Note: Descriptions are shown in the official language in which they were submitted.
' 2149062
Ln/Eur 3457
Title: Immunoassays using a carbon sol label
This invention relates to a method of determining in a test
sample a component of the reaction between a specifically
binding protein and the corresponding bindable substance, using
the mutual reactivity of the component to be determined and a
carbon-labeled component obtained by coupling or adsorbing
particles of an aqueous carbon sol directly to said component,
comprising during, or after the completion of, the reaction,
optionally after the separation of bound and free labeled
component, determining in said test sample, or in one of the
fractions obtained after separation, the presence and/or the
quantity of the carbon label by a method suitable for the
purpose to obtain a qualitative or quantitative indication of
the component to be determined.
The invention further relates to a method of preparing a carbon-
labeled component of the reaction between a specifically binding
protein and the corresponding bindable substance by coupling or
adsorbing particles of a carbon sol directly to the component,
and to a composition useful for the determination of an immuno-
component in an aqueous test sample.
As used herein, the phrase "component of the reaction between a
specifically binding protein and the corresponding bindable
substance" means substances, or parts thereof, such as receptor
proteins and antigenic determinants, which may be present at the
surface of cells, and immunochemical substances such as haptens,
antigens, and antibodies, which may be present in various media,
in particular body fluids, such as blood plasma, serum, and the
like, or culturing media of cells.
The invention is accordingly concerned with a plurality of
fields of histology, such as tissue and cell staining, in which
< <
2149062
2
- immunochemical reactions take place, but also couplings or
adsorptions may take place between the colloidal carbon label
and other (macro)molecular structures, such as enzymes, strept-
avidin, DNA and/or RNA, etc. In addition to these fields, the
invention is also concerned with the field of immunoassays for,
e.g., diagnostic purposes (determination of antibodies, antigens
or haptens in aqueous test samples).
In the part of the specification which follows, the invention
will be described in more detail with particular reference to
the application of the invention to the field last mentioned,
i.e. diagnostic immunoassays, but the invention should not be
construed as being limited to such application as it is equally
applicable to histological and histochemical examination
methods.
Methods as defined above have been described in EP-A-0 321 008
(Van Doorn et al). As disclosed therein, aqueous sols of non-
metallic elements and inorganic compounds not containing any
metallic element can be used as a label, and one of the non-
metallic elements mentioned therein is carbon.
U.S. Patent No. 4,760,030 (Peterson et al) discloses a method
for determining the presence of a specific binding pair member
(sbp member) in a sample. The method involves an agglutination
assay using opaque particles capable of agglutinating in the
presence of the sbp member. The opaque particles may be derived
from carbon particles having a particle size of from 0.2 to
5.0 dun. The carbon particles are conjugated to a specific
binding partner of the sbp member to render them capable of
agglutinating in the presence of the sbp member. For example, if
the sbp member to be determined (i.e. "the analyte") is
rheumatoid factor~(i.e. a heterogeneous population of auto-
antibodies binding to the Fc portion of IgG), a suspension
containing carbon particles and IgG is prepared. The test result
is read by comparing the optical density (measured at a wave-
length of 350 to 800 nm) of the assay medium after the test with
the optical density of the assay medium before the agglutination
2149062
3
t~
test. A change of optical density indicates the presence of the
analyte in the sample. In order to avoid self-agglutination of
' the carbon particles, they are suspended in an aqueous solution
of an amino acid, such as glycine, before coating them with the
specific binding partner of the sbp member, and the assay is
carried out in an assay medium which contains such an amino acid
in an amount sufficient to reduce self-agglutination of the
opaque particles.
Also U.S. patent No. 5,252,496 (Kang et al) teaches that it is
preferable to pretreat particulate carbon blacks with stabili-
zing agents such as polyalkylene glycol or polysaccharides like
dextran to maximize the dispersibility of these carbon blacks in
an aqueous medium. After this pretreatment with a stabilizing
agent, the sbp member is linked to the carbon particle/stabili-
zing agent complex via a semi-covalently linking reagent such
as, e.g., fluorescein-isothiocyanate. The resulting immuno-
chemical label has to be treated subsequently with at least one
ionic or non-ionic surfactant in order to render the label
suspendible in an aqueous medium such as water or a buffer of
low ionic strength.
Bergquist and Waller, J. Immunol. Meth. 61, 339-344 (1983)
disclose a carbon immunoassay (CIA) using carbon particles as
contained in India ink to determine the presence, if any, of IgG
antibodies in a sample. India ink has specific binding
characteristics. It binds, e.g., to rabbit IgG and can be used,
therefore, in an assay to detect rabbit IgG antibodies to a
particulate antigen. It also binds to the membranes of
staphylococci. Said membranes contain protein A which is known
to bind human IgG antibodies. These properties can be utilized
for a rapid detection of human IgG antibodies to the parasite
Toxoplasma gondii. The test comprises mixing active India ink
with protein A to prepare a labeled reagent which is then mixed
with T, gondii tachyzoites (functioning as the particulate
antigen) and a sample suspected of containing human IgG
antibodies to T. gondii. The test result may be read under a
light microscope. The T. gondii tachyzoites appear black due to
_ 2149062
4
adherent carbon particles in the case of a positive CIA
reaction, and otherwise remain white. The CIA test is quite
insensitive and claimed to be attractive only because of its
simplicity.
It is desirable that the particulate carbon label used has such
properties as a low cost price, ease of (bulk) preparation,
light absorbance at a broad wave-length range, optimal features
in view of contrast towards a light coloured background in e.g.
solid phase-, dipstick- or agglutination (inhibition) sol
particle (immuno)assays and capability of adsorbing a wide range
of totally differing binding proteins and/or bindable substances
at the surface of the colloidal carbon particles. In order to be
able to develop assay systems having controllable test
performances with respect of sensitivity, specificity and
reproducibility, it is important to have a thorough knowledge of
the surface properties of the colloidal carbon-label particles.
