Note: Descriptions are shown in the official language in which they were submitted.
~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.
:
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.
.
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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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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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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Table 1: Nephelometric determination of AFP in 15 sera
containing rheumatoid factors.
:
'~ 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
474 3 16 7
, 578 3 19 7
~ 251 3 7 7
,~ 15543 3 7b) 7
2760 3 13 7
, 4~16 ~3 23 *7
178 3 20 *7
2078 *3 46 ~7
'~ 20191 3 18 7
' 1626 3 50 7
158 3 31 7
'~ 248 3 10 7
513 3 57 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
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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.
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-............. 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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~329~1~
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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
15474 44 102 47
2475 120 486 179
652 115 547 178
1501 342 515 293
2332 147 494 202
2û478 57 126 62
400 32 171 74
1940 124 483 176
1750 37 150 38
199 32 304 35
25696 176 326 211
74 9 47 *35
202 1n3 187 98
53 1 35 35
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.
..
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
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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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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.
~.~
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.
~.
-~ 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.
, ,;
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
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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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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.
S
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
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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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