IE66674B1 - Method for the quantitative determination of serum proteins in body fluids and agents for carrying out the process - Google Patents

Abstract

The method for detecting or determining a participant in a particle-enhanced immunological reaction by a nephelometric, turbidimetric or particle-counting method comprises carrying out the detection or determination in the presence of an antiserum which contains no antibodies which are specific for one of the immunological reactants.

Description

The Invention relates to a method for the detection or determination of a participant in a particle-amplified immunological reaction by a nephelometric» turbidimetric or particle-counting method, an agent suitable for this and the use of an antiserum with a specificity which deviates from the immunological reaction in such a method» It is known that the sensitivity ©fi serological or immunological determination methods can be increased by using indicator particles or carrier particles charged with the corresponding immunological reagent (an antibody or antigen) » Red blood corpuscles or cells of a cell culture, for. example, can be used as the carrier material» Latex particles with a diameter of 0,02 to 5 /.oa are also used for this» Such &. 'particle-amplified®' nephelometric or turbidimetric test can reliably detect proteins up to concentrations of about 5 ng/xal. Antibodies or antigens bound to polymers in particle form (89 solid phase) are used in such a particle-amplified test» A solid phase-bound antibody is used to determine an antigen, and a solid phase-bound antigen is used to determine an antibody. In both cases, agglutination of the polymer particles occurs as a result of the immune reaction» Thi® results - in- an increase In the size of the agglutinates and the scattered light signal or the turbidity of the reaction batch increases» Patent Abstract of Japan (Volume 9, Noi 186 (P-377) (1909] 2 August 1985) describes the suppression of nonspecific reactions of sans samples in the agglutination method by addition of heat-degenerated IgG.

In document DE-A-2, 126,128 gamma-globulin of aonhwnaa origin Is used for this.

As inhibitor substance, GB-A-2,052,059 uses aoaiaaniae sera of that animal species which was used for the production of the detection antibodies.

EP-A-0,083,869 uses antibody fragments of specific type as inhibitor substance.

A latex agglutination method using gaama-globulins without antibody specificity in respect of one reaction participant involved in the reaction Is known from EP 0,087,728» These are carried out by mixing a latex reagent with th© sample to be detected in a drop on a platelet of glass or plastic. A positive reaction causes the latex reagent to agglomerate and flocculate, and the milky appearance of the latex suspension disappears. Such methods allow a qualitative conclusion. They can be evaluated subjectively by the eye, but not by an optical system. In addition, they cannot be automated.

Latex particles which contain acetal functions bonded via acid amide groups ar© known from European Patent Application EP-A-0,080,614. Such particles can be used for the nephelometric determination of C-reactive protein. For this, serum samples are diluted with buffer, usually Is 100, whereupon the Influence of Interfering serum proteins which would otherwise lead to false results becomes negligible. This procedure is possible because in general concentrations of C-reactive protein of more than 5 mg/1 must ba present for diagnostic purposes. However, if th© concentration of trace proteins In the range from 1 pxg/l to 5 μ%/1 is to be measured, the samples should not be correspondingly diluted with buffer, because otherwise the concentration of the protein to be detected becomes so low that the detection sensitivity is not sufficient. EP~A 0,080,614 describes the use of TWEEN (0.2% and 1.7%).

An Increase in the detection sensitivity in latex preparations according to the prior art from sera diluted only 1:5, for exampl©, is not readily possible, however, and for the determination of alpha-fetoprotein (AFP) or immunoglobulin B, for example, does act give a test which functions satisfactorily.

Ia the particle-amplified nephelometric or turbidimetric test»· Immune reactions take place oa solid phases. It is a property of these solid phases to he able fo adsorb non-specifically proteins which may interfere from the body fluids investigated, another difficulty which may ax'ise in such methods is that human serum contains the protein Clq and rheumatoid factors (RF). These bond to antibodies. The amounts of rheumatoid factor and Clq in human sera can also vary within wide limits, which is why it is usually necessary first to subject the sera fo treatment for inactivation of Clq or to remove rheumatoid factors. False concentration values for trace proteins in body fluids otherwise result. Reliable determination with the aid of nephelometric or turbidimetric methods is therefore impossible.

