CA1329118C - Method for the quantitative determination of serum proteins in body fluids and agents for carrying out the process - Google Patents

Abstract

Abstract of the disclosure:
A method for the detection or determination of a partner of a particle-amplified immunological reaction by a nephel-ometric, turbidimetric or particle-counting method, which comprises carrying out the detection or determination in the presence of an antiserum which contains no antibodies specific for one of the reaction partners, and an agent suitable for this method are described.

Description

~329~8 FiEHRINGWERKE AKTIENGESELLSCHAFT 87/B 014 - Ma 616 Dr. ~a/Bn Method for the quantitative determination of serum pro-teins in body fluids and agents for carrying out the process ~, The invention relates to a method for the detection or determination of one partner of 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 pro-cess.
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It is known that the sensitivity of serological or immuno-logical determination methods can be increased by using indicator particles or carrier particles charged with the ~;- corresponding immunological reagent tan antibody or anti-gen). 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 ~m are aLso used for this.

~ Such a "particle-amplified" nephelometric or turbidimetric'~ test can reliably detect proteins up to concentrations - 25 of about 5 ng/ml. Antibodies or antigens bound to poly-mers in particles formed ~"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 35 a result of the immune reaction. This results in an increase in the size of the agglutinates and the scattered light sig-nal or the turbidity of the reaction batch increases.

A latex agglutination method using gamma-globulins with-out antibody specificity in respect of one of the reaction ,`
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pArt~ers part~cip~ting ~ the re~ tion i8 ~no~ ~rom C~n~dlan Patent ~o.
1,199,270 (Iasued 14 J~nu~ry 1986). This i~ csrried out by mixi~g a latex re~gent with th~ ~mple to be det~ct~d in ~ drop o~ ~ pl~tolet of glas~ or pl~atic. A positive r~ction c~use~ the latex reagent to agglomer~te And ~ floc~ul~te, ~d the ~llky Appe~rAnc~ of the l~tex s~spenslon dlsappe~rs.
- S~ch method~ allou ~ qual~t~tive con~lus~on. Th~y ~an b~ evaluat~d ~ub~ectively by th~ eye, but ~ot by ~n op~l~al ~yst~. In ~ddition, thsy c~nnot be sutomated.
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Latex particles which cont~in ~cetal unct~ons bonded v~a ~cid a~ide groups ~re kno~ from Canadi~ Patent 1,206,656 ~I~stled 24 June }986). Su~h part1cle~ can b~ used for the ~ph~l~metr~c determln~tion o~ C-reactlv~
protei~. Fo~ this, serum ca~pl~ ar~ diluted ~ith buf~er, usually 1:100, whereupon the influence of interfering serum pro-teins which would otherwise lead to false results becomes negligible. This procedure is possible because in general concentrations of C-reactive protein of more than S mg/l ~ust be present for diagnostic purposes. However, if the concentration of trace proteins in the range from 1 ug/l-to 5 ugtl is to be measured, the samples should not be correspondingly diluted with buffer, because otherwis~
the concentration of the protein to be detected becomes so low that the detection sensitivity is not suf~icient.

An increase in the detection sensitivity in late~ prepara-tions according to the prior art from sera diluted only 1:S, for example, is not readily possible~ however, and for the determination of alpha-fetoprotein (AFP) or immunoglobulin E, for example, does not give a test which functions satisfactorily.

In the particle-amplified nephelometric or turbidimetric test, immune reactions take place on solid phases. It is a property of the solid phases to be able to adsorb non-specifically proteins which may interfere from the body fluids investigated. Another difficulty which may " ~
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arise in such methods is that human serum contains the protein C1q and rheumatoid factors (RF). These bond to antibodies. The amounts of rheumatoid factor and C1q in ; human sera can also vary within wide limits, which is why it is usually necessary first to subject the sera to treat-ment for inactivation of C1q 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 there-fore imPossible.

` The addition of gamma-globulins ~ithout antibody speci-ficity in respect of one of the reaction partners partici-pating in the reaction, which is proposed in EP 0,087,728 for latex agglutination methods wh;ch can be read visualLy, is not sufficiently effective for nephelometric and turbidimetric measurements for the interference of sera with a high content of rheuma factors to be sup-; pressed.
20It has now been found, surprisingly, that the abovemen-tioned difficulties caused by non-specific agglutination of the particles in such a test system can be prevented ` by carrying out the test in the presence of a dilute antiserum which reacts pr re3c-~ neither with the antigen r or antibody to be determined nor with the partner bound to the particles, and if appropriate in the presence of RTween 20.

