DE2934756C2 - Composition for the determination of beta 2 -human microglobulin and process for its preparation and its application - Google Patents

Description

The invention relates to a composition for the determination of ß-human microglobules, consisting of Carrier particles and a / V human microglobulin antibody applied to the surface of the carrier particles, a method for their production and a method for the determination of /? j-human mlkroglobulln under Use of the composition.

/ j-human microglobulin is a protein having a relatively low molecular weight of about 12,000 and it is found in serum, urine, cerebrospinal fluid, saliva, milk or the like of humans. The in vivo effect of /? ₂ -Mlkroglobulln has not yet been fully clarified. However, it has recently been found that in certain diseases such as inflammation, carcinoma and neural diseases, the level of /? _: -Mikroglobulin in the patient's body fluids increases. For example, kidney diseases are divided into those caused by glomerular disorder and those caused by disorders in seminiferous tubules. In the latter case, the amount of ^ 2 microglobules increases first in the blood, more precisely in the serum, and then in the urine.

The / 3: -microglobulin is a protein that penetrates the glomerular basement membrane and is then released from the Nlerenkanüichen or Hamkanüichen is absorbed. However, if there is a disturbance of the tubal tubules, This protein seeps into the urine without being reabsorbed. Accordingly, the ^ 2-Mlkroglobulln level One of the best parameters for the effect of the tubules in the urine. This catfish can be a disorder of the Kidney or urinary tubules by determining the level of ß-Mlkroglobulln in the serum and urine to be diagnosed.

so Up to now no suitable means for assessing a disease of the kidney or urinary tubules was available. However, it can now be easily diagnosed by determining the ^ -Microglobullnsplegels in the serum and urine, thereby opening the way for an appropriate treatment of the disease. This is extremely important and valuable for clinical use.
There has been an interest in developing a method by which ^ human microglobules in serum or in ur: n can be determined with a high degree of accuracy and reliability. However, none of the conventional means for the determination of ^ human microglobules have been found to be satisfactory. A known measure is a method of a single radial immunodiffusion, in which wells are created in an agar plate which contains a ^ -Humanmlkroglobulln antibody. ₂ -Milkroglobulln occurs, the / 32-HumanmlkrogIobulln is determined. Since this method has a low sensitivity, the ^ -Microglobulln in the test sample can only be determined in a concentration above a certain limit.

The Radiol Immunoassay Method is also a common method by which / Jj microglobules measured using a radioisotope-indexed ^ human globule antibody (see Scand. J. Elin. Lab. Invest., Vol. 28, 1971. pp. 439-443). However, this procedure is dangerous and troublesome as it involves the use of a radioactive substance. It can therefore not be carried out if not adequate control and facilities for the application of the radioactive substance To be available.

Another example is a method using a passive reverse hemagglutination reaction (RPHA), according to which ft-microglobules with a sensitivity equivalent to that of the radio immunoassay method can be easily determined, see Jap. J. Clin. Path., Vol. 25, 1977, Suppl., Report No. 54. Da If red blood cells from an animal are used as a carrier, this method has the disadvantage that during production of antibody-sensitized red blood cells Pretreatments of the red blood cells, e.g. B. the fixation red blood cells and treating red blood cells with binding agents such as glutaraldehyde or tannic acid required are. In addition, since a non-specific reaction, which an antibody for the als Red blood cells used in carriers is attributed to the practical determination frequently encountered, it is necessary to use an absorption treatment with the red blood cells and a control experiment from tanned cells.

Finally, DE-OS 22 04 684 also describes a method for the detection of antibodies or antigens in a body fluid, synthetic polymer latex particles being used instead of red blood cells (see in particular page 4, last paragraph and page 16, lines 4 to 6 ). However, nothing is mentioned in this document either about a composition for the determination of ft-human microglobulin or about a method for its determination. The object of the invention is therefore to provide a new composition for the determination of ft-human glycoglobulin (both qualitatively and quantitatively) which has superior storage stability and which, with good reproducibility and a high degree of accuracy, quickly provides reliable results without interference from other proteins, which can be contained in the experimental or test samples, whereby a simple determination procedure and simple determination instruments can be used. Furthermore, the invention aims to provide a method for producing the composition mentioned, and a method for determining / _{I 2} -human microglobu-Hn in human urine or serum using the composition mentioned.

