GB2030293A - Composition for determination of human immunoglobulin G, process for its preparation and its use - Google Patents

Abstract

A composition for use in the qualitative or quantitative determination of human immunoglobulin G (IgG) comprises synthetic polymeric latex particles having an anti-human IgG antibody coated on the surface of the latex particles. This composition may be prepared by contacting a synthetic polymeric latex having a solids concentration of 0.05 to 5% with an anti-human IgG antibody having a concentration of 0.0001 to 1% in a buffer at a pH of from 5 to 10 at a temperature of from 4 to 40 DEG C. The composition can be used to determine IgG in human body fluids by, for example, the microtiter method.

Description

SPECIFICATION Composition for determination of human immunoglobulin G, process for its preparation and its use This invention relates to a composition for determining human immunoglobulin (to be abbreviated to IgG), a process for its production, and to its use. Specifically, it relates to a composition for determining human IgG (both qualitatively and quantitatively) which has superior storage stability and which can rapidly give reliable results with good reproducibility and a high degree of accuracy without being hampered by other proteins that may be contained in an assay sample, by using an easy measuring operation and a simple measuring instrument.

More specifically, this invention relates to a composition for determining human IgG, which is composed of particles of a synthetic polymeric latex, preferably particles of a styrene-type synthetic polymeric latex selected from the group consisting of styrene polymer, styrene copolymers and the carboxylated products or amino-containing carboxylated products thereof; to a process for preparation of the composition; and to use of the composition.

Human IgG is a protein having a molecular weight of about 160,000, and is present in a concentration of from 1.1 to 1.7 g/dl in the sera of healthy humans. It is known to be one of the important components which constitute a main factor of the humoral immunity of an animal.

Since the level of human IgG in the blood or urine varies in hypogammagloburinemia, G-type myeloma, glomerular disorder, etc., determination of the human lgG level is very important for diagnosis. The human IgG is a protein which scarcely permeates the glomerular basement membrane, but as the degree of disorder of the glomerular basement membrane is larger, the human IgG leaks in a larger amount in the urine. Accordingly, the degree of disorder of the glomeruli of the kidney can be known, for example, by determining the concentration of the human IgG in the urine. By determining the ratio of albumin/lgG, this can be more exactly judged. Thus, the albumin/lgG ratio is an essential information for the diagnosis of function of the glomeruli of the kidney.As is clear from the above, the determination of human IgG is essential for diagnosis of renal diseases, especially glomerulonephritis, tracing of the clinical courses of these diseases, evaluating of the efficacy of drugs, and for prognosis.

It has been desired therefore to develop a method which can determine human IgG in the serum or urine with high accuracy and reliability.

Procedures heretofore utilized for the determination of human IgG have not proved to be satisfactory. These prior procedures include, for example, a single radial immunodiffusion method which comprises providing wells in an agar plate containing a human IgG antibody, filling an assay sample into the wells, and measuring the level of human IgG from a precipitation band generated with the diffusion of human IgG in the assay sample, electrophoresis on various supports, and a method which comprises salting out of an assay sample and subsequent determination of the human lgG level by a Kjeldahl method.

The single radial immunoglobulin method has low sensitivity, and therefore, has the defect that it cannot determine human IgG in a concentration below a certain limit in an assay sample. The electrophoretic method requires that the concentration of human IgG should be above a certain limit, and expensive equipment including an electrophoretic device and a detecting device is necessary. The Kjeldalhl method has the defect that the operation is complicated.

Particularly, in determining human IgG in the urine, the urine must be concentrated to 100 to 1,000 times when these conventional methods are used, because the content of human IgG in the urine is small. This requires great labor and time.

Another example of the prior art is a method using a reverse passive hemagglutination reaction (RPHA) which can easily determine human IgG with an equivalent sensitivity to radioimmunoassay.