The invention relates to a method for the detection or determination of a partner of a particle-amplified immuno-logical reaction by a nephelometric, turbidimetric or particle-counting method, which comprises carrying out the detection or determ;nation in the presence of an antiserum which contains no antibodies specific for one of the immunological reaction partners.

Animal gamma-globulins or heat-aggregated human gamma-~' .
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, r~ 1329118 gLobulins are suitabte as such an 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 pro-cesses, 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 partners of an immunological reaction for use in the method according to the invention in order to obtain so-called "reagents" for this purpose are particles - of latex dispersions.
, , , Such latex particles should consist of "non-filmforming"
polymers. Non-filmforming is to be understood as meaning ; polymer latex particles which do not form a film under the use conditions in quest;on here and do not run to-gether. Polymers of carbocyclic aromatic monovinylidene monomers, such as styrene, vinyltoluene or vinylnaphtha-lene and mixtures of these monomers with one another and/
or with methyl methacrylate and acrylonitrile, are pre-ferred. Particularly preferred seed dispersions are polystyrene latices.

If appropriate, the method according to the invention can be carried out in the presence of 0.05 to 2 9/100 ml of RTween 20 and/or 0.5 to 3 g/100 ml of a neutral salt, preferably sodium chloride.
ij A partner of an immunological reaction can be applied to the particles by adsorption or by covalent bondin~ and the particles can be "charged" in such a manner~ Coupling ~il of the particles with an antigen or antibody can be !~ carried out by a known method.
The particles are preferably charged with antibodies against serum proteins, such as alpha-fetoprotein tAFP), m~oglobin, beea-2-microglr,bulin or i~munoglobulin E, human .~

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~ - 5 -hormones, such as human choriogonadotropin, enzymes, such as pancreas lipase, or animal hormones, such as pregnant mare serum gonadotropin.

S A method according to the invention wherein antibodies against alpha-fetoprotein (AFP) or immunoglobul;n E (IgE) are covalently bonded to latex particles is particularly preferred.

If AFP is used by the method of the prior art, such as is described, for example, in EP-A-0,080,614, that is to say w;thout the addition of an antiserum, clearly mea-surable apparent values of AFP are obtained for sera con-.; taining rheumatoid factors (Table 1), whereas the test pro-cedure according to the invention, especially if anti-: sheep erythrocyte serum from rabbits is used, gives results which coincide completely with the enzyme ;mmunoassay (EIA).

The addition of anti-sheep erythrocyte serum from rabbits does not interfere with the measurement of the AFP actually present in corresponding serum samples, es sho.n in TabLe .,, ,., , . :, . :
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- 6 - ~ 32 9II 8 Table 1: Nephelometric determination of AFP in 15 sera containing rheumatoid factors.
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'~ RF content Alpha-fetoprotein content (IU/ml) , S (IU/ml) nephelometric in EIA apparentmeasurement according to according to prior artthe invention ,~ .
10711 3a) 19 7b) 2768 *3 96 *7 , 578 3 19 7 ~ 251 3 7 7 ,~ 15543 3 7b) 7 , 4~16 ~3 23 *7 178 3 20 *7 2078 *3 46 ~7 '~ 20191 3 18 7 ' 1626 3 50 7 '~ 248 3 10 7 ~', denotes "less than"
a) The AFP content is very low and therefore not measur-able by enzyme immunoassay.
b) Cannot be evaluated, but value below the measurement i~ 30 range correctly found.

,~ All the sera were also tested in an enzyme immunoassay lEIA) and in the method according to the invention. In ! the sera tested, the concentration of AFP is low, and A 35 ~w~n,! cannot be measured by EIA. As Table 1 shows, - ,~
nephelometric determination according to the prior art gives apparent concentrat;ons of AFP lfalsely positive values). When tested by the method according to the .
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1329~18 invention, all the sera gave vaLues below the measurement range, that is to say no falsely positive values, in agreement with the EIA.