The addition of gamma-globulins without antibody specificity in respect of one reaction participant Involved in the reaction, which is proposed in EP 0,087,728 for latex agglutination methods which can be read visually. Is not sufficiently effective for nephelometric and turbidimetric measurements for the interference of sera with a high content of rheumatoid factors also to be suppressed.

It has now been found, surprisingly, that the abovementioned difficulties caused by non-specific agglutination of the particles in such, a test system can be prevented by carrying out th© test In the presence of a dilute antiserum which reacts or react neither with the antigen or antibody to be determined nor with th® participant bound to th© particles, and in the presence of ®Tweea 20.

The invention relates to a method for the detection or determination of a participant in a particle-amplified immunological reaction by a nephelometric, turbidimetric or particle-counting method, in the presence of an antiserum which contains no antibodies specific for one of the immunological reaction participants, which comprises carrying out the detection or determination in the presence of 0.05 to 2 ml/100 ml of ®Tween 20.

Animal gamma-globulins or heat-aggregated human gammaglobulins are suitable as antiserum. Such an animal gamma-globulin is, for example, an anti-sheep erythrocyte serum from a mammal and preferably an anti-sheep erythrocyte serum from rabbits. Gamma-globulin fractions which can be obtained by means of known processes, for example ammonium sulfate precipitation or ion exchange chromatography, are also such gamma - globulins. Examples of suitable particles which can be charged with one of the participants in an immunological reaction fox’ use in the method according to the invention in order to obtain so-called "reagents'5 for this purpose are particles of latex dispersions.

Such latex particles should consist of "'non-fiilmformingB polymers. Non-filmforming its to be understood as meaning polymer latex particles which do not form a film under the use conditions in question here «and do not run together. Polymers of carbocyclic aromatic monovinylidene monomers, such as styrene, vinyl toluene or vinylnaphthalene and mixtures of these monomers with one another and/or with methyl methacrylate and acrylonitrile, are preferred. Particularly preferred seed dispersions are polystyrene latices.

The method according to the invention is carried out in the presence of 0.05 to 2 g/100 ml of ®Tweea 20 and/or 0.5 to 3 g/100 ml of a neutral salt, preferably sodium chloride.

A participant in an immunological reaction can be applied to the particles by adsorption or by· covalent bonding and, th© particles can to© Hcharged® in such a manner. Coupling of th© particles with aa antigen or antibody can toe carried out by a known method.

Th© particles are preferably charged with antibodies 5 against serum proteins, such as alpha-fetoprotein (AFP), myoglobin, beta-2-microglobulin or immunoglobulin S, human hormones, such as human choriogonadotropin, enzymes, such as pancreas lipase, or animal hormones, such as pregnant mare serum gonadotropin.

A method according to th© invention wherein antibodies against alpha-fetoprotein (AFP) or immunoglobulin E (IgB) ar© covalently bonded to latex particles Is particularly preferred.

Xf AFP Is measured, by the method o£ the prior art, such as Is described, for example, in EP-A-0,080,614, that is to say without the addition of an antiserum, clearly measurable apparent values of AFP are obtained for sera containing rheumatoid factors (Table 1), whereas the test procedure according to the Invention, ©specially if anti20 sheep erythrocyte serum from rabbits Is used, gives results which coincide completely with the enzyme iseaunoassay (EXA).

Th© addition of anti-sheep erythrocyte serum from rabbits does not interfere with the measurement of AFP actually present in corresponding serum samples, as shown, in Table 2» - 6 Table Is Nephelometric determination of AFP ia 15 asra containing rheumatoid factors.