Since red blood cells of an animal are used as a carrier, this method has the disadvantage that in the preparation of antibody-sensitized red blood cells, pretreatments of the red blood cells, such as the fixation of the red blood cells and treatment of the red blood cells with coupling agents such as glutaraldehyde or tannic acid, are necessary. Furthermore, because a non-specific reaction attributed to an antibody for the red blood cells used as a carrier often occurs in actual determination, it is necessary to perform an absorption treatment by the used red blood cells and a control experiment using tanned cells.

The present inventors have made investigations in order to provide a means for determining human IgG which is free from the defects and disadvantages of the conventional methods described hereinabove, and which can give reliable results with a high level of accuracy by using a simple instrument and a simple measuring operation.These investigations have led to the discovery that a composition comprising particles of a synthetic polymeric latex, preferably particles of a styrene-type synthetic polymeric latex, and a human lgG antibody coated on the surface of the particles has superior storage stability, and is very useful in measuring the level of human IgG in an assay sample rapidly with high accuracy and reliability and good reproducibility by using a simple measuring instrument and an easy measuring operation without being hampered by other proteins that may be contained in the assay sample. It has also been found that the aforesaid composition can be produced by an easy means, and can be stored not only as a latex but also as a dried product.

It is an object of this invention therefore to provide a novel and excellent composition for determining human IgG, a process for preparation thereof, and use of the aforesaid composition.

The above and other objects and advantages of this invention will become more apparent from the following description.

Human IgG is a major component of a serum protein, and is also found in urine. The starting human IgG used for the preparation of a human IgG antibody in the composition of this invention can be prepared by means known per se For example, it can be obtained in a purified form by a conventional method which comprises treating an ethanol fraction from the human serum with trichloroacetic acid, and ammonium sulfate, and by ion exchange chromatography, etc. Commercially available human IgG can also be used.

An antibody from the human IgG that can be obtained by utilizing the aforesaid known means can be prepared by administering the IgG as an antigen to an animal having the ability to produce an antibody, such as guinea pigs, rabbits or goats, in a customary manner to immunize it taking the blood from the immunized animal, and separating the human IgG antibody from the resulting blood.

The animal used in this method may be any animal which has the ability to produce antibodies.

However, to obtain a iarge quantity of an antibody, the use of a large animal is preferred. Usually, rabbits or goats are suitable, but the animal is not limited thereto. Separation of a human IgG antibody from the antiserum containing it can be performed by utilizing known means. For example, the antiserum is salted out, and adsorption-elution using an insoluble antigen is repeated.

Various synthetic latex particles can be used for the production of the composition for determining human IgG of this invention. Examples of polymers or copolymers that form such latex include, polystyrene, carboxylated polystyrene, amino-containing carboxylated polystyrene, polyvinyltoluene, styrene-butadiene copolymer, ca rboxylated styrene-butadiene copolymer, styrene-divinylbenzene copolymer, vinyltoluene-tertiary butyl styrene copolymer, polyesters, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyrrolidone, and vinyl chloride-acrylate copolymer.

Preferred particles of synthetic polymeric laticesare particles of styrene-type synthetic polymeric latices selected from the group consisting of styrene polymer, styrene copolymers such as a copolymer of styrene with a monomer selected from the group consisting of chlorostyrene, methyl methacrylate and vinylidene chloride, and carboxylated or amino-containing carboxylated products thereof. These synthetic polymeric latex particles can be used after pre-treating their surfaces with a nonionic surfaceactive agent. For example, it is preferred to use these particles after causing an ethylene oxide-type nonionic surfactant to be adsorbed thereto in accordance with the method described in Japanese Laid Open Patent Publication No. 9716/76 laid open on January 26, 1976.Examples of suitable nonionic surfactants of the ethylene oxide type are a block copolymer of ethylene oxide and polyoxypropylene glycol, polyoxyethylene alkyl ethers, and polyoxyethylene alkylaryl ethers.

