acetic acid for the lower and elution buffer of the system. To prepare a mixture, 1 ml of 40% sucrose and 3 drops of methyl green dye were added to the 4 ml of the concentrated dialysate from part B. The mixture was then placed on the 7.5% acrylamide gel of the Blichler system and electrophoresed is started (1000 volts, 200 milliamps for about 16 hours). Mixtures are eluted using the lower/elution buffer, collecting 10 ml fractions. The fractions obtained are examined with regard to PAP and protein activity. Fractions containing PAP activity are combined and dialyzed overnight twice against 0.01 M citrate buffer with pH 6.0, during which very pure PAP is obtained for the immunization method according to the following example 2. The very pure PAP can be stored at -2 0°C. ;! in EXAMPLE 2 } ! j i- ;Immunization of goats with purified PAP for production of the first antibody I ;i ' ;; For the preparation of the immunogen for immunization of the goats j ;. the very pure PAP prepared according to example 1 is mixed in 0.01 M citrate buffer with pH 6.0 with an equal volume of Freund's complete adjuvant (a mineral oil suspension containing killed M tubercle bacilli) whereby a mixture containing 0 .5 mg PAP per ml. For the formation of the water/oil emulsion which constitutes the immunogen, the resulting mixture is homogenised. Goats are immunized for three consecutive weeks with a subcutaneous injection of 2 ml of immunogen in two places in the shoulder area, each place receiving one ml of the immunogen. The second injections of incomplete Freund's adjuvant are given at two-week intervals after the first injection, after which further injections of incomplete Freund's adjuvant are carried out over several months. The goats are bled one to two weeks after immunization to obtain antiserum. ;EXAMPLE 3;Preparation of the globulin fraction from donkey anti-goat IgG serum ;The globulin fraction from donkey anti-goat IgG serum which is needed for coupling to the amylose is prepared using the following ingredients and methods: ; 500 ml of donkey anti-goat IgG serum is brought into a suitable container with magnesium stirrers. To saturate the container contents to 37.5%, add 300 ml of saturated ammonium sulphate solution (point b) with gentle stirring. After stirring for one hour at 4°C, the precipitated antibodies are collected by centrifugation at 1200 Xg for 30 minutes at 4°C as a lump. The lump dissolves in approx. 350 ml f o.sf atpuf f is (point c ) , after which the original volume of 500 ml is again produced with the same puff. In order to bring the mixture to a 33% strength, 250 ml of saturated ammonium sulphate solution are added with light stirring at 4°C. (item b) . The mixture is stirred for one hour at 4°C. The precipitated antibody is retained after 90 minutes in a centrifuge. in% at 1208 Hg eg 4°C as a lump - The lump dissolves in approx. 200 ml of the phosphate buffer are then placed in a dialysis vessel and dialyzed against the phosphate buffer at 4°C. The buffer is changed after a minimum of 4 hours of dialysis three times (4 liters per change). The dialysate obtained is dialysed again and then against 4 litres; 1 carbonate bicarbonate buffer (point d). The protein concentration in this dialysate is determined according to the Lowry method, and one j ; dilute to 12 mg/ml in a final volume of 800 ml, h \or j one obtains the globulin fraction from donkey anti-goat IgG serum,| which is suitable for coupling to the activated amylose according to; . in example 4. ;<!>;EXAMPLE 4;i i Activation of amvlose ' ' : * i

In order to couple the globulin fraction from donkey anti-goat IgG serum according to example 3 to amylose, the amylose is activated according to the following steps 1-16 first:

' i

1. 20 g of amylose is slowly added to 4 1-liter centrifuge containers containing 500 ml of deionized water. Stir with a magnetic stirrer during and after the addition. The container is filled to full volume with deionized water. 2. During one hour of stirring at room temperature, the amylose is allowed to swell in each container. 3. The swollen amylose is centrifuged in each individual container for 5 minutes at 2500 Xg. 4. The supernatant in each container is poured off and the clump is kept for step 5. 5. In each container, the clumps are resuspended in one liter of deionized water.

6; Steps 3, 4 6q -5 are repeated twice, which in total

provides 4 washing operations.