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

~:: AFP content in EIA AFP content determined according to the invention . (IU/ml~ (IU/ml) ~;., S0.9 53.0 168.2 173.0 1556.4 58.1 68.8 67.9 44.6 42.0 169.0 149.0 46.3 41.5 2027.0 25.4 19.8 14.4 11.3 11.2 29.4 26.5 14.6 10.0 25 33.2 31.4 ~' 89.4 103.0 127.0 154.0 ~, -; All the sera were also tested by an enzyme immunoassay .~,! 30 (EIA) and in the method according to the invention. In i' the sera tested, the concentration of AFP in both methods is largely in agreement in the context of the slight deviations, familiar to the expert, of different test methods.
~ 35 -............. Falsely high IgE values for sera of rheumatic pat;ents are similarly found in the IgE test carried out according to the prior art (Table 3). Th;s Table also shows the ,~

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results for the test procedure according to the invention.
The advantageous effect of an addition of anti-sheep 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: Nephelometric determination of IgE in 15 sera containing rheumatoid factors.

~ 10 RF content Immunoglobulin E (IgE) content (lU/ml~
; (lu/ml) nephelometric in EIA apparent according according to to prior artthe invention 2û478 57 126 62 74 9 47 *35 202 1n3 187 98 140 4 ~3 35 denotes "Less than"

Results:

Values which are much too high are found nephelometri-cally according to the prior art. The average deviation of the results of the nephelometric test from the values of the enzyme immunoassay is about 370%.

1329~ 18 The values found by the method according to the invention are usually correct. The average deviation of the results of the nephelometric test according to the inven-tion from the values of the enzyme immunoassay is in this case about 25%.

The 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 E, human hormones, such as human choriogonadotropin, enzymes, such as pan-creas lipase, or animal hormones, such as pregnant mare serum gonadotropin.

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

~, Examples are: anti-human IgG serum from rabbits, anti-human IgM serum from rabbits, anti-sheep erythrocyte serum from rabbits, anti-human IgG serum from sheep and Z5 anti-rabbit gamma-globulin serum from sheep. Anti-sheep erythrocyte serum from rabbits is particularly suitable.
The immunization is carried out by known methods. The immunization dose and time are obtained from the immuno-genicity and the molecular weight of the protein.
., The antibody soLutions used contain no antibod;es ,~ against the animal antiserum bound to the latex particles.
They are usually from the same animal species.

-I 35 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.

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Such an antiserum is added in an amount such that it is present in the test mixture in a 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 a nonionic ~ detergent, such as eicosa oxyethylene sorbitan laurate `~ 10 (RTween 20) in the measuring cell is additionally advan-tageous.

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

For the 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 containing no antibodies specific for one of the immunological reaction partners.
The agent particularly advantageously contains 20 to 1 ml/100 ml of the antiserum mentioned. The agent also contains 50 to 0.1 ml/100 ml of RTween 20, particularly advantageously 20 to 0.5 ml/100 ml of RTween 20.
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Example 1 J 30 1. Preparation of the seed polymer for the latex 310 ml of nitro~en-saturated double d;stilled water were introduced into a cylindrical glass vessel fitted with a gas inlet and a gas outlet tube and a ma~netic stirrer rod. 50û mg of sodium stearate were added and ,¦ dissolved by stirring. 1.5 ml of ammonia (25 9/ 100 ml) were ~urthermore added. The pH was checked and was 11.09. The polymerization vessel was rendered , ," , ' ' ` ~

~ ~ L 3 2 911 18 oxygen-free by evacuating and filling with nitrogen several times. The detergent solution was heated to +70C with the aid of a waterbath, with continuous stirring. ~0 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 +70C for a further 15 minutes for emulsification of ; the styrene. The temperature was then raised to +90C
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 +90C for 130 minutes.
The polystyrene was then passed through a fluted filter.
The filtered polystyrene was dialyzed against 10 liters of ammonium bicarbonate solution ~0.01 9/100 ml of ; NH4HC03; 0.01 9/100 9 of NaN3; brought to pH ~û
with 10.5 ml of ammonia (25 g/100 ml in 10 l~ for 50 hours. After the dialysis, 410 ml of polymer with a dry weight of 17.9 9/100 ml were obtained.