In the present invention, the synthetic polymer latex particles preferably have an average particle diameter of 0.01 to 10 microns, more preferably 0.1 to 1 micron. To increase the reproducibility of the results of measurement, it is preferred to use particles having a relatively narrow range of size distribution. The specific gravity of the latex particles is preferably about 0.9 to 1.4, and more preferably about 1.1 to 1.3. In the case of determination utilizing an agglutination reaction on a slide glass plate, latex particles having a broad range of specific gravity can be used. In the case of a microtiter method, it is preferable to use latex particles having a specific gravity of at least 1.1.

The human IgG determining composition of this invention can be produced by a simple means. For example, it can be produced by contacting a synthetic polymeric latex preferably having a concentation of 0.05 to 5% with a human IgG antibody having a concentration of 0.0001 to 1% in a buffer at a pH range of preferably about 5 to about 10, more preferably about 6.5 to about 8, at a temperature of preferably about 4 to about 400C. The contacting can be performed with gentle stirring for about 30 minutes to about 24 hours.

The buffer used may, for example, be a phosphate-buffered saline (M/60 Phosphate buffer (pH 7.2) containing 0.1 so NaCL, to be abbreviated PBS), and a glycine-buffered saline. If desired, 0.01 to 0.1% of a protein such as bovine serum albumin (to be abbreviated BSA) may preferably be added to an antibody solution in order to prevent non-specific agglutination. After the coating reaction, the reaction mixture is washed several times by centrifugation in a neutral salt solution such as a glycine-buffered saline or PBS. Finally, the latex particles having the human IgG antibody coated thereon can be stored in the form of a suspension in a diluent. The diluent is preferably a mixture obtained by adding about 0.1% of BSA to a glycinebuffered saline or a PBS. It is further preferred to add 0.01 to 0.5% of sodium azide (NaN3) to the diluent The human IgG determining composition of this invention may be in the form of a latex 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 such as an amino acid (e.g. glycine, or sodium glutamate) or dextran to the diluent described hereinabove, suspending the latex composition having human IgG antibody coated thereon in the diluent, and lyophilizing the suspension. The amounts of the amino acid and dextran are about 1.2 to about 4 parts by weight, and about 1.6 to about 6 parts by weight, respectively per part by weight of the latex particles.

The human IgG determining composition of this invention has good stability both in the form of a latex and in the form of a dried product. The dried product can be stored stably for a longer period of time, for example for as long as several years.

According to this invention, there is provided a process for determining human IgG, which comprises qualitatively or quantitatively determining human IgG in the human urine or serum using a composition comprising synthetic polymer latex particles and a human lgG antibody coated on the surface of the latex particles.

For the determination, means knownperse can be utilized, and for example, the level of IgG in the serum or urine can be easily determined by a microtiter method. According to this means, a certain amount of a diluent of the type exemplified hereinabove is poured portionwise onto a microplate, and then a certain amount of an assay sample such as the serum or urine is introduced into the first well of the plate and successively diluted with a diluter. On the other hand, a dilution series is prepared in the same way using a standard antigen. A certain amount of a human IgG antibody-sensitized latex is added to each of them and mixed. After standing for a certain period of time at room temperature, the end point of agglutination is observed, and the absolute amount of IgG can be determined by comparison with the standard antigen series.

According to another embodiment of measurement in accordance with this invention, a certain amount of an assay sample is dropped to a slide glass plate, and separately, a certain amount of a human IgG solution of a known concentration is added dropwise. A certain amount of the human IgG antibody-sensitized latex is added to each of them, followed by gentle oscillation. By evaluating the resulting agglutination pattern, the concentration of the human IgG in the assay sample can be qualitatively determined within a few minutes.

The human IgG measuring composition in the form of latex or dried product is superior to compositions obtained by coating human IgG on red blood cells or another carrier in that because of the lack of antigenicity of the carrier itself, non-specific reactions other than the desired antigen-antibody reaction do not take place; that a human IgG antibody can be coated directly on a latex easily by simply dipping an antibody solution in the latex without using a binder; and that it has excellent storage stability.