This object is achieved according to the invention by creating a composition for Determination of ft-human globules, consisting of carrier particles and one on the surface of the FT-human microglobulin antibody applied to carrier particles, which is characterized in that the Carrier particles made of synthetic polymer latex particles of the styrene type having a specific gravity of 1.1 to 1.4 and from the group of particles of a styrene polymer, styrene copolymer and carboxylated or amino group-containing carboxylated products thereof.

Furthermore, according to the invention, a method for the determination of ß-human microglobules in human Urine or serum using a composition consisting of carrier particles and one on / Jj-Humanmlkroglobulln antibody applied to the surface of the carrier particles characterized in that the carrier particles are made of synthetic polymer latex particles of the styrene type with a specific gravity from 1.1 to 1.4 and from the group of particles of a styrene polymer, styrene copolymer and carboxylated or amino group-containing carboxylated products thereof are.

The means or methods for determining /} ₂ human microglobules are free from the drawbacks and deficiencies of the conventional modes of operation described above and provide reliable results with a high degree of accuracy using a simple apparatus and a simple determination procedure.

They have an excellent shelf life and are able to quickly determine the concentration of / 32-human microglobulin in a test sample with a high degree of accuracy and reliability and a good reproducibility is very valuable, with a simple measuring instrument and an effortless one Determination procedure are used, and with no interference from other proteins, which may be contained in the test sample occurs. It has also been found that the above said composition produced in a simple manner and not only as a latex, but also as a dried one Product can be stored.

/ J ₂ -Human microglobules Is present in various human body fluids. So can urine, milk, serum and the like as starting /? ₂ -Human microglobulin for the preparation of the / V human microglobulin antibody can be used in the composition according to the invention. Most expediently, iii, the starting material is taken from the urine of a patient who is attending one of the above-described illnesses

ten is ill, made. The separation of the / J ₂ human microglobules can be carried out using

different, well-known work worlds are carried out. Examples of such arbeltswels include a zone electrophoretic process, the Berggärd et al. (I. Berggärd et al: J. Blol. Chem., ij; 243, 4095, 1968) by ion exchange chromatography, gel filtration, and the method of Ohsawa et al. (M.

Ohsawa et al: Experlentla, 29, Ϊ179, 1973).

Ii The Exit S '/ Jj-Human Molecular Globule Is expediently a sample of a single protein, since the inclusion

| - ^r ! the subsequent work cycle is made more difficult by impurities.

! i; An antibody of the ft-human microglobulin, which using the above-mentioned known

% th working modes can be obtained by administering the /? ₃ -Mlkroglobullns as an antigen

1 $ an animal capable of producing an antibody, e.g. B. guinea pigs, rabbits or goats, in ω

conventional manner of immunizing the same, drawing blood from the immunized animal and separating it of the /! 2-human glycoglobulin antibody can be produced from the blood thus obtained.

The animal used in this method can be any animal that has the ability to produce Owns antibodies. However, in order to obtain a large amount of antibody, the use of a large animal preferred. Usually rabbits or goats are suitable, but whichever is to be used Animal not limited to this 1st. The separation of a human glycoglobulin body from this containing antiserum can be carried out using known means. For example the antiserum is salted out and an absorption elution using an insoluble antigen

will be repeated.

As synthetic latex particles can be used for the preparation of the composition for the determination of ß ₂ - human microglobulin according to the invention, for. B. polystyrene, carboxylated polystyrene, amino-containing carboxylated polystyrene, styrene / butadiene copolymer, carboxylated styrene / butadiene copolymer, styrene / divinylbenzene copolymer and acrylonitrile / Butadie.i / styrene copolymer can be used.

Preferred particles of synthetic polymer latex are those from the group of styrene polymers, Copolymers of styrene with a monomer from the group of chlorostyrene, methyl methacrylate and vinylidene chloride and carboxylated or amine-containing carboxylated products thereof. These synthetic polymer latex particles can after pre-treating their surfaces with a non-ionic surfactant ίο be used. For example, these particles are preferably used after a non-ionic Ethylene oxide type surfactant was made to adhere to it by a process (cf. JP-OS No. 9716/76), examples of suitable ethylene oxide type nonionic surfactants are a block copolymer of ethylene oxide and polyoxypropylene glycol, polyoxyethylene alkyl ether and polyoxyethylene alkyl aryl: here.