RF content (lu/ml) Alpha-fetoprotein content (lu/ml) nephelometric in EIA apparent according prior art to measurement according to the invention 711 *3a) 19 *7b) 2768 *3 96 *7 474 *3 16 *7 578 3 19 *7 251 '3 7 *7 543 *3 *7b) *7 2760 *3 13 *7 446 *3 23 *7 178 *3 20 *7 2078 *3 46 *7 191 *3 18 ’7 1626 *3 50 "7 ' 158 "3 31 *7 248 3 10 *7 513 *3 57 ’7? denotes l ) The AFP less than1·’ content ia very low and therefore not measurable by enzyme immunoassay, b) Cssnot be evaluated, but. value below the measurement range correctly found. .-7All the sera were also tested in an ensyme immunoassay (SIA) and in the method according to the invention. In the sera tested, the concentration of AFP is low, and cannot even he measured by EIA. .As Table . 1 shows, nephelometric determination according to the prior art gives apparent concentrations of AFP (falsely positive values) . When tested by the method according to the invention, all the sera gave values below the measurement range, that is to say no falsely positive values, in agreement with the ΞΙΑ.

Table 2:. Nephelometric determination of. AFP in 15 sera containing AFP.

AFP content in EIA AFP content determined according to the invention (Ιυ/ml) (TU/ml) 50.9 53.0 168.2 173.0 56.4 58.1 68.8 67.9 44.6 42.0 169.0 149.0 46.3 41.5 27.0 25.4 19.8 14.4 11.3 11.2 29.4 26.5 14.6 10.0 33.2 31.4 89.4 103.0 127.0 154.0 All the sera were also tested by an easyme iaBnmoassay (HIA) and in the method according to the invention. In the sera tested, the concentration of AFP in both methods is largely ia agreement in the context of the slight deviations, familiar to the .expert, of different test methods.

Falsely high IgS values for sera of rheumatic patients are similarly found ia the IgE test carried out according to the prior art (Table 3) . This Table also shows the results for the test procedure according to the invention. The advantageous effect of an addition of antisheep erythrocyte serum from rabbits Is likewise found here. Results which show good agreement with those of the enzyme immunoassay are obtained in this manner.

Table 3 s Nephelometric determination of IgE In 15 sera containing rheumatoid factors.

RF content Immunoglobulin E (IgE) content (IU/ml) (lU/ml) nephelometric In EIA apparent according according to to prior art the Invention 474 44 102 47 2475 120 486 179 652 1X5 547 178 1501 342 515 293 2332 147 494 202 478 57 126 62 400 32 171 74 1940 124 483 176 1750 37 150 38 199 32 304 35 696 176 326 211 74 9 47 *35 202 103 187 98 53 1 *35 *35 140 4 83 *35 denotes "less than® Result: Values which are much too high are found nephelometrically according to th© prior art. Th® average deviation of the results of the nephelometric test from the values of the enzyme immunoassay is about 370%.

The values found by the method according to the invention ar® usually correct.· The average deviation of the results of th© nephelometric test according to the invention from the values of the enzyme immunoassay Is In this case XO about 25%.

Th© reagent particles mentioned can be coupled with the antigens or antibodies by a known method.

Preferably, the latex is charged with antibodies against serum proteins, such as alpha-fetoprotein, myoglobin, beta-2-microglobulin or immunoglobulin IS, human hormones, such as human choriogonadotropin, enzymes, such as pancreas lipase, or animal hormones, such as pregnant mare serum gonadotropin.

The antisera used are prepared by immunization of ani20 male, in particular rabbits, sheep and goats, with a protein of human or animal origin, which should not contain the protein to be determined ia the test.

Examples ares anti-human IgG serum from rabbits, antihuman IgM serum from rabbits, anti-sheep erythrocyte serum from rabbit®, anti-human IgG serum from sheep end anti-rabbit gamma-globulin serum from sheep. Anti-sheep erythrocyte serum from rabbits is particularly suitable. The immuni ration «as carried out by known methods. The immunization dose and time are obtained from th© immunogenic ity and the molecular weight of the protein.

