GB2058083A - Composition and method for determining antibody - Google Patents

Abstract

A composition for determining streptococcal nicotinamide adenine dinucleotidase antibody is composed of synthetic polymeric latex particles and streptococcal nicotinamide adenine dinucleotidase coated on the surface of said latex particles. Streptococcal nicotinamide adenine dinucleotidase antibody is determined by contacting a humor sample with said composition. The antibody can be determined, quantitatively and qualitatively, with accuracy and precision using the composition, without the need to resort to enzymic assay methods.

Description

SPECIFICATION Composition and method for determining antibody This invention relates to a composition for determining streptococcal nicotinamide adenine dinucleotidase (NADase) antibody and a method for determining same.

Specifically, the invention relates to a composition for determining streptococcal NADase antibody, the use of which, as contrasted with the extremely complicated, time-consuming and costly enzymic assay means that has been practiced hitherto, makes it possible to determine, both quantitatively and qualitatively, with accuracy and precision the streptococcal NADase antibody contained in a human sample by an easy assay means using a simple instrument. In addition, not only an accurate analysis of the hemolytic streptococcus infection can be made, but a differentiation from these disorder of other diseases that are diagnosed as false positive is also made possible. This invention also relates to a method of determining NADase antibody.

More specifically, this invention relates to a composition for determining streptococcal NADase antibody, the composition being composed of synthetic polymeric latex particles and NADase coated on the surface of said particles.

It has been known that hemolytic streptococcus, especially the group A hemolytic streptococcus, specifically Streptococcuspyogenes, was communicated from person to person to cause such serious diseases as scarlet fever, rheumatic fever, acute glomerulonephritis, etc.

However, excepting for a part of the diseases that are caused by hemolytic streptococcus, such as scarlet fever, which exhibits typical symptoms, clinical symptoms characteristic of the disease are not usually seen in the case of the disorders caused by hemolytic streptococcus. Hence, for diagnosing these diseases an examination means utilizing a serological reaction for determining the streptococcus antibody is relied upon at present.

In the serological examination method presently employed the value of the antibody to streptolysin 0 (SO), the exo-toxin produced by hemolytic streptococcus, is measured, i.e., the value of antistreptolysin 0 (ASO) is measured. However, the shortcoming of this method resides in the fact that, as is well known, even though the disease is one caused by hemolytic streptococcus, the frequency with which the ASO titer shows a significant rise is not necessarily high. On the other hand, there are times when a high ASO value is exhibited non-idiosyncratically, i. e., a false positive indication is shown.

Hence, there has been a desire for a means by which the disorders caused by hemolytic streptococcus can be diagnosed with still greater sensitivity and reliability. Again, for precluding an erroneous diagnosis ascribable to a false positive indication, it is recommended that measurements be made of the antibody to extracellular products of hemolytic streptococcus other than SO, e.g. such enzymes as hyauronidase and NADase, i.e. the hyauronidase antibody (AH), the antibody to hyauronidase, and the NADase antibody (ANADase), the antibody to NADase, and that the diagnosis be made of the disorders caused by hemolytic streptococcus from these two measurement values.A marked improvement in the reliability and accuracy of the diagnosis can thus be achieved by carrying out the diagnosis on the basis of these two measurement values, and it becomes possible to accurately distinguish between the diseases that are caused by hemolytic streptococcus and other disorders that are diagnosed as false positive.

NADase is one of the most important enzymes of those that participate in the antigen-antibody reaction and is a protein that is produced in the culture fluid of hemolytic streptococcus. This NADase splits the nicotinamide adenine dinucleotide (NAD) into nicotinic acid amide and adenosine diphosphate ribose.

The enzymic method is the only method that is used at present in determining NADase antibody. A method that is based on a true immunological reaction is not known at all. That is to say, there has been no proposals whatsoever as regards the determination of NADase antibody utilizing passive latex agglutina tion.

The foregoing method of determining ANADase (which is the only one used presently) is mentioned in Peterson, K. F., Z. Hyg. Infectionskr. 147:357 (1961). This method is carried out in the following manner. After mixing NADase with the antibody in test tubes, a part of the NADase is neutralized, and the NADase remaining is reacted with the substrate. The amount of remaining NADase is measured, and the amount of ANADase is calculated. The drawback of this method resides in the fact that the NAD used as the substrate and the alcohol dehydrogenase (ADH) used for measuring the NAD are expensive reagents. In addition, the 7measuring operation itself is extremely complicated and time-consuming and hence cannot possibly be used for routine examination. As a consequence, it is hardly being used.