According to the invention, the synthetic polymer latex particles preferably have an average particle diameter from 0.01 to 10 μm, in particular 0.1 to 1 μm. To increase the reproducibility of the measurement results it is preferred to use particles having a relatively narrow range of size distribution. That specific gravity of the latex particles is 1.1 to 1.4, preferably 1.1 to 1.3. The determination can be made under Application of an agglutination reaction carried out on a slide plate or as a microtiter method will. The composition for the determination of / ^ - human microglobules according to the invention can be found in can be produced in a simple manner. According to the invention it is made by using synthetic polymer latex particles of the styrene type with a specific gravity of 1.1 to 1.4 from the group of particles of a styrene polymer, a Siyrene copolymer and a carboxylated or amino group-containing carboxylated one Products thereof in a concentration of 0.05 to 5% with a / ^ - human microglobulin antibody In a concentration of 0.0001 to 1% in a buffer at a pH in the range of about 5 to 10, in particular 6.5 to 8, at a temperature of 4 to 40 ° C. That can bring into contact with gentle stirring for about 30 minutes to about 24 hours.

The buffer used can, for. B. a phosphate-buffered saline solution [M / 60 phosphate buffer (pH 7.2) with a content of 0.15 M NaCl, abbreviated to PBS] and a glycol-buffered saline solution. If desired, 0.01 to 0.1% of a protein, for example bovine serum albumin (abbreviated BSA), can preferably be added to an antibody solution in order to prevent non-specific agglutination. After the Beschlchtungs- or coating reaction, the reaction mixture is repeatedly centrifuged in a neutral salt solution, e.g. B. glycine buffered saline or washed in PBS. Finally, the latex particles with the coating applied thereon can be removed from the /? ₂ -Humanmlkroglobuliri Antibodies To be stored in the form of a suspension in a diluent. The diluent is preferably a mixture obtained by adding about 0.1% BSA to glycine buffered saline or PBS. It is also preferred to add 0.01 to 0.5% sodium azide (NaN _{3) to the diluent.}

The composition for the determination of / 2-human nikroglobulin according to the invention can be in the form of a latex suspension, as described above, and also in the form of a dried product. The composition in the form of a dried product can be obtained by adding a stabilizer, e.g. B. an amino acid (z. B. glycine or sodium glutamate) or dextran is added to the diluent described above, the latex mass with the coating of the ^ human microglobulin antibody thereon is suspended in the diluent and the suspension is freeze-dried. The amounts of amino acids and dextran are about 1.2 to about 4 parts by weight and about 1.6 to about 6 parts by weight, respectively, for every 1 part by weight of latex particles.

The composition for the determination of /) ₂ -Humanmlkroglobulln according to the invention has a good stability both in the form of a latex suspension and in the form of a dried product. The dried product can be stored stably for a longer period of time, for example for several years.

so For the determination, measures known per se can be used; for example, the Concentration of ß-Mlkroglobulln in serum or urine determined effortlessly by a micro-filtering method will. According to this mode of operation, a certain amount of a diluent becomes the above The type described is partially poured onto a microplate and then a certain amount of a Test sample, e.g. B. of serum or urine, introduced into the first bore or the first floor and gradually-Hch diluted with a thinner. Likewise a dilution series is made in the same catfish below Made using a standard antigen. A certain amount of a human body with /?: Dissolved latices are added to and mixed with each dilution series. After standing the end point of agglutination is observed over a period of time at room temperature and the absolute amount of /? 2-Mlkroglobulln can be determined by comparison with the Standaidantlgenrelhe will.

With the common method of single radial immunodefusion for the determination of / ^ - Mlkroglobulln the / ^ - Mlkroglobulln can not be determined, if its amount is not at least 5 μg per ml of a test sample, if the test sample is serum or urine. Since errors tend to occur with a / ^ - Mlkroglobulln content of about 5 to 10 μg, a somewhat higher concentration is preferred. On the contrary, in the method for determining / _{I 2} -molkroglobulin according to the invention using the latex detected with the ^ -human microglobulin antibody, it is possible to make a determination when the amount of A -mycoglobulin is more than 1 ng per ml amounts to. Thus, the method according to the invention shows a very high sensitivity, and the sensitivity is equivalent to that

or higher than that of the radio immunoassay method, considered to be a method with the highest sensitivity is considered in common analyzes.