Though the literature suggests the use of carbon particles as a
label in immunoassays (Peterson et al, Kang et al, Bergquist and
Waller), none of these examples match the qualifications posed
on the carbon particles, neither in the sense of carbon particle
properties, nor in the sense of carbon particle size. All,
Peterson et al, Kang et al, and Bergquist and Waller, show that
prior to the supposed coupling of binding protein to the carbon
particles in an aqueous medium, these particles must already
have been stabilized by an amino acid like glycine (Peterson et
al), a polyalkylene glycol or a polysaccharide like dextran
(Kang et al), or other stabilizers like arabic gom and resins
which is the case when India ink carbon particles are used
(Bergquist and Waller). Without the addition of such stabilizers
the carbon particles described by Peterson et al, Kang et al,
and Bergquist and Waller do not form a stable, non-self aggluti-
nating colloidal suspension in aqueous media such as pure water
or buffer solutions of low ionic strength. These stabilized
aqueous carbon sols of the prior art therefore need the addition
or presence of stabilizing agents to the carbon particles in an
zi49os2
- aqueous medium prior to the addition of a specifically binding
protein or the corresponding bindable substance.
The use of such stabilized aqueous carbon sols as a label system
5 in immunoassays has several drawbacks:
- Because the particles tend to agglutinate spontaneously, the
sols are hard to handle.
- There is no proof for an actual coupling of binding protein to
the surface of the carbon particles. Both Peterson et al and
Bergquist and Waller use their stabilized aqueous carbon sols
solely in an agglutination device. The formation of an
antibody-antigen precipitate in the presence of suspended
colloidal particles of the size 200-500 nm would force such
(large) particles to co-precipitate anyway.
- Because the surface of the carbon particles is first enveloped
with an amino acid, a polyalkylene glycol, a polysaccharide
like dextran, or a resin to stabilize the colloidal suspension
in an aqueous medium, it is most likely that the binding
protein will be hindered in coupling directly via hydrophobic
interaction to the actual surface of the carbon particles in
the following coupling step. In fact, Kang et al need a time-
consuming coupling step in which the binding protein is linked
to the carbon label with the aid of an extra linking reagent.
Such coupling procedures are tedious and will lead to varying,
non-reproducible results in respect of sensitivity and
accuracy of the test methods.
- Stabilized aqueous carbon sols are complex mixtures of
stabilizing components and carbon particles in water with a
large batch-to-batch variation. Due to this variation the
handling of such sols is not straightforward and, from an
economical point of view, at least unfavourable.
- Treatment of the immunochemical carbon label with ionic or
non-ionic surfactants (Kang et al) can lead to reduced
(immuno)reactivity (denaturation of binding protein, or
decrease of the interaction forces between e.g. a sbp member
and its corresponding bindable substance), or to desorption of
immobilized bindable substances from the carbon particles
[Gershoni and Palade, Anal. Biochem. 131, 1 (1983): Spinola
CA 02149062 2004-11-26
6
and Cannon, J. Immunol. Meth. 81, 161 (1985): Wedege and
Svenneby, J. Immunol. Meth. 88, 233 (1986); Wedege et al, J.
Immunol. Meth. 113, 51 (1988); Bird et al, J. Immunol. Meth.
106, 175 (1988); Stott, J. Immunol. Meth. 119, 153 (1989);
Tyllianakis et al, J. Immunol. Meth. 162, 273 (1993)].
The present invention may advantageously provide aqueous carbon
sols which are essentially non-stabilized and are useful as a
label in a method of determining in a test sample a component of
the reaction between a specifically binding protein and the
corresponding bindable substance.
The invention may advantageously provide a method of deter-
mining in a test sample a component of the reaction between a
specifically binding protein and the corresponding bindable
substance, wherein an essentially non-stabilized aqueous carbon
sol is, used as a label.
The invention may advantageously provide a (method of
preparing a) carbon-labeled component of the reaction between a
specifically binding protein and the corresponding bindable
substance, wherein an essentially non-stabilized aqueous carbon
sol is used for labelling said component.
SUMMARY OF THR INVENTION
We studied whether the characteristics and properties of various
carbon types have any predictive value with respect to their
dispersibility in aqueous media. We evaluated, in relation to
experimental data on essentially non-stabilized aqueous carbon
sols made by us, characteristics and properties such as jetness
index, surface area-N2, primary particle diameter, dibutyl-
phthalate value, tinting strength, volatile content, density,
etc., as given by the manufacturers of carbon blacks.
Surprisingly, we found that the dispersibility of a particulate
carbon black in aqueous media as an essentially non-stabilized
carbon sol can be modeled as a function of the parameters
. .
- ~ 2149~fi2
7
dibutylphthalate (DBP) value, volatile content (VC) and primary
particle diameter (PPD).
The estimated model formula for the linear predictor value (V)
is: V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD.
A positive linear predictor value (V > 0) of a particulate
carbon black indicates that it is suited to prepare an essen-
tially non-stabilized aqueous carbon sol. A linear predictor
value V <_ 0 of a particulate carbon black indicates that it is
not suited to prepare an essentially non-stabilized aqueous
carbon sol. These latter carbon blacks need the addition or
presence of stabilizing agents in order to form stabilized
aqueous carbon sols (Peterson et al, Bergquist and Waller,
Kang et al).
So we have surprisingly found that it is possible to predict
in a reliable manner whether a certain carbon grade or type is
suited to be used as starting material for the preparation of
an essentially non-stabilized aqueous carbon sol or not. Said
prediction can be made on the basis of three different
parameters, which characterize the carbon type in question.
Therefore, according to this invention it has been found that
the above objects of the invention can be realized, more in
particular that essentially non-stabilized colloidal carbon
particles can be made which can be used as a label and have
advantages over and above other labels.