2. Polymerization of Z-hydroxypropyl methacrylate (HPM) on polystyrene nuclei The polymerization was 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 9/100 g, 56.7 ml of distilled water and 50 mg of sodium dodecylsulfate was prepared. This was intro-duced into the polymerization vessel and the oxygen was removed. 1 ml of d potassium peroxydisulfate solution (16 mgiml in distilled water) was furthermore added and the batch was heated to +7ûQC. A mixture of 3S 0.4 ml of styrene, 0.4 ml of methacrylamidoacetalde-hyde di-n-pentyLacetal, 0.025 ml of methacrylic acid and 0.2 ml of 2-hydroxypropyl methacryLate (HPM) ~as prepared. The monomer mixture was slowLy added dropwise '- :
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~2~1g to the vigorously stirred polystyrene latex suspension at +70C for 60 minutes. Stirring was then continued at the same temperature for a further 4 hours.
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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/l, pH 8-8.2) for about 20 hours.
~;~ 87 ml of a latex dispersion with a solids content of 5.1 9/100 9 were obtained.
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-~ 3. ~inding of AFP antibodies to a polymer ~,~
. AFP antibodies were bound, as described below, to a polymer, using 2-hydroxypropyl methacrylate prepared according to Z. The particular polymer used was diluted ~, with distilled water to a solids content of 4 9/100 g.
An antiserum obtained by immunization of rabbits with ~ .:
~ purified AFP was pur;f;ed by affinity chromatography : 20 by known methods. It was then concentrated until a protein content of 10 mg/ml was reached.

i~ 3.4 ml of the above polymer were mixed with 0.34 ml ., of the AFP ant;body solut;on. 0.17 ml of a 20 ml/
100 ml aqueous solution of eicosaoxyethylene sorbitan laurate (RTween 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 ar, incuba-~,!1, tion time of 30 minutes at room temperature, 0.85 ml ; 30 of saturated aqueous sodium 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.
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l 35 This charged batch was then centrifuged at about 50,000 9 for 30 minutes. The supernatent was discarded.
The residue was resuspended in 5 ml of a glycine-NaCl , buffer (0.1 mol/l of glycine, 0~17 mol/l of NaCl and ~, ~i ~.:' ' ' ~ ' .

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0 5 ml/100 ml of eicosaoxyethylene sorbitan Laurate t Tween 20), pH 8.2). The suspension was then sub-jected to ultrasonic treatment for 2 seconds. The reagent redispersed in this way was diluted with the abovementioned glycine-NaCl buffer in a volume rat;o of 1:60.

Examp~e 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 dis-tilled water to a solids content of 4 9/100 9. An anti-serum obtained by immunization of rabbits with purified IgE was purif;ed by affinity chromatography by known methods. It was then concentrated until a protein con-tent of 10 mg/ml was reached.

3.4 ml of the abovementioned poLymer were mixed with 0.34 ml of the anti-IgE antibody soLution. 0.17 ml of a 20 ml/100 ml aqueous solution of eicosaoxyethylene sor-bitan laurate (RTween 20) was then added and the entire batch was m;xed again. O.û5 ml of 1 N HCl was added so that a pH of about 2 was reachedO After an incubation time of 30 minutes at room temperature~ 0.85 ml of satura-ted aqueous sodium hydrogen phosphate solution (pH 6.5) and 0.85 ml of aqueous sodium cyanoborohydride solution 30 (25 mg/ml) were added and the batch was mixed thoroughly.
Incubation was then carried out for one hour at room temperature.

This charged batch was then centrifuged at about 50,000 9 35 for 30 minutes. The supernatent was discarded. The residue was resuspended in 5 ml of a glycine-NaCl buffer (0.1 mol/l of glycine, 0.17 moL/l of NaCl and 0.5 ml/
1Q0 ml of eicosaoxyethylene sorbitan laurate (R~ween 20), ." ' '' ' , ' ' ~ ~ " , , .:

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~^ - 14 -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 ylycine-NaCl buffer in a vo~ume ratio of 1:80.
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Example 3 Measurement of AFP concentrations in serum samples The reagent for determination of AFP prepared according to Examp~e 1 by binding anti-AFP antibodies to latex pre-; parations was used for measurement of AFP in sera. Alpha-fetoprotein standard serum (human) for immunoprecipitation with an AFP concentration of 322,000 ng/ml (~ehringwerke AG, Marburg, FRG) was used as the standard. This standard was diluted to 100û ng/ml in 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 ser;es with decreasing AFP concentrations was thus ob-tained. The patient sera to be determined were diluted 1:5 in a phosphate-sodium chloride buffer (1.2 9/100 ml of NaCl, 1.3 9/100 ml of Na2HP04 and 0.2 gtlOO ml of NaH2P04.
For the measurement, 80 ~l of the patient serum dilution or standard serum dilution were incubated with 160 ~l of ,3 25 a reaction buffer (1.2 9/100 ml of NaCl, 1.3 9/100 ml of Na2HP04, 0.2 9/100 ml of NaH2P04 and 5.6 9/100 ml of polyethylene glycol 6000) and 30 ~l of the antiserum against sheep erythrocytes from rabbits diluted 1:8 in `I phosphate-sodium chloride buffer (1.2 9/100 ml of NaCl, !~j 30 1.3 g/10û ml of Na2HP04 and û.2 9/100 ml of NaH2P04) with 5 ml/100 ml of RTween 20 and 60 ~l of AFP reagent (Example 1.3~ at room temperature for 12 minutes. The results ~ere then measured in a nephelometer (for example that of ~ehringwerke AG).
3 The reference curve for the measurement of the standard;l serum ~as plotted and the mE~sured values for the ~ patient s~ra ~ere evalu~ted thereon.