In another embodiment of the determination according to the invention, a specific amount of a test sample is dropped onto a slide glass plate, and separated therefrom, a defined amount of a _2- human globule solution of a known concentration is added dropwise. A certain amount of the latex sealed with the human microglobulin antibody is added to each of the solutions, whereupon a gentle rocking motion is applied. When evaluating the resulting agglutination pattern, the concentration of the / _{I 2} -human croglobulin in the test sample can be qualitatively determined within a few minutes. A semi-quantitative determination can also be carried out if the test sample is diluted in a test glass and the reaction is observed with reference to the respective dilution.

The composition for the determination of ^ -human microglobulin in the form of a latex or in the form of a dried product is superior to the compositions obtained by coating red blood cells or other carrier materials with /? ₂ -Human microglobulin were obtained because, due to the lack of antigenicity of the carrier, non-specific reactions other than the desired antigen-antibody reaction do not take place, and because a / i ₂ -Human microglobulin antibody can be applied directly to a latex easily by simply dipping an antibody solution into the latex can be applied as a coating without the use of a binder, and because it has good storage stability.

The novel composition according to the invention does not react at all with proteins other than the /? 2-human microglobulin and the carrier also does not react with ft microglobulin.

The invention is explained in more detail below on the basis of production games and work tables.

Manufacturing area 1
Production of / V human microglobulin:

One kilogram of ammonium sulfate became about 2 liters of urine in a patient with kidney inflammation and the solution was left to stand at 4 ° C. overnight. The precipitate formed was collected and dissolved in water. The insoluble material was separated by centrifugation and the precipitate was dissolved in saline and applied to a Sephadex GIO column (a product of Pharmacia Fine Chemicals Co., Ltd., Sweden) (5 × 100 cm), buffered with a 0.02M Tris buffer solution (pH 8.0) containing IM sodium chloride applied. The buffer was allowed to flow at a flow rate of 40 ml / h, to perform gel filtration. 400 ml of elulener fractions were collected, desalted and made up to volume of 10 ml concentrated. Then the product was applied to a DEAE cellulose column (a product of Brown Company, USA) (2.5 χ 40 cm) buffered with a 0.01M phosphate buffer (pH 7.5) and the Elution was carried out while the concentration of sodium chloride in the buffer increased linearly to 0.2 mol increased. 70 ml of a protein fraction eluted in the first place was collected, desalted and up 10 ml concentrated. The liquid was applied to an SP-Sephadex C 50 column (Pharmacia Fine Chemicals Co., Ltd., Sweden) (2 × 35 cm), buffered with a 0.04M phosphate buffer (pH 5.9), and the elution was carried out at a flow rate of 25 ml per hour, the concentration of the buffer increasing linearly 0.02 mole was increased. 100 ml of fractions from 75 ml to 175 ml of eluate were collected, desalted, concentrated and freeze-dried to give 60 ml of a white powder.

The product showed a single band on disk electrophoresis.

Production example 2

Production of A-human microglobulin antibodies:

The / ^ - human microglobulin obtained in Preparation Example 1 was dissolved in a physiological saline solution in a concentration of 4 mg / ml of the latter. The solution was mixed with an equal amount of Freund's complete tool. The mixture was in an amount of 0.1 m! Injected three times a week into each foot pad of 4 healthy rabbits weighing approximately 2.3 to 2.5 kg. One week later, 1 ml of a 0.1% Ig / 32 human glycoglobulin solution was injected. Whole blood was drawn from the rabbits' carotid artery three weeks after the last injection. The blood was centrifuged in the usual manner and the resulting serum was kept at 56 ° C. for 30 minutes , thereby producing about 200 ml of anti-serum.

From the fact that a single band of precipitation with an antigen was formed by an Ouchterlony method and immunoelectrophoresis, it was found that the resulting anti-serum was a single antibody.