Our researches were carried out with the view of developing a method for determining ANADase in which these drawbacks of the conventional method have been completely overcome. As a consequence, we succeeded in developing for the first time a way of measuring ANADase by the passive agglutination method.

It has been found in accordance with our studies that by utilizing purified NADase obtained from a culture filtrate of an NADase-producing strain, such as Streptococcuspyogenes, it is possible to sensitized the synthetic polymeric latex particles with said purified NADase and further that by using this composition composed of synthetic polymer latex particles and the purified NADase coated on the surface of said latex particles it is possible to determine ANADase accurately with a high degree of sensitivity and reliability and moreover promptiy with an extremely simple operation by means of the passive latex agglutination reaction in accordance with the microtiter method or the slide glass plate method.

There is also the advantage that this method can be carried out at far lesser cost than the conventional method in which the use of the costly reagents such as NAD and ADH was indispensable.

An object of the present invention is therefore to provide a novel and excellent composition-for use in determining ANADase. Another object is to provide a novel and excellent method for determining ANADase.

These and other objects and advantages of the present invention will become apparent from the following description.

Various synthetic latex particles can be used for the production of the composition for determining ANADase of this invention. Examples of polymers or copolymers that form such a latex include the polymers or copolymers derived from a monomer or monomers selected from the group consisting of styrene, chlorostyrene, alkylstyrene, vinyltoluene, divinylbenzene, vinylpyrrolidone, acrylic acid, alkyl acrylate, methacrylic acid, alkyl methacrylate, acrylonitirile, vinyl chloride, vinyl acetate, vinylidene chloride and butadiene, or the carboxylated or amino-containing carboxylated products of these polymers or copolymers.

As specific examples of such polymers or copolymers, there can be named say polystyrene, carboxylated polystyrene, amino-containing carboxylated polystyrene, polychlorostyrene, polyvinyltoluene, styrenebutadiene copolymer, carboxylated styrene-butadiene copolymer, styrene-divinyl benzene copolymer, vinyltoluene-tertiary butyl styrene copolymer, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyrrolidone and vinyl chloride-acrylate copolymer.

Preferred particles of synthetic polymeric latices are the particles of styrene-type synthetic polymeric latex selected from the group consisting of styrene polymer, chlorostyrene polymer, styrene copolymers such as a copolymer of styrene with chlorostyrene, methyl methacrylate and vinylidene chloride, and the carboxylated or amino-containing carboxylated products thereof.

These synthetic polymeric latex particles can be used after pretreating their surfaces with a nonionic surface active agent. For example it is preferred to use these particles after causing the absorption thereto of an ethylene oxide-type nonionic surfactant in accordance with the method described in Japanese Laid-Open Patent Publication No. 9716176 laid open on January 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 alkyl-aryl 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, and especially 0.3 to 0.9 micron, to achieve a reduction in the measurement time. The specific gravity of the latex particles is preferably about 0.9 to 1.4, more preferably about 1.1 to 1.3, and especially 1.2 1 0.05. In the case where the determination is performed utilizing an agglutination reaction on a slide glass plate, latex particles having a broad range of specific gravities can be used. In the case of the microtiter method, it is preferred to use latex particles having a specific gravity of at least 1.1.

As the NADase to be used for sensitizing the synthetic polymeric latex particles such as described hereinabove, there can be used, for example, that which has been separated from a culture filtrate of a NADase-producing strain such as Streptococcuspyogenes and purified. Alternatively, usable is also that which has been separated from a commercially available NADase-containing material such as VARIDASE (a product of Lederle, Ltd., Japan) and purified.

The NADase-producing strains belonging to the genus Streptococcus, their method of cultivation, and the method of collecting the NADase from the culture filtrate are known, and these methods can be used in the present invention.

As is well known, NADase is produced from practically all of the strains of the groups A, C and G of the genus Streptococcus. Hence, NADase can be obtained by using any one of these strains. Needless to say, it is preferred to use one whose amount of accumulation of NADase is the greatest. Examples of such strains are that which can be obtained as Streptococcuspyogenes, 058, (Richards) from the Institute of Medical Science, University of Tokyo, Japan (located at Shiroganedai, Minato-ku, Tokyo, Japan) and that which can be obtained as Streptococcus pyogenes ATCC 10389 from the American Type Culture Collection (located at Rockville, Maryland, U. S. A.).

NADase can be produced by cultivating such known freely available streptococci in say such known culture media as the Todd-Hewit medium, veal infusion medium, trypticase soy medium, etc. under such known cultivation conditions as temperature ca. 37"C for a cultivation time of about 16 to about 24 hours.

