Note: Descriptions are shown in the official language in which they were submitted.
2a~3~
Merck Patent Gesellschaft
mit beschr~nkter ~aftung
6100 Darmstadt
Method and agent for the turbidLmetric or nephelometric
determination of analytes
The invention relates to method and agent for the
turbidimetric or nephelometric determination of analytes
in liguids with the aid of an antibody-binding reacti~n.
Both immunoturbidimetry and immunonephelometry
are based on the interaction between antibodies and the
antigen to be detected. This interaction re~ults in the
formation of high molecular weight aggregates which act
as centres for scattering incident light. The scattering ~
of the light is recorded as an increase in extinction in
immunoturbidimetry and a~ an incr~ase in the intensity of
scattered light at an angle > 0 - s 90 relative to the
incident light in immunonephelometry. Since the extent of
the light scattering is approximately proportional to
1~4, the wavelength regions typically selected for
immunoturbidimetry and immunonephelometry are of short
wavelength (320-360 nm). The theoretical ba~es of immuno-
precipitation are described, for example, in Immunology
32, 445-457 (1977).
The optimal conditions for carrying out such
determinations, ~uch as buffers, detergents, neutral
salts, accelerators, etc., are described ln the litera-
ture and vary slightly depending on the antibodies used
in each case and on the antigens to be detected tAnn.
Clin. Biochem. 20, 1-14 (1983)).
The great advantage of the immunoturbidimetry and
immunonephelometry described to date i~ that the methods
can be automated relatively simply. Immunoturbidimetry
and immunonephelometry are described for many serum
proteins (The Plasma Protein~, Vol. 2, Academic Press,
New York, pages 375-425 (1975), Automated Immunoanalysis,
Part 1+2, R.F. Ritchie (ed.), Marcel Dekker Inc., New
- 2
York, Basel (1978)~.
The detection limits reported in the state of the
art differ and vary depending on the antibody and anti-
gen. The detection limits for immunoturbidimetry are at
about 5 mg/l, and for immunonephelometry are at about
1 mg~l. The upper limits for the measurement range for
immunotuxbidimetry and immunonephelometry are at about
200-500 mg/l, depending on the op~imisation of the assay.
It i5 not possible to determine lower concen-
trations of analytes by immunoturbidimetry and immuno-
nephelometry. Employed for thi~ is, for example, latex
agglutination which use~ relatively large particles
(diameter about 0.05-3 ~m) which are coated with
antibodies or antigens depending on the principle of the
assay. Detection limits < 1 ~g/l can be achieved with the
latex agglutination technique. The typical upper limits
for the measurement range are then, however, as much as
200-1000 ~g/l (0.2-1 mg/l).
The present invention i8 ba~ed on the ob~ect of
providing method~ and agents for the turbidimetric or
nephelometric determination of analytes which are more
sensitive than the known methods and which permit detec-
tion of antigens in the concentration range 0.1
> 100 mg/l.
The invention relates to a method for the tur-
bidimetric or nephelometric determination of analytes in
liquids with the aid of an antibody-binding reaction,
which $~ characterised in that a polypeptide is reacted
with~ antibodieEI or fragments thereof, the coupling
product resulting therefrom is incubated with the ana~
lyte, and the resulting turbidity is measured.
The invention further relates to an agent for the
turbidimetric or nephelometric determination of analytes
in liquids, which contains a coupling product composed of
a polypeptide and antibodies or fragments thereof,
preferably a coupling product composed of a globular
protain and Fab' fragments.
- -
It has been found, surpri~ingly, that polymerised
antibodies which have been obtained, for example, by acid
precipitation from anti~erum provide a higher measu~ement
signal than monomeric antibodies in the turbidimetric
assay. However, there is variation in the degree of
polymerisation of these antibodies and thus in the
measurement signal during storage. To avoid this disad-
vantage, a defined, ~table complex with multiple binding
sites has been prepared by coupling natural IgG or
antibody fragments, preferably Fab' fragments, to a
molecular carrier.
Suitable molecular carriers are polypeptides such
as protein~ with a molecular weight of at least 10,00~ D.