7 ■. The lump is resuspended in water and allowed to stand overnight in water at room temperature. The next day, the containers are centrifuged, after which the clumps can be stored for the later step 9 after decantation. 8. Dissolve 25.68 g of sodium metaperiodate in 700 ml of ion-i

■ " j In free water and fill to a volume of 800 ml.

in

9. The lump according to step 7 is resuspended in each of four; in containers with 200 ml of 0.15 M sodium metaperiodate according to step 8. i : • ; r 10. The resuspended lump according to step 9 is gently stirred for two hours at room temperature. 11..49.66 g of ethylene glycol (liquid) is mixed with 300 ml of deionized water. With deionized water, you fill up to a volume of 400 ml, and no centrifugation takes place before the addition of ethylene glycol.

in

in 12. At the end of the 2-hour stirring according to step 10, 100 ml of 2 M ethylene glycol solution according to step 11 are placed in each of the four containers, after which they are stirred carefully for 30 minutes at room temperature. 13. The containers are centrifuged for 5 minutes at 2500 Xg.

After the decaitization, the lumps are held back separately from each other. 14. The clumps according to step 13 are resuspended separately in up to 1 liter (measurement content) of carbonate bicarbonate buffer (point d from example 3), after which one stirs for 15 - 30 minutes.

15. Steps 13 and 14 are repeated twice.

16. When decanting, lumps are obtained which contain activated amylose; which are ready for coupling to the globulin fraction from donkey anti-goat IgG serum prepared according to

EXAMPLE 5

i Connection of globulin fraction from donkey anti- goat IgG serum i -

■ ' i to activated amylose The coupling of the globulin fraction from the donkey anti-goat IgG serum (prepared according to the procedure in example 3) to activated• o i • amylose (prepared according to the method according to example 4)! is carried out using the following components and j j procedures. |

Approach

1. To each of 4 containers containing a lump of activated amylose (20 g dry weight, swollen as indicated in example 4 to 100 ml) add 200 ml of donkey anti-goat IgG (prepared according to example 3; 12 mg/ml) for .linking to the amylose on IgG. 2. The coupling takes place at room temperature for 36 - 48 hours with stirring. 3. The container is then centrifuged for 5 minutes at 2500 Xg. 4. The protein in the supernatant of each container is tested - on the O.D. at 280 . 5. Add 0.8 g of sodium borohydride to 800 ml of phosphate buffered saline solution. in ii

'I

i 6. To each of the four centrifuged containers from step 31

<1> which contains amylose protein, add 200 ml of sodium

in borohydride solution. The resulting suspensions are gently stirred for one hour at room temperature.

i i 7. • After stirring the suspension according to step 6, the resulting reduced amylose protein gel is centrifuged

in each container as in step 3, after which the supernatant;

thrown away.

8. The gel in each container is resuspended with phosphate buffered saline solution and then filled up to 1 liter with phosphate buffer. The resuspended gel is stirred for 10 - .20 minutes;

at room temperature.

9.. After stirring, the gel is centrifuged as in step 3. The supernatant is discarded, and steps'3 and 9 are repeated twice. 10. The four resulting amyl<p>se-<p>rote clumps are resuspended in 200 ml of PBS and then brought by washing into a 2-liter container, during which an amylose-protein suspension is obtained. 11. 4.8 g of bovine serum albumin and 1.6 g of sodium acid are brought into 100 ml of PBS with stirring. This solution is added to the amylose-protein suspension. 12. The amylose protein suspension is then brought into a 5% aqueous suspension which is achieved by adding PBS to a final volume of 1600 ml.