The antibody solutions used contain no antibodies against the animal antiserum bound to the latex particles. They are usually from the same animal species.

Such an antiserum or such a gamma-globulin solution is added to the solution in which an antigen or an antibody is to be determined or detected.

Such an antiserum is added in an amount such that it is present in th® test mixture in & concentration of between 50 and 0.05 ml/100 ml. A concentration In the test batch of . between 10 and 0.1 ml/100 ml is more advantageous. A concentration of between 2 and 0.2 ml/100 ml in the test batch is particularly advantageous.

A concentration of 0.05 to 2 ml/100 ml of tha nonionic detergent eicosa oseyethylene sorbitan laurate (®Tween 20) in the measuring cell is additionally [lacuna].

The method according to the invention can be used for the detection of ail immunologically active substances which are contained in the blood (sens or plasma) of mammals, in particular humans. Examples of such isonunologically active substances are serum proteins.

For th© method according to the invention, an agent is added to the test cell. This agent contains 90 to 0.1 ml/100 ml of an antiserum which contains no antibodies specific for one of the immunological reaction participants. The agent particularly advantageously contains 20 to 1 ml/100 ml of the antiserum mentioned. Th© agent also contains 50 to 0.1 ml/100 ml of ®Twsea 20, advantageously 20 to 0.5 ml/100 ml of ®Tween 20.

Example 1 1. Preparation of seed polymer for the latex 310 ml of nitrogen-saturated’ double distilled water were introduced Into a cylindrical glass vessel fitted with a gas inlet and a gas outlet tub© and a magnetic stirrer rod. 500 mg of sodium stearate were added and dissolved by stirring. 1.5 ml of ammonia (25 g/100 ml) were furthermore added. The pH was checked and was 11.09. The polymerization vessel was rendered oxygen free by evacuating and filling with nitrogen several times. The detergent solution was heated to *70°C with th® aid of a waterbath, with continuous stirring. 90 ml of freshly distilled styrene were then introduced into the polymerization vessel under nitrogen, with the aid of a dropping funnel with pressure compensation. The mixture was stirred at *70°C for a further 15 minutes for emulsification of the styrene. The temperature was then raised to *9Q°C and the mixture was stirred for a further hour. 67.5 mg of potassium peroxydisulfate, dissolved in 50 ml of nitrogen-saturated distilled water, were then added. The mixture was stirred at *90 °C for 130 minutes. The polystyrene was passed through a fluted filter. The filtered polystyrene was dialyzed against 10 liters of ammonium bicarbonate solution (0.01 g/100 ml of NH4HCO3; 0.01 g/100 g of NaN3,brought to pH 10 with 10.5 ml of ammonia 25 g/100 sal in 10 1) for 50 hours. After the dialysis, 410 ml of polymer with a dry weight of 17.9 g/100 ml were obtained. 2. Polymerization of 2-hydroxypropyl methacrylate (ΞΡΜ) oa polystyrene nuclei The polymerization wae carried out in a vessel in a similar way as described in Example 1. A mixture of 22.4 ml of polystyrene latex with a solids content of 17.9 g/100 g, 56.7 ml of distilled water and 50 mg of sodium dodecyl sulfate was prepared. This was introduced into the polymerization vessel and the oxygesn was removed. 1 ml of a potassium peroxydisulfate solution (16. mg/ml in distilled water) was furthermore added and the batch was heated to *70°C.