Preferably used are globular proteins such as albumin,
thyroglobulin, transferrin, haemocyanin or else poly-
peptides such as polylysine. It is likawise possible to
employ the known avidin~biotin or streptavidin/biotin
system, in which case biotinylated antibodies or frag-
ments thereof form a stable complex with avidin or
streptavidin.
The coupling to the antibodies or fragments
thereof can be carried out in a variety of ways. Prefer-
ably employed for this purpose are bifunctional hetero-
crosslinkers, for example succinimidylmaleimide cross-
linkers and related substances. The advantage of thesecrosslinkers is that unwanted coupling products are
avoided, antibodi.es or antibody fragments are introduced
stepwise, the entibodies or antibody fragment~ are
coupled end-on, and the coupling products are easy to
purify by chromatography.
The coupling products are generally prepared by
the following stepss
1. cleavage of the antibodies, for example with pep6in,
2. purification of the F~ab')z fragments by
chromatography,
3. reduction of the F(ab' )2 fxagments,
4. isolation of the Fab' fragments by chromatography,
5. coupliny of the crossli;nker onto the carrier,
- 4 - ~7~3l~
6. purification of the carrier-crosslinker conjugate by
chromatography,
7. coupling of the Fab~ fragments onto the carrier-
crosslinker con~ugate,
8. purification of the coupling product by
chromato~raphy.
The carrier-crosslinker con~ugate can be prepared
without problems in a batch process and be stored frozen
in solution or lyophilised. It is likewise possible for
the F(ab' )2 fragments to be prepared in a batch process
and to be ~tored as described above. Only steps 3, 4, 7
and 8 are then necessary for the actual preparation.
Compared with known reagents, in the turbidi-
metric and nephelometric a~say the reagents prepared in
thi~ way give distinctly higher measurement signals an~
thus allow the sensitivity of detection to be increased.
The sensitivity of detection can be further optimised by
the presence of known accelerators (for example poly-
ethylene glycols). The reagents prepared in this way are
stable in solution, they can be employed without problems
in automatic analysers, and they are thus also suitable
for automated immunoturbidimetric determination of
analyte~.
Both the immunoturbidimetric and the immuno-
nephelometric del:ermination i8 carried out at 320-400 nm,
preferably at about 340 nm. The determination can be
carried out klnetically but preferably a~ endpoint
reactlon.
The method and agents according to the present
invention can be u~ed to determine all antigen~, that is
to say substance~ which have a plurality of binding sites
for antibodies. Examples of proteins of thi~ type are
transferrin, CRP, ~l-microglobulin, RF, TBG, RBP, C3, C4,
Igs etc. which occur in body fluids and are of diagnostic
relevance. These proteins are measured in the concen-
tration range of 0.05 - > 100 mg/l, preferably 0.5 -
100 mg/l, without further prior dilution. Haptens which
nave only one binding site are amenable to competitive
- 5 ~ 3 ~ 3
determination using so-called polyhaptens.
Example 1
Preparation of an anti-transferrin Fab~-albumin conjugate
3.2 g of sheep anti-tran ferrin IgG are taken up
in 150 ml of 0.9 % NaCl solution. The solution is
adjusted to pH = 4.5 with 1 M acetic acid. 80 mg of
pepsin are added to this ~olution, and the mixture is
incubated at 37C for about 16 h. The reaction solution
is ad~usted to pH = 7.0 with 1 M TRIS solution. The
reaction ~olution is purified by affinity chromatography
using a Sepharos~4~ column to which human transferrin is
covalently bonded. The bound F(ab')2 fragments are eluted
with 0.1 M glycine/HCl (pH = 2.8). The protein-containing
fractions are pooled and ad~usted to pH = 7.0 with 1 M
TRIS solution. The solution is concentrated to 30 ml in
an ultrafiltration cell. After photometric determination
376 mg of specific F(ab' )2 fragments are obtained.