This invention therefore provides a method for determining the
presence or amount of an analyte in a sample comprising
contacting said sample with a carbon-labeled constituent
consisting of an aqueous carbon sol having directly conjugated
to the surface of the colloidal carbon particles a binding
component capable of specifically recognizing said analyte and
determining the presence or absence of a resulting analyte/
carbon particle complex as an indication of the presence or a
measure of the amount of analyte in said sample, said method
2~~9Q6~
being characterized in that a carbon black having a linear
predictor value V > 0, wherein
V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD;
DBP is the dibutylphthalate adsorption in ml/100g, as determined
according to DIN 53601;
VC is the volatile content in ~, as determined according to DIN
53552; and
PPD is the average primary particle diameter in manometers;
is used to prepare an essentially non-stabilized aqueous carbon
sol which is used to prepare said carbon-labeled component.
This invention further provides a composition useful for the
determination of an analyte in a sample, said composition
comprising an aqueous carbon sol having directly conjugated to
the surface of the colloidal carbon particles a binding
component capable of specifically recognizing said analyte,
characterized in that said aqueous carbon sol is an essentially
non-stabilized aqueous carbon sol derived from a carbon black
having a linear predictor value V > 0, wherein
V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD;
DBP is the dibutylphthalate adsorption in ml/100g, as determined
according to DIN 53601;
VC is the volatile content in ~, as determined according to DIN
53552; and
PPD is the average primary particle diameter in manometers.
This invention also provides a method for preparing a composi-
tion useful for the determination of an analyte in a sample
comprising preparing an aqueous carbon sol and conjugating
directly to the surface of the colloidal carbon particles a
binding component capable of specifically recognizing said
analyte, characterized in that said aqueous carbon sol is an
essentially non-stabilized aqueous carbon sol derived from a
carbon black having a linear predictor value V > 0, wherein
V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD;
DBP is the dibutylphthalate adsorption in ml/100g, as determined
according to DIN 53601;
VC is the volatile content in ~, as determined according to DIN
_ , . . ~ 2149062
9
_ ~_
53552; and
PPD is the average primary particle diameter in manometers.
In one embodiment of the invention, the analyte is selected from
the group consisting of receptor proteins and epitopes present
on the surface of cells. In another embodiment of the invention,
the analyte is selected from the group consisting of haptens,
antigens, enzymes, antibodies and antibody fragments, DNA and
RNA. The binding component conjugated to the colloidal carbon
particles is preferably selected from the group consisting of
haptens, antigens, enzymes, antibodies and antibody fragments,
receptor proteins, epitopes, DNA and RNA. It is preferred that
the primary colloidal carbon particles have an average particle
size within the range of from 1 to 100 mm and that the
essentially non-stabilized aqueous carbon sol does not contain
sol-stabilizing agent. Preferred embodiments of the method are
solid phase immunoassays and immunochromatographic assays (such
as dipstick immunoassays).
DETAILED DESCRIPTION OF THE INVENTION
According to this invention, the aqueous carbon sol used for
preparing the carbon-labeled constituent is an essentially non-
stabilized aqueous sol of a carbon black having a linear
predictor value V > 0, wherein
V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD;
DBP is the dibutylphthalate adsorption in ml/100g, as determined
according to DIN 53601;
VC is the volatile content in ~, as determined according to DIN
53552; and
PPD is the average primary particle diameter in manometers.
The words "essentially non-stabilized aqueous carbon sol" or
"essentially non-stabilized colloidal carbon particles" refer to
a carbon sol in an aqueous medium, such as pure water or water
containing a buffer system of low ionic strength, which carbon
sol does not require any added stabilizing agent to be stable
and preferably does not contain any stabilizing agent. The words
~i~~o~~
"essentially non-stabilized" intend to cover aqueous carbon sols
containing a substance which may have a sol-stabilizing effect
but are stable also in the absence of said substance. Most
preferably, however, said essentially non-stabilized aqueous
5 carbon sol does not contain sol-stabilizing agent.
Characteristics and properties of particulate carbon blacks
(such as jetness index, surface area-N2, primary particle
diameter, dibutylphthalate value, tinting strength, volatile
10 content, density, etc.) were evaluated in relation to the
experimentally ascertained utility of a certain particulate
carbon black as a starting material for the preparation of an
essentially non-stabilized aqueous carbon sol. As the
dispersibility is measured as a binary variable - particulate
carbon blacks are either dispersible or not dispersible as
essentially non-stabilized aqueous carbon sol - the stochastic
part of the model is assumed to follow a binomial distribution.
The deterministic part of the model, a linear combination of the
variables DBP, VC and PPD, is related to the dependent variable,
i.e. the dispersibility of particulate carbon blacks in aqueous
media as an essentially non-stabilized aqueous carbon sol, by
the logit link function (McCullagh and Nelden, Generalized
Linear Models, 2nd edition, 1989, chapter 4, Chapman and Hall,
ISBN 0-412-31760-5). Using a Generalized Linear Model the
dispersibility was modeled as functions of all possible combi-
nations of the aforementioned characteristics and properties of
various particulate carbon blacks. The models were fitted to the
data using the statistical computer package Genstat (Payne and
Lane (eds.), 1987, Genstat 5 Reference Manual, Clarendon Press,
Oxford) .
Surprisingly, it was found that of these fitted models a linear
combination of three variables, i.e. DBP value, volatile content
(VC) and primary particle diameter (PPD) is related to the
dispersibility of particulate carbon blacks as essentially non-
stabilized aqueous carbon sols.
214906
;:
11
- The first of said three parameters, the DBP value, is the
dibutylphthalate adsorption according to DIN53601 (DBP, in
' ml/100g), which is a measure of secondary particle structure.
The second parameter, the Volatile Content VC (in ~), is
determined by maintaining the carbon sample at a temperature
of 950°C for 7 minutes according to DIN53552. Higher volatile
contents indicate surface oxidation with more polar groups.