From the standpoint of the amount produced, the Todd-Hewit medium is preferred. These media are available commercially. For example, the Todd-Hewit medium can be obtained from Difco Laboratories (U.

S. A.). The collection of the NAdase from the culture broth can be performed by known means. For example, the crude NADase can be obtained by separating the bacterial cells from the culture filtrate using a centrifuge at same about 3000 - 5000 rpm for about 10 minutes to about 30 minutes.

The crude NADase may contain DNase B (deoxyribonuclease B), hyauronidase, steptokinase, proteinase, etc. Hence, the so obtained crude NADase is purified before using it. The purification may be performed say buy a salting out method with a half-saturation of ammonium sulfate, a gel filtration method with Sephadex G-200 (product of Pharmacia Fine Chemicals, Sweden), a DEAE-Sephadex (a product of Pharmacia Fine Chemicals, Sweden) a chromatography method with Sephadex A-50 (a product of Pharmacia Fine Chemicals, Sweden), a column chromatography method with 2',5' ADP-Sepharose 4B (product of Pharmacia Fine Chemicals, Sweden) column, or a combination of these methods. The chromatography method with 2',5'ADP-Sepharose is preferably employed.Either this method alone, or a combination of the method with any of the other above-exemplified methods can be recommended.

The above chromatography utilizing ADP-Sepharose can be performed say in the following manner. The crude NADase obtained in the manner described above is applied to a column of ADP-Sepharose and caused to be selectively adsorbed thereon. The impurities are then washed out by flowing down say, a 0.02 M tris-buffer having a pH about 7.4, following which a buffer similar to the foregoing buffer but containing about 1 M of sodium chloride is used, and the NADase is eluted. Or, it is possible to utilize a commercially available produce such as Varidase (Product of Lederle Ltd.) after purifying it in like manner.

The ANADase determining composition of this invention can be produced by a simple procedure. For example, it can be obtained by contacting the latex particles and the antigen (NADase) in a suitable aqueous medium such as water, a physiological saline, various buffer solutions, etc.

The buffer used may, for example, be a phosphate-buffered saline such as a M 60 phosphate buffer (pH 7.5) containing 0.15 M NaCI (PBS) ora glycine-buffered saline.

The above contact can be carried out in the following manner. The NADase and a suspension of the latex particles are contacted by mixing them in an aqueous medium at say a pH of about 6.4 to about 7.6, more preferably a pH 7 1 0.5, and particularly a pH about 7, at about 4 to about 30'C, preferably about 4 to about 20"C for about 30 minutes to about 24 hours, and if desired, with stirring or shaking. The contact is carried out at preferably a concentration of the latex particles of about 0.05 to about 30o by volume, more preferably about 0.5 to about 1% by volume, and preferably a concentration of the NADase of about 100 to about 1000 u ml.

After having sensitized the latex particles with the NADase in the above manner, it is also possible to saturate the unadsorbed portion of the NADase on the surface of the latex particles by treating the latex particles similarly with say a bovine serum albumin.

After the coating reaction, the reaction mixture is washed with say PBS. The NADase not absorbed to the latex particles can be completely removed by washing.

The thus obtained composition composed of synthetic polymeric latex particles and NADase coated on the surface of said latex particles can be used for the determination of ANADase in a state of suspension in a diluent. The composition is preferably used as a suspension in the diluent in a concentration of the order of about 0.25 to about 0.5% by volume. As diluent, that obtained by adding about 0.1 > O of BSA to say a glycine-buffered sodium chloride solution or a phosphate-buffered sodium chloride solution to which has been further added 0.01 - 0.5SO of sodium azide (NaN3) may be used.

The NADase-coated latex particles obtained are stable and may be kept stored in a refrigerator suspended in the diluent, or they may be lyophilized.

In carrying out the lyophilization, various amino acids, preferably such as glycine, sodium glutamate and or dextran in such amounts as 0.2 - 20c by weight in the case of the amino acid and 0.3 - 30o by weight in the case of dextran may be added to the diluent as stabilizers, after which the NADase-coated latex particles may be quick-frozen in liquid nitrogen or liquid air and then lyophilized. The preservation period is further prolonged by lyophilization, and the antigen-coated latex particles that have been lyophilized can usually be stored stably for about two years or more. And the lyophilized product can be used in the same manner as in the case with a fresh product by just adding a diluent and dissolving it.

Since the NADase-coated latex particles of this invention is agglutinated by ANADase, the latex particles are added to a human humor sample such as human serum or its dilution and allowed to react with the ANADase. The agglutination pattern can then be read.