16 ml of this solution are ad~usted to pH = 4.9
with 1 M acetic acid, deaerated and blanketed with argon
and mixed with 0.9 ml of an 11% solution of cysteamine
hydrochloride in water. After incubation at 37C for 2 h,
the Fab' fragments are purified by gel chromatography on
Sephadex G-25 (150 mM NaCl, 20 mM phosphate buffer, pH =
7.0).
The protein-containing fractions are pooled.
After photometric determination, 190 mg of Fab' fragments
are obtained in 34 ml of ~olution. The ~olution i~ stored
in ~he cold under argon gas.
40.5 mg of bovine ~erum albumin are dissolved in
7 ml of water, snd 1 ml of a ~olution of 30 mg of suc-
cinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate
(SMCC) in dioxane i~ added. The reaction solution is
incubated at 30C for 1 h and then purified by gel
chromatography on Sephadex~G-250 The protein-containing
fractions are pooled. After photometric determination,
38 mg of the coupling product are obtained in 14.8 ml of
solution. The Fab' solution is mixed under argon gas with
- 6 _ s~7~
the solution of the coupling product and incubated at
room temperature for 17 h.
The reaction mixture is purified by gel fil-
tration chromatography (for exampls Superdex@~
Pharmacia/L~B, 150 mM KCl, 20 mM pho~phate buffer, 1 mM
EDTA, 0.02 ~ NaN3, pH = 7.0). The fractions which contain
the required product are combined.
Example 2
TurbidLmetric Lmmunoassay with anti-tran~ferrin Fab~-
albumin con~ugate.
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6~ polyethylene glycol
6000, 0.1~ CHAPS, O.1~ TRITON~ X-100, 0.02~ NaN3.
Fab' albumins
Prepared as in Example 1.
Transferrin:
Human transferrin was dissolved in 0.1 M TRIS
HCl buffer, pH = 7.6. Concentrations in the range to
100 mg/l transferrin were prepared by serial dilution.
Apparatus ad~u~tments
The aB8ay i8 carried out a4 endpoint assay in an
EPOS 5060 automatic analyser for clinical chemical
assay~. Firstly 40 ~1 of sample (tran~ferrin ~olution)
and-200 ~1 of re~action bufer are pipetted, mixed and
preincubated at 37~C for 64 sec. Then 30 ~1 of Pab'-
albumin con~ugate are pipetted in, mixed and the re~ult-
ing extinction i~ measured after 288 sec. The apparatus
cycle is set at 16 sec. The extinction at the end of the
preincubation time, before addition of the start reagent,
i~ u8ed to determine the sample blank. The resulting
calibration plot (extinction/concentration) i~ depicted
in Fig. 1.
- 7 _ 2~43~
Example 3
Preparation of an anti-CRP Fab~-albumin conjugate
250 mg of lyophilised sheep anti-CRP IgG are
dissolved in 20 ml of a 150 mM NaCl solution. The 601u-
tion is mixed with 4 ml of 1 M sodium acetate buffer,pH - 4.9. The pH of the solution is ad~usted to pH = 4.35
with 1 M acetic acid. 6.3 mg of pep~in are added, and the
mixture is incubated at 37~C for about 16 h. The result-
ing F(ab' )2 fragments are purified by gel filtration
chromatography (for example Superdex~ Pharmacia/LRB,
20 mM potassium phosphate buffer, pH = 7.0, 150 mM RCl).
The combined fractions contain 122 mg of F~ab' )2 frag-
ments. The pool is concentrated to 12 ml in an ultrafil-
tration cell (12.3 mg/ml F(ab') 2 ) '
6 ml of this solution (73.3 mg F(ab' )2 are mixed ~
with 1 ml of 1 M sodium acetate buffer, pH = 4.9. The pH
is ad~usted to 5.0 with 1 M acetic acid. 0.35 ml of an
aqueous 11~ cysteamine hydrochloride solution is added to
this solution. The reaction mixture is blanketed with
argon gas and incubated at 30C for 2 h. Tho resulting~
Fab' fragment~ are purified by gel filtration on Sephadex
G-25. 53 mg of Fab' fragments are obtained.