Production example 3 Manufacture of insoluble / 82-human microglobulin:

5 mg of ft-human microglobulin was dissolved in 5 ml of a 0.2M acetic acid buffer solution (pH 5.0) and about 0.5 ml of a 5% IgE bovine serum albumin solution was added. Then was 5% glutaraldehyde added dropwise until a precipitate was formed. The precipitate was collected, homogenized and washed with PBS and then with a glycine-hydrochloric acid buffer (pH 2.8) and further with PBS and with

stored below -20 ° C. iff

Production Example 4 jfi

Purification of ft-human glycoglobulin antibodies:; '|

An equal amount of a saturated solution of ammonium sulfate was added to the anti-serum, iw

after which it was completely mixed. The mixture was left to stand at room temperature for 30 minutes. j. #

The precipitate that formed was collected by centrifugal separation, with 0.5 saturated solution "!";

washed by ammonium sulfate and then diluted against PBS. Then that was in the manufacturing bowl 3 fe ";

insoluble /? 2-prepared Humanmikroglobulln added to the dialysate and the mixture was anschlle- fi

Let stand at room temperature for 30 min. The mixture was separated into a supernatant ^

Liquid and centrifuged into a deposition. Insoluble $ again became the supernatant.

^ -Humanmlkroglobulln added and the mixture was treated in the same way. The two precipitates-! $!

genes were combined and washed with PBS. Then a glycine-hydrochloric acid buffer (pH 2.8) was added and S

the mixture was shaken for 5 minutes and then centrifuged. The precipitation was similar to S.

Treated catfish with a glycine-hydrochloric acid buffer and disperse the resulting supernatant fluids

nlgt. The mixture was dialyzed against PBS to obtain a purified ft-Humanmikroglobulin antibody gebil- ψ

was det. U

i

Working example 1 ff

Latex sensitized with ft-human microglobulin antibodies: M

Polystyrene latex (a product of Takeda Chemical Industry Co., Ltd; SDL 59; specific gravity 1.14; Tell-I

diameter 0.9 μm) was diluted with PBS so that the concentration of the particles reached 0.25%, whereupon an equal amount of a purified antibody diluted up to 60 times with PBS, was admitted. The mixture was kept at 37 ° C for two hours, followed by centrifugation. The latex particles were collected and washed with PBS and then with a diluent. Thereafter the Laiex particles were reduced to a concentration of 0.25% using a diluent suspended, whereby a with ^ -Humanmicroglobulln-Antibodies senslbllislerter latex was formed.

When a human granule solution was added to this sensitized latex, agglutination occurred a. However, no agglutination took place when albumin, IgG, IgM and IgA solutions, respectively were added to this.

The diluent used was PBS containing 0.08% BSA and 0.1% sodium azide.

Working example 2
Determination of /? ₂ -Human microglobulin:

A diluent in an amount of 0.025 ml was in each case in a well of a microplate from V-type poured and 0.025 ml of serum or a serum diluted with a suitable diluent were added to the first well and diluted with a serial double dilution process gradually thinned. Furthermore, 0.025 ml of a standard solution with a content of 0.01 μg ft-human microglobulin was added per ml of the first hole in a further row and in the same way diluted. Then 0.025 ml of the latex sensitized with the ft-human mlc globule antibody was added to each of the Drilled holes are poured and, after sufficient mixing, left to stand at room temperature for more than 10 hours, to observe the end point of agglutination. If an anti-antibody reaction has taken place, the latex particles were dispersed over the entire surface of the bottom of a well. If no

so the antigen-antibody reaction took place, the latex particles collected in the center of each hole. on this catfish can be assessed as the end point of agglutination. Following the procedure given above pre-determined the amount of-Mlkroglobulin with respect to seven samples. Serjm, that is 100 times was diluted, and seven samples of urine diluted 50 times were determined. The results are in in Table I below.

Table 1: Latex agglutination value

sample

Concentration of
Standard Zi-Mlkroglobulln (ng / ml)
Agglutination pattern

No.