The third parameter is the average primary particle diameter
PPD (in nanometers), which is calculated from number and size
measurements taken under an electron-microscope.
The estimated model formula for the linear predictor value (V)
is: V = - 138.954 - 0.987xDBP + 15.609xVC + 3.994xPPD.
The distribution of different particulate carbon blacks into
carbons which are dispersible in aqueous media as essentially
non-stabilized carbon sols (Dispersibility = 1) and particu-
late carbon blacks which are not dispersible in aqueous media
as essentially non-stabilized carbon sols (Dispersibility = 0)
is defined by the inverse of the logit link function:
Disp. (~) - 100 x [e°/ (1 + a°) ] .
A positive linear predictor value (V > 0) and hence a
Disp.(~) > 50~ of a certain particulate carbon black indicates
that it is suited to prepare an essentially non-stabilized
aqueous carbon sol. A linear predictor value V ~ 0 and hence a
Disp.(~) ~ 50~ of a certain particulate carbon black indicates
that it is not suited to prepare an essentially non-stabilized
aqueous carbon sol. These latter carbon blacks need the
addition or presence of stabilizing agents in order to form
stabilized aqueous carbon sols (Peterson et al, Bergquist and
Waller, Kang et al).
Kang et al use e.g. Cabot particulate carbon blacks with V-
values varying between -28.90 and -171.40 and hence Disp.(~)
values « 50, indicating that their carbon blacks are not
. 2149Qfi2
12
suited to prepare essentially non-stabilized aqueous carbon
sols.
Table 1 and Figure 1 both show the distribution of a variety
of particulate carbon blacks into dispersibility = 1 carbons
(Disp.l carbons dispersible in aqueous media as essentially
non-stabilized carbon sols) and dispersibility = 0 carbons
(Disp.O carbons; not dispersible in aqueous media as
essentially non-stabilized carbon sols) on the basis of both
experimental data and predicted values (V and dispersibility).
many r
carbon rade ~ ex erimental results redicted results
dis ersibilit V dis ersibilit
De uaaa
Farbruss FW200 1 100.51 1
Farbruss Fw2 1 35.20 1
Farbruss FW1 0 -161.17 0
S ezial schwarzSS6 1 51.99 1
S ezial schwarzSS5 1 46.76 1
S ezial schwarzSS4 1 78.66 1
Printex 150T 1 19.46 1
Printex 95 0 -111.63 0
2 S ezial schwarzSS550 0 - 32.83 0
5
S ezial schwarzSS350 0 - 24.48 0
S ezial schwarzSS250 1 91.96 1
S ezial schwarzSS100 1 15.79 1
Printex G 0 - 15.96 0
Cabot
Black earl 0 -373.53 0
2000
Monarch 1000 0 - 30.40 0
Monarch 700 0 -159.13 0
Mo 1 L 0 - 19.33 0
Elftex 485 0 -133.94 0
Elftex 285 0 - 79.02 0
It follows that, of the particulate carbon blacks investigated,
the Degussa particulate carbon blacks Farbruss FW200, Farbruss
FW2, Spezial-schwarz SS6, Spezial-schwarz SSS, Spezial-schwarz
SS4, Printex 150T, Spezial-schwarz SS250 and Spezial-schwarz
SS100 are dispersible in aqueous media as essentially non-
stabilized carbon sols.
Use of essentially non-stabilized colloidal carbon particles
as a label in a method of determining in a test sample one or
z~4so~~
13
more components of the reaction between a specifically binding
protein and the corresponding bindable substance has several
advantages over and above the use of other labels, such as a
low cost price, ease of (bulk) preparation, light absorbance
at a broad wave-length range, optimal features in view of
contrast towards a light coloured background in e.g. solid
phase-, dipstick- or agglutination (inhibition) sol particle
(immuno)assays and capability of adsorbing a wide range of
totally differing binding proteins and/or bindable substances
at the surface of the colloidal carbon particles.
The essentially non-stabilized carbon sol particles to be used
according to the present invention have a number of advantages
over the stabilized colloidal carbon particles and also over
other colloidal particle labels. Surprisingly we found that it
is very easy to produce stable colloidal carbon suspensions in
aqueous media without using any stabilizing agents or other
components, i.e. non-stabilized aqueous carbon sols. Examples of
suitable carbons are several particulate channelblack/furnace-
black carbon types.
When used as a label in a method of determining in a test sample
one or more components of the reaction between a specifically
binding protein and the corresponding bindable substance, these
non-stabilized aqueous carbon sols have several advantages over
the stabilized aqueous carbon sols as described by Peterson et
al, Kang et al, and Bergquist and Waller.
These advantages include:
- The aforementioned carbons can form stable, colloidal
suspensions in aqueous media such as pure water or buffers of
low ionic strength by themselves, without the need of addition
of complex mixtures of stabilizers (such as amino acids,
polyalkylene glycols, polysaccharides such as dextran, resins
or detergents), and preservatives.
- The aforementioned carbons are delivered in prescribed,
strictly defined size classes, covering the whole colloidal
particle range, without any need for crude grinding of carbon,
- , . . . 2I4gp~2
14
- after purification using EDTA and HC1, in a mortar, as
described by Peterson et al. In their initial stage of ~carbon-
- label preparation, also Kang et al mention grinding of a
mixture of raw carbon material and a stabilizing agent.
- The aforementioned carbon starting material is available in
very large bulk quantities problems connected to batch to
batch variation (as with India ink, see Bergquist and Waller)
are diminished. Therefore, reproducibility of carbon sols in
respect of characteristics and properties of particulate
carbon blacks has been found not to be a matter of concern.
- The non-stabilized aqueous carbon sols are non-expensive, can
easily be prepared in very large quantities, are very stable
and therefore have a long shelf-life.