The method for determining IgG using the human IgG antibody-sensitized latex or its composition permits measurement of more than 1 ng (1 x 10-9 g) of IgG per ml, and can achieve an equivalent or higher sensitivity to or than the radioimmunoassay which is considered to show the highest sensitivity among ordinary analyses.

In view of the fact that the novel composition of this invention comprising latex particles and a human IgG antibody coated thereon does not at all react with proteins other than the human IgG and the unsensitized latex does not at all react with IgG, it can be said that the human IgG antibody is bonded to the latex.

The following Preparation Examples and Working Examples illustrate the present invention in more detail. However, the present invention is not limited to these examples so long as it does not depart from the scope and spirit thereof.

PREPARATION EXAMPLE 1 Preparation of a human ígG antibody: Human IgG produced by Miles Laboratories Inc., U.S.A. was used as an antigen. Human IgG was dissolved in physiological saline in a concentration of 4 mg per ml. The solution was mixed with an equal amount of complete Freund's adjuvant The mixture was intramuscularly injected in an amount of 0.2 ml to five parts of each of four healthy rabbits weighing 2.0 to 2.5 kg, and three weeks later, was injected in the same way. After two weeks, 1 ml of a 0.2% human IgG saline was injected intraveneously to each of the rabbits. Three weeks after the final injection, the whole blood was taken from the carotid artery of the rabbits, and centrifuged in a customary manner. The resulting antiserum was maintained at 560C for 30 minutes to obtain about 20 ml of antiserum.The resulting antiserum was determined to be a single antibody from the fact that it forms a single band with an antigen by an Ouchtalony method and immunoelectrophoresis.

PREPARATION EXAMPLE 2 Preparation of insoluble human IgG:- 250 mg of Human IgG was dissolved in 5 ml of a 0.1 M phosphate buffer (pH 7.0), and 2.5% glutaraldehyde was added dropwise to the IgG solution with stirring until a yellowish gel formed. The mixture was allowed to stand for 3 hours at room temperature. The precipitate was collected, homogenized, washed with PBS, then with a glycine-hydrochloric acid buffer (pH 2.8) and further with PBS, and then stored at below -200C.

PREPARATION EXAMPLE 3 Purification of a human IgG antibody: An equal amount of a saturated ammonium sulfate solution was added to antiserum, and they were mixed well. The mixture was allowed to stand for 30 minutes at room temperature. The resulting precipitate was collected by centrifuge, washed with 0.5 saturated ammonium sulfate solution, and then dialyzed against PBS. Then, insoluble human IgG was added to the dialyzate, and the mixture was allowed to stand at room temperature for 30 minutes and then centrifuged into a supernatant liquid and a precipitate. To the supernatant liquid was again added insoluble human IgG, and the mixture was treated in the same way. The two precipitates were combined, and washed with PBS. Then, a glycinehydrochloric acid buffer (pH 2.8) was added. The mixture was shaken for 5 minutes and centrifugally separated.The precipitate was again treated with the glycine-hydrochloric acid buffer. The resulting supernatant liquids were combined, and dialyzed against PBS to form a purified antibody.

WORKING EXAMPLE 1 Preparation of a human IgG antibody-sensitized latex: Polystyrene latex (a product of Takeda Chemical Industry Co., Ltd.; SDL 59; specific gravity 1.14; particle diameter 0.9 micron) was diluted with PBS so that the concentration of the particles became 0.25%, and then an equal amount of a purified antibody diluted to 60 times with PBS was added. The mixture was maintained at 370C for 2 hours, and then centrifuged. The latex particles were collected, and washed with PBS and then with a diluent. Then, the latex particles were suspended to a concentration of 0.25% using a diluent to form a human IgG antibody-sensitized latex.