3 4 5 6 7 8 Concentration

in the rehearsal
2.5 1.25 0.625 0.312 0.156 0.078 0.039 (ng / ml)

Healthy adult (32 year old male)

Healthy adult (43 year old male)

Healthy adult (22 year old male)

Leukemia patient (9 years old, male) year old man, suffering from stomach cancer Year old man, suffering from nerve failure. Year old woman, suffering from SLE

1
0.5

over 16

Healthy adult +

(32 year old man)

Healthy adult +

(43 year old man)

Healthy adult +

(22 year old man

Year old man +

with bone marrow disease (myeloma)

Years old, male +

got leukemia

Year old man, +

suffers from nerve failure

Year old woman, +

suffers from SLE

Note: + = positive; - = negative

0 .25 0 .13 0 .25 4th 4th over 8 4th

Working example 3 like this

By carrying out treatment in the same manner as in Working Example 1, a latex was sensitized.

washed and containing in a diluent
Product of Pharmacia Fine Chemicals Co. Ltd., Sweden) so that the concentration of the latex particles reached 1.25%. The suspension was then freeze-dried in the usual manner to give a composition of latex particles having a sensitivity to / 82 human microglobulin antibody.

Working example 4

A diluent was added to the latex particle composition obtained in Working Example 3 with a Sensitivity to ft-human microglobulin antibody added. When running the same Working as in Working Example 2, the amount of ft-microglobulin in serum and urine was then determined certainly. Similar results were obtained.

It is clear from the results obtained in the above Examples that the amount of / S ₂ -mcroglobulin in the serum and urine of patients afflicted with various diseases was apparently higher than that in the sera and urinary fluids of healthy adults . Accordingly, the determination of the amount of ft-microglobulin in blood and urine is extremely valuable to the

Diagnosis of these diseases.

Separately, the amount of / ij-microglobules in the serum and in urine of humans was measured, wherein a latex sensitized with / Ji human microglobulin antibody made from goat antiserum Using the same procedure as in Preparation Example 4 and In the Working Examples below, were used. The results were exactly the same as in the tables listed above.

Using the latev sensitized with / ij-human microglobulin antibody or the composition containing the sensitized latex according to the invention, the amount of /? ₂ -Mlcroglobulin Measured within a shorter period of time when a sample (human serum or urine) is present in a very small amount, less than 0.03 ml. The sensitivity is extremely high and a ng of ^ 2 microglobules per 1 ml of the test sample can be determined. Accordingly, the composition according to the invention provides important and useful information for the diagnosis of leukemia, kidney disease, inflammation or the like, and its scope is very broad. Furthermore, since the amount of the test sample can be very small. The composition according to the invention is particularly suitable for children, in particular for newborns and infants from whom only a small amount of blood can be drawn.

Claims (6)

Patent claims: 1. Composition for the determination of ft-human globules, consisting of carrier particles and a / fe-human microglobulin antibody applied to the surface of the carrier particles, thereby characterized in that the carrier particles are composed of synthetic polymer latex particles of the syrole type with consist of a specific gravity of 1.1 to 1.4 and from the group of particles of a styrene polymer, Styrene copolymers and carboxylated or amino group-containing carboxylated products thereof are selected. 2. Composition according to claim 1, characterized in that the latex particles have an average ίο have particle diameters of 0.01 to 10 μm. 3. Composition according to claim 1 or 2, characterized in that it further comprises a stabilizer from the group of amino acids and dextran. 4. Method for the determination of / ^ - human microglobulin in human urine or serum under Use of a composition consisting of carrier particles and one on the surface of the FT-human microglobulin antibodies applied to carrier particles, characterized in that the carrier particles of synthetic polymer latex particles of the styrene type having a specific gravity of 1.1 to 1,4 and consist of the group of particles of a styrene polymer, styrene copolymer and carboxylated or amino group-containing carboxylated products thereof. 5. The method according to claim 4, characterized in that the determination using a Micro-filtering process is being carried out. 6. A process for the preparation of a composition for the determination of ft-human microglobules according to one of claims 1 to 3, characterized in that synthetic polymer latex particles of the styrene type with a specific gravity of 1.1 to 1.4 from the group of particles of a styrene polymer, Styrene copolymers and carboxylated or amino group-containing carboxylated products thereof in a concentration of 0.05 to 5% with a ^ -human microglobulin antibody in a concentration of 0.0001 to \% in a buffer at a pH in the range from 5 to 10 at a temperature of 4 to 40 ° C.