According to this invention, there is provided a method for determining ANADase by utilizing the foregoing reaction. The method consists in qualitatively or quantitatively determining ANADase in human serum using a composition comprising synethetic polymeric latex particles and NADase coated on the surface of the latex particles.

In performing the determination, means known per se can be utilized, and for example, the level of ANADase in the serum can be easily determined by the microtiter method According to this method, a given 'amount of a diluent is poured portionwise into a microplate, and then a given amount of an assay sample such as the serum is introduced into the first well of the plate and made serial twofold dilutions with a diluter.

This is followed by the addition and mixing of a given amount of the NADase-coated latex particles. After allowing the mixtures to stand for a given period of time at room temperature, the end points of agglutination are observed.

As the diluent or diluter to be used in forming the foregoing dilution series, there can be used that obtained by adding about 0.1"0 of BSA to say a glycine-buffered sodium chliride solution or a phosphate-buffered sodium chloride solution to which has been further added 0.01 to 0.5SO of sodium azide (NaN3).

The ANADase determining composition of this invention in the form of a latex or dried product has the following extremely superior advantages. Specifically, an antibody against carrier cannot possibly be present. In fact, it has not been found. Hence, no pretreatment whatever need be administered to the humor to be tested, not is it necessary to carry out a test to confirm such a pretreatment. The operations for the determination consists of only placing the humor or its dilution in a well of a microplate and thereafter adding the latex dropwise. Thus, the determination of the ANADase can be performed very easily with a simple procedure, and no special technique is required. Moreover, since the NADase is one that has been highly purified, it is extremely specific. In addition, the sensitivity of the test is fully satisfactory for measuring human humor.It is more than 10 times sensitive than the aforementioned enzymic method, the only known conventional method. Again, it is possible to carry out the qualitative and quantitative determinations of a number of samples simultaneously.

The determination of the concentration of ANADase in the humor, which had to be performed hitherto by the complicated enzymic method, can now be carried out easily in a short period of time and at low cost by means of a simple procedure when the NADase-coated latex particles of this invention are used. It thus becomes possible to conduct an accurate etiologic analysis of patients, as well as to make evaluations of the results of treatments administered and a prognosis of the disease. In addition, the determination of ANADase, which could not be carried out customarily or routinely by the conventional enzymic method, can now be performed readily and inexpensively anywhere as a routine test. Hence, the contribution made by this invention to diagnosis is extremely great.

From the fact that this NADase-coated latex only reacts with the ANADase antibody and does not react at all with antibodies other than ANADase, and that a latex uncoated does not react at all with the ANADase antibody and antibodies other than ANADase, it can be said that NADase is bonded to this latex as a coating.

The invention will now be more fully described by reference to preparation and working examples. It is to be understood that this invention is to be in no way restricted to these examples except as defined in the appended claims.

Preparation ofNADase A 5-liter Erlenmeyerflask containing 1 liter of Todd-Hewit Broth (product of Difco Laboratories, U.S.A.) was inoculated with Streptococcuspyogenes, Richards strain, and the cultivation was carried out by allowing the inoculated medium to stand at 37"C for 18 hours. After completion of the culture, the broth was centrifuged at 10,000 rpm for 30 minutes followed by taking the supernatant, which was designated the culture filtrate.

Ammonium sulfate was added to this filtrate to an 0.5 saturation to form a precipitate, which was separated, dissolved in distilled water and dialyzed. This was then applied to a column (1.5 x 33cm) packed with 2',5'ADP-Sepharose 4B (product of Pharmacia Fine Chemicals, Sweden) and washed with an 0.02 M Tris buffer (pH 7.4) after which the elution was carried out with 0.02 M Tris buffer containing 1.0 NaCI. After separating the NADase fraction gel filration was further performed with Sephadex G-200 to separate an NADase fraction. Salting out was then carried out with ammonium sulfate of 0.8 saturation, and the precipitate was dissolved in distilled water followed by dialysis to give 10 ml of a solution containing 2.8 x 105 units of NADase. The presence of hyauronidase, streptokinase, streptolysin 0 and DNase could not be detected in this sample.

EXAMPLE 1 Sensitization of latex particles with NADase (1) A 5% suspension of Latex SDL 59 (product of Takeda Chemical Company) was diluted to an 0.25% suspension with M 60 PBS1' of pH 7.2. One volume of NADase diluted to 500 u'ml with PBS was then mixed with the suspension, and the sensitization was carried out at room temperature for 3 hours. The mixture was lightly shaken frequently during the meantime.

(2) The latex was then washed three times with PBS followed by suspending it in one volume of a diluent2' to prepare the sensitized latex.