When a human IgG solution was added to the sensitized latex, agglutination occurred. But no agglutination occurred when albumin, 2-micrnglobulin, immunoglobulin M and immunoglobulin A solutions were respectively added to it.

The diluent used was PBS containing 0.07% BSA and 0.1% sodium azide.

WORKING EXAMPLE 2 Determination of human IgG:- A diluent in an amount of 0.025 ml was poured into each well of a V-type microplate, and 0.025 ml of serum or serum diluted with a suitable diluent was added to the first well, and diluted successively with a diluter by a serial twofold dilution method. On the other hand, 0.025 ml of a standard solution containing 0.1 y9 of human IgG per ml was added to the first well of another row, and similarly diluted.

Then, 0.025 ml of the human IgG antibody-sensitized latex was poured in each of the wells, and after sufficient mixing, was allowed to stand at room temperature for more than 10 hours to observe the end point of agglutination. When an antigen-antibody reaction occurred, the latex particles were dispersed on the entire surface of the bottom of a well. When no antigen-antibody reaction occurred, the latex particles gathered at the center of each well. Thus, the end point of agglutination can be judged.

In accordance with the aforesaid procedure, the amount of IgG was measured with regard to six examples of urine diluted to 100 times. The results are shown in Table 1.

Table 1: Agglutination value of latex

l No. 1 2 3 4 5 6 7 8 Standard lgG concentration sa (ng/ml) o 25 12.5 6.25 3.12 | t ration in the ci s pattern + l + + + + + + Healthy adult (32 years old male) + + + - - - - 2 tD adult ED years old male) + + + + + + Healthy adult (22 years old male) + + + + - - - - 2 In N years + + + t eo l +++ + + + 32 cd with SLE (32 years old Patient with glomerulonephritis (53 years old male) + + + + + + + - 16 Ê Ê E o o 4 X n X 6O c .o X > e o O 2 t N &commat; J E CD E ' ' < ,c O .eD (a . > X ~ I I I Z E Z ao < : aU!n +: positive; -: negative WORKING EXAMPLE 3 Determination of human IgG with slide aggregation:- Polystyrene latex (a product of Dow Chemical; particle diameter 0.22 + 0.006 micron; specific gravity 1.05) was diluted to 1% with a glycine-sodium chloride buffer (pH 8.4), and an equal amount of a purified antibody was added. The mixture was allowed to stand overnight at 40C, and then centrifuged to separate the latex. The latex vivas washed once with a glycine sodium chloride buffer, and suspended tc a concentration of 1% in a glycine sodium chloride buffer containing 1% BSA to form a sensitized latex.

50 ,ul of a human IgG solution having a concentration of 10 yg/ml, 5 ssg/ml, 1 ,ugiml 0.5 ,ug/ml, or 0.1 yg/ml, was added dropwise separately on a slide glass. Then, 50 ,ul of the resulting sensitized latex was added dropwise on top of it, and with gentle stirring for a few minutes, the reaction was observed.

An agglutination pattern appeared when the concentration of the human lgG solution was 10 g/ml.b g/ml, and 1 g/ml, but no agglutination pattern appeared at a concentration of 0.5 g/ml and 0.1 Hg/ml.

By the above procedure, the content of human IgG was estimated with regard to 6 examples of urine diluted to 10 times. The results are shown in Table 2.

Table 2: Agglutination value of latex

Agglutina- Concentration in Assay sample tion pattern the sample (mg/ss} Urine (diluted to 10 times) Healthy adult (32 years old male) - less than 5 Healthy adult (43 years old male) Healthy adult (22 years old male) Nephrotic patient (47 years old male) + at least 10 Patient with SLE (32 years old female) + Patient with glomerulo nephritis (55 years old male) + " Control Standard lgG concentra tion ( g/ml) 10 5 1 0.5 0.1 Agglutination pattern ++ + + -+ + WORKING EXAMPLE4 By treating in the same way as in Working Example 1, a latex was sensitized, washed, and suspended in a diluent containing 0.5% of glycine and 0.7% of Dextran T-1 0 (a product of Pharmacia Fine Chemicals Co., Ltd., Sweden) so that the concentration of the latex particles became 1.25%. The suspension was then lyophilized in a customary manner to obtain a composition of latex particles sensitive to a human IgG antibody.