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Example 4 .~
Measurement of IgE concentrations in serum samples The reagent prepared according to Example 2 was used for the measurement of IgE in patient sera. This IgE standard contained 1000 IU/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 concentra-tions was thus obtained.

., The patient sera to be determined were diluted in a phos-'~ phate-sodium chloride buffer (1.2 g/100 ml of NaCl, 1.3 g/
100 ml of Na2HP04 and 0.2 g/10û ml of NaH2P04). For the measurement, 80 ~l of patient serum dilution or standard serum dilution were incubated with 150 ~l of a reaction buffer (1.2 g/100 ml of NaCl, 1.3 g/100 ml of Na2HP04, 0.2 g/100 ml of NaH2P04 and 5.6 g/100 ml of polyethylene glycol 6000) and 20 ~l of the antiserum against sheep erythrocytes from rabbits diluted 1:30 in phosphate-sodium chloride buffer t1.2 9/100 ml of NaCl, 1~3 9/100 ml of ; Na2HP04 and 0.2 g/100 ml of NaH2P04) with 4 mL/100 ml ~ of RTween 20 and 75 ~l of IgE reagent (Example 3) at room ., .
temperature for 12 minutes. The results were then measured ,l~ 25 in a nephelometer (for example that from Behringwerke AG).
, The reference curve for the measurement of the standard '~ serum was plotted and the measurement values for the ~ patient sera were evaluated thereon.
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Claims (11)

1. A method for the detection or determination of an analyte which comprises the steps of:
a. contacting a sample with a specific binding partner bound to a particle for said analyte in the presence of an antiserum, said antiserum substantially containing no antibodies specific to said analyte or said specific binding partner for said analyte, and in the presence of a detergent;
b. allowing said analyte to bind to said particle-bound specific binding partner; and c. detecting the decrease of the number of said particles by nephelometric, turbidimetric or particle-counting methods to detect or determine said analyte.

2. The method as claimed in claim 1, wherein the detergent is eicosa oxyethylene sorbitan laurate.

3. The method as claimed in claim 1, wherein the antiserum is an aqueous solution of a gamma-globulin.

4. The method as claimed in claim 1, wherein the antiserum is an anti-sheep erythrocyte serum from a mammal.

5. The method as claimed in claim 1, wherein the antiserum is an anti-sheep erythrocyte serum from rabbits.

6. The method as claimed in claim 1, wherein the reaction is carried out using a latex reagent with a covalently bound antigen, antibody or hapten.

7. The method as claimed in claim 2, wherein the concentration of eicosa oxyethylene sorbitan laurate is 0.05 to 2 ml/100 ml.

8. The use of an antiserum which contains no antibodies specific for an analyte or specific binding partner for an analyte in a method for the detection or determination of said analyte, said method comprising the steps of:
a. contacting a sample with a specific binding partner bound to a particle for said analyte in the presence of said antiserum and in the presence of a detergent;
b. allowing said analyte to bind to said particle-bound specific binding partner, and c. detecting the decrease of the number of said particles by nephelometric, turbidimetric or particle-counting methods.

9. An agent for carrying out the method as claimed in claim 1, containing 90 to 0.1 ml/100 ml of an antiserum containing no antibodies which are specific to said analyte or said specific binding partner for said analyte and 50 to 0.1 ml/100 ml eicosa oxyethylene sorbitan laurate.

10. An agent as claimed in claim 9, wherein said agent contains 20 to 1 ml/100 ml of said antiserum.

11. An agent as claimed in claim 9 or 10, wherein said agent contains 20 to 0.5 ml/100 ml of said eicosa oxyethylene sorbitan laurate.