- Due to the absence of any stabilizing agents in non-stabilized
aqueous carbon sols before and during coupling of macromole-
cules (such as proteins, antigens, DNA/RNA) to the colloidal
carbon particles, there is an actual, direct interaction
between these macromolecules and all potential, active (e. g.
hydrophobic) binding sites at the surface of the colloidal
carbon particles.
Moreover, the conditions for coupling macromolecules onto
colloidal carbon particles via e.g. physical adsorption are
known and can be strictly defined and controlled. (The
composition of most India inks is not specified (Bergquist and
Waller) and also the interactions between glycin or dextran,
carbon particles and a binding protein remain more or less
obscure (Peterson et al, Kang et al)).
In conclusion it is very easy to couple macromolecules onto non-
stabilized aqueous carbon sols. The resultant carbon/macro-
molecule conjugates give results which in terms of sensitivity,
specificity and accuracy are not only reliable, but also very
reproducible.
The aforementioned carbons are delivered in prescribed, strictly
defined size classes. The primary carbon particles preferably
have average particle diameters ranging from 1 to approximately
100 nm. Due to the production/manufacturing process comprising
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heating, evaporating, burning and cracking of hydrocarbons used
as a starting material, it might happen that during cooling some
primary particles fuse together into larger, higher structured
particles, called "secondary particles". The surface properties
5 of primary and secondary particles of a certain particulate
carbon black are the same and as a consequence they behave
similarly in view of their colloid-chemical stability in aqueous
media such as water or buffers of low ionic strength. Preferably
the average particle size of the secondary particles does not
10 exceed 400 nm. More preferably, the colloidal carbon particles
have an average particle size within the range of from 1 to
200 nm.
These particle sizes of the non-stabilized aqueous carbon sols
15 render them very suitable for application as a label in e.g.
immuno-chromatographic assays. A preferred example of such an
immunochromatographic assay is a dipstick assay in which the
solid phase carrier (strip) consists of porous or fibrous
materials such as natural or synthetic polymers and derivatives
like nitrocellulose or nylon with pore sizes between 0.20 Fun and
15 Fun and a strip thickness of about 100 Fun. On the strip, e.g.
antibodies or antigens are immobilized by adsorption, absorption
or covalent bonding. Sample materials containing an analyte
specifically reactive with the immobilized member of the binding
pair are applied to the carrier material and move chromato-
graphically through the strip, where the analyte is immobilized
by reaction with its corresponding binding pair member. The non-
reacted sample materials are then removed by e.g. a washing step
after which, in the case of a sandwich-type assay, the carbon-
labeled reagent is applied to the carrier material.
Said carbon-labeled reagent is chromatographically easily mobile
as a consequence of the relatively small carbon particle size
and is capable of reaction with, and immobilization by the
immobilized analyte.
The carbon-labeled reagent can be applied to the carrier
material in a liquid form but, alternatively, it can be sprayed
214906
16
and dried onto a chromatographic medium in the presence of e.g.
a meta-soluble protein and/or polysaccharide. In this case, the
carbon-labeled reagent can be rapidly resolubilized in the
presence of an appropriate solvent such as the sample or a
chromatographic transport solvent.
P~Ppa_ration and use of carbon sole
Production of essentially non-stabilized aqueous carbon sols is
very easy and non-expensive. After selection of a well-suited
particulate carbon black according to a Linear Predictor Value
V > 0 and addition of pure water or a buffer of low ionic
strength to an amount of the selected dry carbon powder, a
stable black sol can be obtained by several methods such as for
example by means of a sonifier. The essentially non-stabilized
(though stable!) carbon sol can be strongly diluted in water or
buffers of low ionic strength. The diluted sol flocculates after
adding an excess of NaCl and within a few minutes a black
flocculated pellet and a clear, colorless supernatant are
formed. This flocculation phenomenon can be used as a tool for
monitoring physical adsorption of macromolecules onto carbon
particles only in the case of non-stabilized aqueous carbon
sols.
Addition of, for example, a suspension of a macromolecule in a
buffer of low ionic strength to a diluted non-stabilized aqueous
carbon sol will, under the proper conditions and after gentle
mixing, result in coupling of the macromolecule onto the surface
of the colloidal carbon particles via, amongst others, hydro-
phobic interaction. As a result of this macromolecule coating,
the colloidal carbon particles will now be protected against
flocculation by addition of an excess NaCl to the carbon/
macromolecule-conjugate suspension.
In a systematic experimental set up in which increasing amounts
of a certain macromolecule are incubated with a fixed amount of
non-stabilized aqueous carbon sol under strictly defined and
controlled conditions, addition of excessive amounts of NaCl
will no longer lead to flocculation of the sol when a certain
21490fi2
(~ ~ I 1'
macromolecule/colloidal carbon particle ratio has been reached.
This amount of macromolecule, the so called "minimal protective
' amount" (NBA) is an important parameter in the coupling
procedure of macromolecules onto non-stabilized aqueous carbon
sols and the value of the 1~A depends amongst others on the
nature of the macromolecule to be coupled, on the nature and
amount of the colloidal carbon particles and on the coupling
conditions in respect of pH and ionic strength.
Coupling of macromolecules to non-stabilized aqueous carbon sols
can be ascertained therefore by performing a flocculation test
and determination of the MPA, but can also be (double) checked
by measurement of the light-absorbance of the supernatant after
the carbon/macromolecule-conjugates have been pelleted by
centrifugation. After addition of a minimal protective amount of
a macromolecule to a non-stabilized aqueous carbon sol and a
short incubation under proper conditions, repeated
centrifugation of the carbon-macromolecule conjugates followed
by repeated resuspending the successive pellets in an aqueous
medium without any (other) macromolecule, addition of NaCl to
the suspended pellet does still not cause flocculation. This
indicates that an irreversible macromolecule-carbon bond has
been formed.