14.6 mg of bovine serum albumin are dissolved in
1.8 ml of water. 7.2 mg of SMCC in 0.18 ml of dioxane are
added to thi~ solution and incubated at 30C for 1 h. The
resulting BSA-SMCC con~ugate is purified by gel chroma-
tography on Sephadex~G-25. 14.3 mg of BSA-SMCC con~ugate
are obtained. T~he solution of the Fab' fragments is
com~ined under argon gas with the BSA-SMCC con~ugate
solution and incubated at room temperature for 18 h.
0.25 ml of an 11% cysteamine hydrochloride solution i~
subsequently added. The reaction mixture is concentrated
to 8 ml in an ultrafiltration cell. The reaction mixture
i~ purified by gel filtration chromatography ~for example
Superdex~ Pharmacia/LRB, 150 mM RCl, 20 mM phosphate
buffer, 1 mM EDTA, 0.02~ NaN3, pH = 7.0). The fractions
which contain the required product are combined ~21 ml).
6~7 ~3l~3
-- 8 --
Example 4
TurbidLmetric immunoassay with anti-CRP Fab'-albumin
conjugate
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6% polyethylene glycol
6000, 0.1~ CHAPS, 0.1% TRITONP X-100, 0.02% NaN3
Fab'-albumin:
Prepared as in Example 3
CRP:
Human CRP is dissolved in 0.1 M TRIS HCl
buffer, pH = 7.6. Concentrations in the range to 100 mg/l
CRP are prepared by serial dilution.
Apparatus ad~ustments
The assay is carried out as endpoint assay in an
EPOS 5060 automatic analyser for clinical chemical
assays. Firstly 20 ~l of sample (CRP solution) and 220 ~l
of reaction buffer are pipetted, and the mixture is
preincubated at 37C for 64 sec. Then 30 ~l of Fab'-
albumin con~ugate are pipetted in, mixed and the result-
ing extinction i3 measured after 288 sec. The apparatuCcycle i8 set at 16 sec. The extinction at the end of the
preincubation time, before addition of the start reagent,
iB used to determine the sample blank.
The resu:Lting calibration plot (extinction/con~
centration) is depicted in Fig. 2.
Example 5
Preparation of an anti-transferrin Fab~-thyroglobulin
con~ugate
Anti-transferrin IgG (sheep) i~ treated with
pepsin in analogy to Example 1. The F(ab~ )2 fragments are
purified by gel filtration chromatography. 1.3 ml of a
~olution of 41.5 mg of these F(ab~) 2 fragments in
phosphate buffer (20 mM phosphate, lS0 mM KCl, pH = 7.0)
~7~
are mixed with 0.2 ml of 1 M acetate buffer, pH = 5.0,
and blanketed with argon gas. 0.1 ml of an 11% cysteamine
hydrochloride solution in degassed water is added and
incubated at 37C for 2 h. The resulting Fab~ fragments
are purified by gel chromatography on Sephade ~G-25. The
yield is 34.6 mg of Fab' fragments in 2 ml of elution
buffer.
25 mg of thyroglo]bulin (pig) are dissolved in
3 ml of phosphate buffer (20 mN phosphate, 150 m~ RCl,
pH = 7.0). 0.1 ml of a solution of 27 mg/ml SMCC in
dioxane i5 added, and the mixture is incubated at 30C
for 30 min. The reaction mixture i8 purified by gel
chromatography on Sephadex~G-25. The solution of the Fab'
fragments is added in portions to the coupling product
and incubated at room temperature overnight.
The rasulting thyroglobulin-(Fab' )n con~ugates
are purified by gel filtration chromatography (150 mM
KCl, 20 mM phosphate buffer, 1 mM EDTA, 0.02% NaN3, pH =
7.0). The fractions which contain the required product
are combined (24 ml).