The determination of the PAP activity in a sample of a biological fluid is carried out as follows: i

• 1 1

Part_A

! in 200 ul test serum sample or control is placed in a test tube, and for the reagent void weight, 200 ul distilled water is added to another test tube. 20 ul of the goat anti-PAP serum obtained according to example 2 is added to each tube. ■ After the last tube has been mixed with anti-PAP serum, the mixtures obtained are mixed and incubated for 15 minutes at room temperature. 200 ul of a well-stirred amylose-protein suspension according to example 5 is then added to each tube. After the last tube has been sorri| to be examined is mixed with the amyiosis protein suspension is mixed, the tubes and incubated for 15 minutes at room temperature. 3.0 ml of a 0.9% salt solution (9.0 g of sodium chloride per 1000 ml of distilled water) is then added to each tube, after which mixing is done. Each tube is then centrifuged for 10 minutes at 1500 Xg, after which the supernatant is removed by

■decanting.- You thus get tubes containing a solid-phase lump for use in part B. in Part_B

The colorimetric PAP substrate solution is prepared by reconstituting a tube of Worthington sodium thymolphthalein monophosphate with 41.0 ml of distilled water. The bottle is gently shaken to completely dissolve the substrate. This substrate solution can be stored for up to 60 days at 2 - 8 C or 2 days at 18 - 26°C. The substrate solution is then equilibrated

at room temperature. The tubes containing the solid-phase lump from part A are placed in a 37°C water bath and equilibrated for 5 minutes. 0.5 ml of coiorimetric PAP substrate solution is then added to each tube containing this lump. Each tube is then mixed and incubated for 30 minutes at 37 °C (after adding the substrate solution to the first test tube). At the end of the 30-minute period, 1.5 ml of alkaline Worthington solution is added to each tube, after which mixing and then centrifugation in 10 minutes at 1500 Xg.. The absorbance of the resulting supernatant in each tube is read at 590 nm in a spectrophotometer..

Part_C

The colorimetric PAP substrate solution. which is used in part Bj..f, F\ £ 7 man aom follows: I

To determine the concentration of PAP in the samples, a standard curve is drawn up using conventional Worthington thymolphthalein dilutions. After the subtraction of the absorbance of the reagent value from the absorbance of the serum samples and the controls, the corrected absorbances are used to read the concentrations of PAP from the standard curve.

Claims (9)

1. Procedure for determining the isoenzyme's acid prostate phosphatase in a sample from a biological fluid; characterized by the fact that one in ' i i •; a) incubating the sample with • j (i) a soluble-first antibody, which selectively I i; binds immunologically to the isoenzyme in the sample without inhibiting the enzymatic activity of this isoenzyme, and (ii) an insolubilized antibody formed by coupling of this second antibody to amylose, the second antibody being selectively bound immunologically logically to the first antibody without inhibiting the enzymatic activity of the isoenzyme, for the formation of a precipitate of a mixture of the immune complex, which contains isoenzyme-first antibody-second antibody and first antibody-second antibody, and that one b.) isolates the precipitates containing the immune complexes and c) measures the isoenzyme activity in the precipitate. 2. Method according to claim 1, characterized .in that the sample of biological fluid is blood serum. 3. Method according to claim 1 or 2, characterized in that the first antibody is goat anti-acid prostate phosphatase and the second antibody donkey anti-goat IgG; '4. ' Method according to one of the claims 1-3, .karak terized in that the isoenzyme activity is determined by spectrophotometric measurement. <!> 5. Method according to claim 4, characterized in that the spectrophotometric measurement uses <1> ! colorlmetric rea <g> enses. ! ' <!> i 6. Method according to claim 5, characterized in that ymolphthalein aminophosphate or a basic addition salt were used as coiorimetric reagents: hence. ' 7. Diagnostic test set for determining the quantity ' i ■ of the isoenzyme acid prostate phosphatase in a sample of ^ a biological fluid, characterized by ; ■ that the test kit system contains the following components: in a) a first container containing an antibody; which is capable of selectively binding immunologically to the isoenzyme, b) a second container containing an insoluble antibody formed by linking this second antibody to amylose, the second antibody being able to selectively immunologically react with the antibody in the first container and c.) a reagent with which the enzymatic activity of the isoenzyme. can be determined. 8. A composition consisting of a protein which is connected to amylose through an amio bridge. 9.. Composition according to claim 8, characterized in that the protein is immunoglobulin.. 10-. Procedure for producing a composition according to ki?av & or 9, k a i? and in? in; and in? i.e. by linking the protein through an amino acid to the amylose.