This strong attachment of a macromolecule (e.g. an antibody) to
the surface of the colloidal carbon particles makes
non-stabilized aqueous carbon sols not only suitable to act as
label in agglutination (inhibition) immunoassays, but makes them
also very suited to act as signal generating label in all kinds
of solid phase immunoassays, such as membrane chromatographic
immunoassays. Considering application as a label in such chroma-
tographic immunoassays, the relatively well defined particle
size distribution of different species of non-stabilized aqueous
carbon sols in the present invention is also an advantage over
the aqueous carbon sols of Peterson et al, Bergquist and Waller,
and Kang et al.
- . . . . 2149062
18
The homogenization step in the preparation of essentially
non-stabilized aqueous carbon sols is advantageously performed
with ultra-sonification, but can also be achieved by shaking or
boiling (with or without stirring) a mixture of carbon particles
and an aqueous medium without stabilizing agents.
Sonification of carbon in pure water or in a buffer of low ionic
strength, followed by mixing this colloidal carbon suspension
with a suspension of a macromolecule in (the same) buffer of low
ionic strength under gentle mixing, is a simple, non-expensive
and fast route towards the development of carbon-sol particle
labels for all kinds of immunoassays.
Even addition of a suspension of a macromolecule (such as a
protein) in a buffer of low ionic strength (final macromolecule
amount at NIPA-level) to a mixture of carbon powder and water
during a (short) homogenization step by sonification leads to
the formation of colloidal carbon-particle labels carrying said
macromolecule, which in turn can be applied in immunoassays.
The immuno components labeled with carbon sol particles are used
as reagents, commonly in combination with other reagents, for
demonstrating and quantifying e.g. haptens, antigens, antibodies
and DNA/RNA in an aqueous test medium, e.g. body fluids such as
blood plasma, serum and the like, or culturing media of cells,
for which all sorts of immunochemical techniques as are in use
in radio-immunoassays and enzyme-immunoassays are suitable.
The invention accordingly also relates to test kits for use with
such immunochemical techniques, and containing as the most
important component an immuno component.
One of the conventional immunochemical techniques is the
competitive immunoassay, which can be used for demonstrating and
determining an immuno component. For demonstrating, for example,
a certain antigen, this method comprises contacting a test
sample containing an unknown amount of antigen with either a
pre-determined quantity of the antigen in question, labeled with
2149062
_ ~ 19
- carbon and an insolubilized antibody against this antigen, or a
pre-determined quantity of insolubilized antigen and an antibody
directed against this antigen, labeled with carbon.
After completion of the reaction the quantity of the carbon is
determined in the bound or free fraction, which can give a
qualitative or a quantitative indication of the antigen to be
determined. Mutatis mutandis, a similar method applies for
determining other immuno components.
Other methods frequently being used are the so-called Sandwich
techniques, which are also particularly suitable for the use of
a component labeled with carbon according to the present
invention. According to these techniques, an immunological
component, for example, an antibody in case an antigen has to be
determined, is insolubilized by coupling it to a solid carrier.
This solid carrier is, for example, the inner surface of the
reaction vessel in which the immunochemical reaction is
conducted. Also dipsticks on the basis of a nitrocellulose
membrane or on the basis of a nylon membrane or polystyrene rods
can be used as a solid phase carrier. After a first incubation,
possibly followed by a washing step, a second incubation is
effected with antibody labeled with carbon, whereafter said
carbon is determined in the bound or the free phase.
The immuno components labeled with carbon also lend themselves
well to the application in so-called homogeneous immunoassays,
i.e. immunoassays in which a separation between the labeled
immunological component bound in the immunochemical reaction and
that which is~still free is unnecessary. Such assays have the
advantage of being simple to perform, providing the desired
information relatively fast, and lending themselves excellently
for automation.
In the actual assay, for example, test sample (or standard
solution) containing the antigen to be determined is incubated
together with the labeled antibody in the wells of a microtiter
2149062
- plate. The immunochemical reaction between antigen and (labeled)
antibody will result in agglutination. The thus induced
agglutination of the particles in a sol of carbon is accompanied
by a change in light absorption, which can be monitored, e.g.
5 spectrophotometrically or with the naked eye.
To determine small antigens, which in immunochemical respect are
monovalent, use is made of an agglutination-inhibition reaction,
which is based on the same principle.
In addition to the techniques mentioned above, there are
countless other immunochemical techniques in which the immuno
component labeled with carbon can be used as a reagent. Most
preferably, however, the method of the invention is a solid
phase immunoassay, more specifically an immunochromatographic
assay, such as a dipstick immunoassay.
The analyte may be a soluble substance present in solution in a
liquid test sample. Preferably, the analyte is selected from the
group consisting of receptor proteins and epitopes present on
the surface of cells, or, particularly in the case of liquid
samples containing a soluble analyte in solution, is selected
from the group consisting of haptens, antigens and antibodies.
Preferably, the binding component conjugated to the colloidal
carbon particles is selected from the group consisting of
haptens, antigens, antibodies, DNA and RNA.
The measurement of the nature and/or the concentration of the
carbon in a certain phase of the reaction mixture can be
effected according to numerous known techniques.
Figure 1 shows the relationship between the Linear Predictor
Value (V) and Degussa/Cabot particulate carbon black dispersi-
bility in aqueous media. Experimental data (Disp.1 or Disp.O)
are plotted against the calculated V.
2~4906~
21
All steps described below are carried out at room temperature
unless otherwise stated.
Method A: ultra-sonification
Stock solution: 1 g carbon (Degussa, Printex 150T) is suspended
in demineralized water to a final volume of 100 ml (1~ (w/v)).
The suspension is homogenized for 15 minutes by means of a
Branson Model 250 Sonifier: Output control 3 ~ 27 Watt, 20 KHz
(this sonification of the suspension can, optionally, occur on
ice) .
A deep black colloidal carbon suspension consisting of spherical
carbon particles having an average primary particle diameter of
29 nm is formed. The stock C-suspension is kept at 4°C.