Example 6
Turbidimetric immunoassay with anti-transferrin Fab'-
thyroglobulin con~ugate
Reaction buffer:
0.1 M TRIS HCl ~buffer, pH = 7.6, 5% polyethylene glycol
6000, 0.1% CHAPS, 0.1% TRITON^, 0.02~ NMN3
-
Fab'-thyroglobuli.ns
Prephred as in Example 5
Tran~ferrins
Human transferrin is dissolved in 0.1 M TRIS -
HCl buffer, pH = 7.6. Concentrations in the range to
100 mg/l transferrin are prepared by serial dilution.
- 10 ~ J~ 3
Apparatus ad~ustment:
The assay is carried out as endpoint assay in an
EPOS 5060 analy er. Firstly 15 ~1 of example (transferrin
solution) and 235 ~1 of reaction buffex are pipetted, and
the mixture is preincubated at 37C for 64 sec. Subse-
quently 60 ~1 of Fab~-thyroglobulin con~u~ate are
pipetted in, mixed and the resulting extinction is
measured after 288 sec. The apparatus cycle is set at 16
sec. The extinction at the end of the preincubation time,
before addition of the start reagent, is used to deter-
mine the sample blank.
The resulting calibration plot (extinction/con-
centration) is depicted in Fig. 3.
Example 7
Comparative experiment to determine transferrin
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5~ polyethylene glycol
6000, 0.1% CHAPS, 0.1~ TRITONa X-100, 0.02% sodium azide
Fab'-albumins
Prepared as in Example 1
Anti-transferrin IgG:
Starting material of Example 1
Tran~ferrins
~ Human transferrin i8 dissolved in 0.1 M TRIS ~
HCl buffer, pH = 7.6. Concentrations in the range to
100 mg/l tran~ferrin are prepared by serial dilution.
Apparatus ad~ustments
The assay i8 carried out as endpoint assay in an
EPOS 5060 analy~er. Pirstly 20 ~1 of sample (transferrin
solution) and 235 ~1 of reaction buffer are pipetted, and
the mixture i8 preincubated at 37C for 64 sec. Sub~e-
quently 50 ~1 of Fab'-albumin con~ugate or anti-
~7 ~
11 --
transferrin IgG are pipetted in, mixed and the resultingextinction is measur~d after 288 sec. The apparatus cycle
is set at 16 sec. The extinction at the end of the
preincubation tLme, before addition of the start reagent,
is used to determine the sample blank.
The resulting calibration plots (extinction/con-
centration) with Fab'-albumin con~ugate and anti-trans-
ferrin IgG are depicted in Fig. 4. Fig. 5 shows a section
of these calibration plotR for low transferrin concen-
trations. The measurement signals obtained with the Fab'-
albumin conjugate are distinctly higher than with natural
anti-transferrin IgG under conditions which are otherwise
the same.
Example 8
Nephelometric immunoassay with anti-CRP Fab'-albumin
con~ugate
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5% polyethylene glycol
fiO00, 0.1% CHAPS, 0.1% TRITON~ X-100, 0.02~ sodium azide.
Fab'-albumin:
Prepared as in Example 3
CRP:
Human C~ is dis~olved in 0.1 M TRIS HCl
buffer, pH = 7.6. ConcentratLons in the range to
77.5 mg/l CRP are prepared by serial dilution.
Apparatu~ ad~ustments:
The assay i~ carried out as manual endpoint assay
in a fluorescence spectrophotometer (HITACHI F 4000). ~he
apparatus measures the intensities of scattered light at
an angle of 90. The intensities are reported as arbi-
trary units (a.u.~. The wavelength~ of excitation and
emission are set at 340 nm. The slit width iR set at
3 mm. After preced.ing zero ad~ustment, an expansion of
12 ~7~3~
0-9999 a.u. is chosen.
200 ~1 of sample and 2500 ~1 of reaction buffer
are pipetted into a 1 cm fluorescence cuvette, mixed and
preincubated at room temperature for 90 sec. Subsequently
300 ~1 of Fab'-albumin con~ugate are pipetted in, mixed,
and the increase in the scattered light inten~ity is
followed for 10 min. The resulting measurements are
plotted again3t the CRP concentrations.
The calihration plot (scattered light intensity
as a.u./concentration) is depicted in Fig. 6.