A mixture of 0.4 al of styrene, 0.4 »1 of methacrylamidoacataldehyde di-n-pentylacetal, 0.025 xal of methacrylic . acid and 0.2 ml of 2-hydroxypropyl methacrylate (HPM) was px'epared. The monomer mixture was slowly added dropwise to the vigorously stirred polystyrene latex suspension at -f-70eC for 60 minutes. Stirring was then continued at the same temperature for a further 4 hours» After cooling to room temperature and filtration through a fluted filter, 73 ml of the polymer were obtained. The polymer was then dialyzed against NaHC03 buffer (0.25 g/1, pH 8-8.2) for about 20 hours. 87 ml of a latex dispersion with a solids content of 5.1 g/100 g were obtained. 3. Binding of AFP antibodies to a polymer AFP antibodies were bound, as described below, to a polymer, using 2-hydroxypropyl methacrylate prepared according to 2. The particular polymer used was diluted with distilled water to a solids content of 4 g/100 g. An antiserum obtained by immunization of rabbits with purified AFP was purified by affinity chromatography by known methods. Xt was then concentrated until a protein content of 10 mg/ml was reached. 3.4 ml of the above polymer were mixed with 0.34 ml of the AFP antibody solution. 0.17 ml of a 20 ml/100 ml aqueous solution of eicosaoxyethylene sorbitan laurate (®Tween 20) was then added and th® entire batch was mixed again. 0.05 ml of 1 NT SCI was added so that a pH of about 2 was reached. After an Incubation time of 30 minutes at room temperature, 0.85 ml of saturated aqueous disodium hydrogen phosphate solution (25 mg/ml) was added and the batch was mixed thoroughly. Incubation was then carried out for one hour at room temperature.

V - 13 This charged batch was then centrifuged at about 50,000 g for 30 minutes. The supernatant was discarded. The residue was resuspended in 5 ml of a glycine-NaCl buffer (0.1 mol/1 of glycine, 0.17 mol/1 . of NaCl and 0.5 ml/100 ml of eicosaoxyethylene sorbitan laurate (®Tween 20), pH 8.2). The suspension was then subjected to ultrasonic treatment for 2 seconds. Th© reagent rediepersed in this way was diluted with the above-mentioned glycine-NaCl buffer in a volume ratio of Is 60.

Example 2 Binding of anti-IgE antibodies to a polymer Anti-IgE antibodies were bound to a polymer, using 2-hydroxypropyl methacrylate, prepared according- to Example 1. The particular polymer used was- diluted with distilled water to a solids content of 4 g/100 g. An antiserum obtained by immunization of rabbits with purified IgE was purified by affinity chromatography by known methods. It was then concentrated until a protein content of 10 mg/ml was reached. 3.4 ml of the ahovementioned polymer were mixed with 0.34 ml of the anti-IgE antibody solution. 0.17 ml of a 20 mI/100 ml aqueous solution of ei cos aoxye thyl ene sorbitan laurate (®Tween 20) was then added and the entire batch was mixed again. 0.05 ml of 1 N HCl was added so that a pH of about 2 was reached. After an incubation time of 30 minutes at room temperature, 0.85 ml of saturated aqueous disodium hydrogen phosphate solution (pH 6.5) and 0.85 ml of aqueous sodium cyaao3 0 borohydride solution (25 mg/ml) were added and th© batch was mixed thoroughly. Incubation was then carried out fox' one hour at room temperature.

This charged batch was then centrifuged at about 50,000 g fox' 30 minutes. The etpexaataat was discarded. The residue was resuspended in 5 ml of a glycine-NaCl buffer (0.1 mol/1 of glycine, 0.17 mol/1 of NaCl and 0.5 ml/100 ml of eicosaoxyefchyleaa sorbitan laurate (®Tween 20) , pH 8.2). The suspension was then subjected to ultrasonic treatment for 2 seconds. The reagent redispersed in this way was diluted with the. abovementioned glycine-N%Cl buffer in a volume ratio of l;80.