WORKING EXAMPLE 5 A diluent was added to the latex particle composition sensitive to human IgG antibody obtained in Working Example 4. Then, by operating in the same way as in Working Example 2, the amount of IgG in the serum and urine was determined. Similar results were obtained.

As is clearly seen from the results obtained in the above Examples, the amount of IgG in the urine of patients affected with various diseases showed an apparently higher value than that of the urine samples of healthy adults. Accordingly, the determination of the amount of IgG in the urine is very useful for diagnosis of these diseases.

Separately, the amount of IgG in the urine of humans was measured by using a human IgG antibody-sensitized latex prepared from the antiserum of a goat in the same way as in Preparation Example 3 and subsequent Working Examples. The results are were quite the same as in the above tables.

By using the human IgG antibody-sensitized latex or the composition containing the sensitive latex in accordance with this invention, the amount of IgG can be measured within a shorter period of time if there is a sample (human serum or urine) in a very small amount of less than 0.03 ml. The sensitivity is very high, and 1 ng of IgG can be measured per ml of the assay sample. Accordingly, the composition of this invention affords significant and useful information for the diagnosis of hypogammagloburinemia, G-type myeloma, glomerular disorder and other various diseases, and its range of application is broad.

In the determination of IgG in the urine, it has previously been necessary to concentrate the urine.

However, the use of the human IgG antibody-sensitized latex in accordance with this invention makes it possible to use urine without concentrating it. Thus, the composition in accordance with this invention is especially suitable for determining IgG in the urine.

Claims (14)

1. A composition for use in the qualitative orquantitive determination of human immuno-globulin G(lgG) comprising synthetic polymeric latex particles having an anti-human IgG antibody coated on the surface of the latex particles.

2. A composition according to claim 1 wherein the latex particles have an average particle diameter of from 0.01 to 10 microns.

3. A composition according to claim 1 or 2 wherein the latex particles have a specific gravity of from 0.9 to 1.4.

4. A composition according to any one of the preceding claims which further comprises a stabilizer which is an amino acid, a salt thereof, or dextran.

5. A composition according to any one of the preceding claims wherein the synthetic polymeric latex particles are particles of a latex of a styrene homopolymer, a styrene copolymer or carboxylated or amino-containing carboxylated products thereof.

6. A composition according to any one of the preceding claims which is in the form of a latex.

7. A composition according to any one of the preceding claims which is in the form of a dried product.

8. A composition according to claim 1 substantially as hereinbefore described with reference to any one of Working Examples 1, 3 or 4.

9. A process for producing a composition as claimed in any of the preceding claims, which process comprises contacting a synthetic polymeric latex having a solids concentration of 0.05 to 5% with an anti-human IgG antibody having a concentration of 0.0001 to 1% in a bufferata pH of from 5 to 10 ata temperature of from 4 to 400C.

10. A process according to claim 10 substantially as hereinbefore described with reference to any one of Working Examples 1,3 or4.

1 A method for the qualitative or quantitative determination of human IgG, which method comprises causing any human IgG contained in a sample of a human body fluid to be brought into contact with composition as claimed in any one of claims 1 to 8 or which has been produced by a process as claimed in claim 9 or 10.

12. A method according to claim 11 wherein the human fluid is urine or serum.

13. A method according to claim 11 or 1 2 wherein the determination is carried out by a microtiter method.

14. A method according to claim 11 substantially as herein before described with reference to any one of Working Examples 2, 3 or 5.