(3) If the sensitized latex is to be lyophilized, this can be accomplished by suspending it in 0.2 volume of a lyophilizing medium3' followed by quick-freezing the suspension in liquid nitrogen and then lyophilizing it.

1) PBS M 15, pH 7.2 phosphate buffer ........................................ 25 ml Physiological saline ........................................ 75 ml Sodium azide .. ........................................ 0.1 g 2) Diluent PBS ........................................ 100 ml Bovine serum albumin ........................................ 0.07 g Sodium azide . 0.1 g 3) Lyophilizing medium Diluent ........................... ......... .................... ....... 100 ml Glycine ................ ....... .. .................. ......... ............ ............. 0.5g Dextran (molecular weight 200,000 - 300,000, Dai-ichi Chemical Co.) . .............................................. ...... 0.7 9 EXAMPLE 2 Passive latex agglutination reaction resulting from the invention latex reagent I. Agglutination reaction by means of the microtiter method (1) When solids are present in the fluid to be tested such as serum or other humors, these are removed by centrifugation, but otherwise the fluid is not treated.

(2) When the latex reagent is a lyophilized product, it is dissolved by adding a diluent.

(3) The determination of the NADase antibody is conducted in the following manner. To a microplate (which may be of either the U-type or V-type) is added portionwise 0.025 ml of a diluent to each of the wells with an 0.025-ml dropper. To the first well is then added 0.025 ml of the fluid to be tested. After performing a serial twofold dilution with a diluter, the latex reagent is added dropwise is an amount of 0.025 ml to each of the wells. After thorough mixing of the mixtures, they are left standing overnight at room temperature. The following day the agglutination patterns of the well bottoms are judged, and the reciprocal of the maximum dilution of the fluid tested exhibiting positive agglutination is designated the antibody titer.

If only qualitative determination is required, it is only necessary to add dropwise 0.025 ml of the latex reagent to the well in which has been poured only 0.025 ml of the fluid to be tested, following which the agglutination pattern can be judged in the same manner as described above.

II. Agglutination reaction on a slide glass One drop of serum is dropped onto a slide glass and, after dropping one drop of the latex reagent on top of this, the two are spread out with say a toothpick to a 2-cm square. The slide is then gently shaken for 3 minutes, and the occurence of agglutination is judged. Agglutination denotes the presence of an antibody, while nonagglutination denotes the absence of an antibody. When the indication is positive, it is preferred that a further determination be made by the microtiter method.

Comparison of the enzymic method with the passive latex agglutination method in accordance with the microtitermethodofthis invention Concentration Antibody value of sensitizing antigen 100 200 400 800 1600 3200 Diluent 800 u.'ml + + + + + - 400u1ml + + + + + 200u.ml + + + + + - - 100uml + + - - - - 50 u ml - - - - - - - +: Agglutination, i.e. a positive indication.

-: Nonagglutination, i.e. a negative indication.

The antibody used in this case was a patient's serum having a titer of 160 by the enzymic method.

As shown in this table, the latex agglutination method exhibited a sensitivity about 10 times that of the enzymic method. Moreover, it was completely negative to the diluent. Again, an unsensitized latex was negative. Further, that this was s specific reaction was confirmed from the fact that the antibody value could not be detected in both the agglutination reaction and the enzymic reaction when the antibody was neutralized in advance by the addition of purified NADase.

Claims (10)

1. A composition for determining Streptococcal nicotinamide adenine dinucleotidase antibody, said composition being composed of synthetic polymeric latex particles and Streptococcal nicotinamide adenine dinucleotidase coated on the surface of said 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 2 wherein the latex particles have an average particle diameter of from 0.1 to 1 micron.

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

5. A composition according to claim 4 wherein the latex particles have a specific gravity of from 1.1 to 1.3.

6. A composition according to any one of the preceding claims wherein the synthetic polymeric latex particles are particles of a polymer or copolymer derived from a monomer or monomers selected from styrene, chlorosytrene, alkylstyrene, vinyltoluene, vinylpyrrolidone, divinylbenzene, alkyl acrylate, alkyl methacrylate, acrylonitrile, vinyl chloride, vinyl acetate, butadiene and vinylidene chloride, or are of carboxylated or amino-containing carboxylated products ofsuch polyers or copolymers.

7. Acomposition according to claim 1 substantially as described with reference to Example 1.

8. A method for determining streptococcal nicotinamide adenine dinucleotidase antibody which comprises contacting a humor sample with a composition as claimed in any one of the preceding ciaims.

9. A method according to claim 8 wherein the determination is carried out by the microtiter method.

10. A method according to claim 8 substantially as described with reference to Example 2.