Example 3 Measurement of AFP concentrations in serum samples The reagent for determination of AFP prepared according to Example 1 by binding anti-AFP antibodies to latex preparations was used for measurement of AFP in sera. Alpha-fetoprotein standard serum (human) for immunoprecipitation with an AFP concentration of 322„000 ng/ml (Behringwerke AG, Marburg, FRG) was used as the standard. The standard was diluted to 1000 ng/ml ia an AFP-free serum pool. This dilution was further diluted stepwise to in each case twice the volume in the AFP-free serum pool. A standard series with decreasing AFP concentrations was thus obtained. The patient sera to be determined were diluted ls5 in a phosphate-sodium chloride buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2HP04 and 0.2 g/100 ml of NaH2PO4) . For the measurement, 80 μΐ of patient serum dilution or standard serum dilution were incubated with 160 μΐ of a reaction buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2HPO.„ 0.2 g/100 ml of NaH,PO4 and 5.6 g/100 ml of polyethylene glycol 6000) and 30 μΐ of the antiserum against sheep erythrocytes from rabbits diluted Is 8 in phosphate-sodium chloride buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of’ Na2HPO4 and 0.2 g/100 ml of NaH2PO4) with 5 ml/100 ml of ®Tween 20 and 60 pi of AFP reagent (Example 1.3) at room temperature for 12 minutes. The results were then measured in a nephelometer (for example ' that of Behringwerke AG) . Tha reference curve for the measurement of th© standard serum was plotted and the measured values IS for the patient sera were evaluated thereon.

Example 4 Measurement of IgE concentrations la sense, samples * Th© reagent prepared according to Example 2 was used for 5 the measurement of IgE in patient sera. This IgE standard contained 1000 ΙΠ/ml. The standard was diluted stepwise to in each case twice the volume in an IgE-free serum, pool.' A standard series with decreasing IgE concentrations was thus obtained.

The patient sera to be determined were diluted in a phosphate-sodium chloride buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2EFO4 and 0.2 g/100 ml of NaH2FO4) . For the measurement, 80 μΐ of patient serum dilution or standard serum dilution were Incubated with 150 μΐ of a reaction buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2Ht?O., 0.2 g/100 ml of NaH2P0A and 5.6 g/100 ml of polyethylene glycol 6000) and 20 pi of the antiserum against sheep erythrocytes from rabbits diluted Is30 in phosphate-eodium chloride buff tar (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2HPO4 and 0.2 g/100 mi of NaH2PO4) with ml/100 ml of ®Tween 20 and 75 pi of IgE reagent (Example 3) at room temperature for 12 minutes. The results were then measured In a nephelometer (for example that of Behringwerke AG) . The reference curve for the measurement of the standard serum was plotted and the measured values for the patient. sera were evaluated thereon.

Claims (7)

1. Patent. Claims

1. A method for the detection or determination of a participant in a particle-amplified immunological reaction by a nephelometric, turbidimetric or particle-counting method, in the presence of an antiserum which contains no antibodies specific for one of the immunological reaction participants, which comprises carrying out the detection or determination in the presence of 0.05 to 2 ml/100 mi of ®Tweea 20 (polyolyoxyethylene (20) sorbitol monolaurate). 2. The method as claimed in claim 1, wherein the antiserum is an aqueous solution of a gammaglobulin. 3 . The method as claimed in claim 1, wherein the antisenna is mammal. an anti-sheep erythrocyte serum from a 4. The method as claimed in claim 1, wherein th© antiserum is rabbit. an anti-sheep erythrocyte serum from a 5. The method as claimed In claim 1, wherein the reaction xs carried out using a latex reagent with a covalently bound antigen, antibody or hapten.

2. 6. Aa agent for carrying out the method as claimed in claim 1, containing 0.1 to 90 ml/100 ml of an antiserum which contains no antibodies specific for one of the immunological reaction participants, and O.i to'50 ml/100 ml of *Tween 20.

3. 7. The agent. as claimed la claim 6, containing 1-20 ml/100 ml of an antiserum which contains no antibodies specific for one of th® immunological reaction participants.

4. 8„ The agent as claimed ia claim 6 or 7 g containing 0.5-3 gf/lGO axl of a neutnal salt.

5. 9„ The agent as claimed ia claim 8, containing NaCl as aeutxal salt.

6. 10. A method as claimed in claim 1, substantially as hereinbefore described and exemplified.

7. 11. An agent as claimed in claim 8, substantially as hereinbefore described and exemplified.