Method B: ultra-sonification
The procedure of Method A was applied to the Degussa particulate
carbon blacks Farbruss FW200, Farbruss FW2, Spezialschwarz 6,
Spezialschwarz 5, Spezialschwarz 4, Spezialschwarz 250 and
Spezialschwarz 100.
Deep black colloidal carbon suspensions consisting of spherical
carbon particles having an average primary particle diameter of
13, 13, 17, 20 , 25, 56 and 50 nm, respectively, were formed. The
stock C-suspensions were kept at 4°C.
vortexing and differential centrifugation
Demi-water is added to 0.05 g carbon (Degussa, Printex 150T) to
an end volume of 5 ml (1$ w/v). The suspension is homogenized by
vortexing for 10 minutes at 2500 rpm in a vortex mixer. The
suspension is washed three times by centrifugation for 10 min at
13,800 x g and resuspending the pellets each time in 5 ml demi-
water. Finally, the suspension is centrifuged for 10 minutes~at
1,000 x g to remove aggregated colloidal carbon particles. The
supernatant is decanted carefully and kept at 4°C.
. , . 2149062
22
- In view of the loss of material occurring in the working-up of
the suspensions, the carbon concentration is standardized to a
spectrophotometric absorption of 1 at a wave length of 500 nm.
Method D: boiling and differential centrifugation
Demi-water is added to 0.25 g carbon (Degussa, Printex 150T) to
an end volume of 25 ml (1% w/v). While stirring with a magnetic
stirrer, the suspension is gently boiled for 15 minutes in a
closed glass vessel on a heating plate. After cooling to room
temperature, the suspension is subjected to differential
centrifugation in accordance with Method C.
Fxam 1p a 1 - Detection of human fibrinogen by means of a linear
dilution series of goat anti-human fibrinogen antibodies spotted
onto nitrocellulose strips.
prPparat;nn of carbon particles-fibrinogen conjugate
Before use (for example, the physical adsorption of proteins)
the stock carbon (C) suspension (made according to Method A) was
diluted 5 times by means of 2.5 mM Tris-HC1, pH 8.5.
15 mg bovine fibrinogen, type i-s (Sigma) was dissolved in 5 ml
of 2.5 mM Tris-HC1, pH 8.5 = solution A;
15 mg human fibrinogen, fraction I (Sigma) was also dissolved in
5 ml of 2.5 mM Tris-HCl, pH 8.5 - solution B.
5 ml of 0.2% (w/v) C-sol in 2.5 mM Tris-HC1, pH 8.5 were added
to both 5 ml of solution A and 5 ml of solution B. The
suspensions were incubated with stirring for 3 to 4 hours.
Subsequently, the conjugates formed were washed 3 times by means
of 5 mM NaCl, 1% (w/v) BSA, 0.02% (w/v) NaN3, pH 8.5 by
centrifugation at 13,636 x g for 15 minutes. The first
supernatant which was formed in each washing step was again
centrifugated for 15 minutes at 13,636 x g, after which the
pellets were combined. This extra centrifugation served to
minimize the loss of material. After the third and last washing
CA 02149062 2004-11-26
23
step the pellets were resuspended in half of the starting volume
(the carbon concentration again was 0.2% (w/v)). The washed
conjugates were kept in the dark at 4°C.
Preparation of the nitrocellulose strips with a linear dilution
series of goat anti-human fibrinogen antibodies spotted ther,~on
The following linear dilution series of polyclonal goat anti-
human fibrinogen antibodies was spotted onto nitrocellulose
membranes (Schleicher and Schuell, type BA 85/23 having a pore
diameter of 0.45 Eun) : 1000ng; 500ng; 250ng; 125ng; 62ng; 3lng;
l5ng; 8ng; 4ng; 2ng.
The series dilution was made with 10 mM PBS, pH 7.2. Per spot,
1 ~1 of solution was used; spot diameter < 2 mm.
After application of the spots the membranes were air-dried for
3 hours. The free positions of the nitrocellulose were blocked
by immersing the membranes for 1.5 hours at 37°C in 10 mM PBS,
2% (w/v) BSA, 0.02% (w/v) NaN3, pH 7.2. The membranes were air-
dried again, whereafter they were affixed onto an adhesive
plastic carrier material (Costar Serocluster platesealers) and
finally cut to size (5 x 50 mm). The strips were kept dry in the
dark and at room temperature.
best Pr~edure
In stoppable polystyrene tubes (Greiner) of 4.5 ml, "goat
antihuman fibrinogen strips" were incubated with:
1 ml C-human fibrinogen 1 ml C-bovine fibrinogen
conjugate conjugate
500 ~tl 2.5 mM Tris-HC1 pH 8.5 500 ~.1 2.5 mM Tris-HC1 pH 8.5
500 ~1 20 mM Tris-HC1 500 ~1 20 mM Tris-HC1
600 mM NaCl 600 mM NaCl
1% (w/v) casein 1% (w/v) casein
0.2% (w/v) Tween-20 0.2% (w/v) Tween-20
0.02% (w/v) NaN3 0.02% (w/v) NaN3
pH 8.5 pH 8.5
2149062
i 24
After 5 minutes of incubation the strip in test A showed five
clearly colored spots (1000-62 ng) decreasing in intensity.
After 1.5 hours of incubation the spots had reached their
maximum color intensities.
In Test B the strip did not show any colored spots.
Example 2 - Isotyping test for determining the isotype of
monoclonal mouse immunoglobulins by means of monoclonal rat
anti-mouse kappa/lambda (RAM k/~,) conjugate.
QPtPrm~nat~on of the "minimal protective amount" (MPA) for
mnnnnlnnai rat anti-mouse ka~na/lambda (RAMx/7~)-antibodies which
are physically adsorbed onto colloidal carbon particles
The minimal protective amount (MPA) is the minimal amount of a
macromolecule that is necessary to protect one liter of a
particular colloidal suspension against electrolytically induced
flocculation by 14.3 g NaCl/1 (end concentration). The MPA is
dependent not only on the conditions in which coupling takes
place, but also on the size of the molecule to be coupled and on
the total available surface of all the particles that are
present in one liter of a specific colloidal suspension.
The test was performed as follows:
The pH of the carbon sol (0.01 (w/v) Degussa Printex 150T in
2.5 mM Tris-HC1, pH 10.5) was checked and, if necessary,
adjusted at pH 10.5. A stock solution of RAMx/~,-protein
containing 0.94 mg/ml in 2.5 mM Tris-HC1, pH 10.5 was prepared
and, using the same buffer, diluted so that a linear concentra-
tion range of 0-0.94 mg/ml RAMx/~,-protein in 2.5 mM Tris-HC1, pH
10.5 was achieved. 500 ~1 of the carbon sol (0.01~(w/v) Degussa
Printex 150T in 2.5 mM Tris-HC1, pH 10.5) was added to 100 ~,1 of
each RAMx/~,-protein solution which was in turn thoroughly mixed
with a vortex mixer. After an incubation period of 10 minutes,
100 ~1 10~(w/v) NaCl solution was added to each carbon sol/
protein suspension, which was thoroughly mixed once again.
Exactly 5 minutes after adding the 10~(w/v) NaCl solution, each
sol/protein-suspension was screened visually for the appearance
2.149062
of black carbon clumps which tended to flocculate rapidly
into/as a black pellet with a clear, colorless supernatant.
Controls of the test were:
5 1. 500 ~l carbon sol + 2 x 100 ~1 2.5 mM Tris-HC1, pH 10.5
imitating a carbon sol which is completely protected by
RAMx/~,-protein;
2. 500 ~1 carbon sol + 100 ~.1 2.5 mM Tris-HC1, pH 10.5 + 100 ~.1
10~(w/v) NaCl solution to determine the visual effects of
10 complete flocculation on the appearance of the reaction
mixture.
The results of this flocculation test show that at least 37.5 ~.g
RAMx/~,-protein was necessary to protect 500 ~.1 of a O.Oig(w/v)
15 Degussa Printex 150T carbon sol in 2.5 mM Tris-HC1, pH 10.5
against flocculation by 14.3 g/1 NaCl.
So, under the described coupling conditions the MPA for RAMx/~,-
protein would be 75 mg to adequately protect 1 liter (1000 ml!)
0.01~(w/v) Degussa Printex 150T carbon sol in 2.5 mM Tris-HC1,
20 pH 10.5 against an electrolytically induced flocculation.
Prey~arat~on of carbon particle-RAM conjugate
The stock carbon (C) solution (prepared according to method A)
was diluted 100 times by means of demineralized water, after
25 which the pH was set at 10.4 with 1 M KZC03 solution.
Starting from a stock solution of RAM (Euroclone) of 4.5 mg/ml
in 20 mM Tris-HC1, 150 mM NaCl, pH 8.0, 167 ~,1 RAM protein
suspension were added to 10 ml sol (-75 ~g RAM/ml carbon sol).
The suspension was incubated with stirring for 60 minutes.
Subsequently, the conjugate formed was washed 3 times by means
of 2.5 mM Tris-HC1, 5 mM NaCl, 1~ (w/v) BSA, 0.05 (w/v) NaN3,
pH 8.5 by centrifugation at 13,800 x g for 15 minutes. The
washed conjugates Were kept in the dark at 4°C.
CA 02149062 2004-11-26
26
Test ,procedu re
In stoppable polystyrene tubes (Greiner) of 4.5 ml HBT-
subisotyping test strips (HBT, no. L10.10/L10.20) were incubated
with:
A. 500 ~tl 20 mM Tris-HCl, 600 mM NaCl, 1% (w/v) casein,
0.2% (v/v) Tween-20, 0.02% (w/v) NaN3, pH 8.5
500 p1 with 10 ~.g/ml IgGl and 10 ~,g/ml IgG3 in RPMI
(Gibco), 10% (v/v) fetal calf serum
1 ml C-RAM conjugate
or
B. 500 ~,1 20 mM Tris-HC1, 600 mM NaCl, 1% (w/v) casein,
0.2% (v/v) Tween-20T;' 0.02% (w/v) NaN3, pH 8.5
500 ~1 RPMI (Gibco), 10% (v/v) fetal calf serum
1 ml C-RAM conjugate.
After 5-30
minutes of
incubation,
meanwhile
(carefully)
continuously shaking the tubes containing the strips, the test
result could be read:
Strip A showed
a specific
coloring of
the spots
at the positions
1.3 and kappa,
Strip B did
not show any
coloring.
E;Kam~le 3 - One- and two-step test strip for determining human
Chorionic Gonadotropin (hCG) by means of a (colloidal) carbon
anti-hCG conjugate
Presargti0ri of carbon particles anti-~-hGG conjugate
Method 1:
Before physically adsorbing the anti-a-hCG mouse monoclonal
antibodies (MAB) the stock-C-suspension (Degussa Spezialschwarz
4) was diluted 5 times by means of 5 mM KFi2P04 buffer, pH 6.2.
To 1 ml of 0.2% (w/v) C-sol in 5 mM KH2P04 buffer, pH 6.2, anti
a-hCG MAB (750 ~g/ml sol) was added. The suspension was incuba-
ted for 3 hours with shaking. Subsequently, the conjugate formed
was washed 3 times with 5 mM NaCl, 1% (w/v) BSA, 0.02% (w/v)
NaN3, pH 8.5 by centrifugation at 13,636 x g for 15 minutes. The
first supernatant which was formed in each washing step was
2149062
. ~ ~ 27
- again centrifugated for 15 minutes a 13,636 x g, after which the
pellets were combined. See also Example I. After the third and
last washing step the pellet was resuspended in the starting
volume. The washed conjugate was kept in the dark at 4°C.
Method 2:
Alternatively, carbon particle anti-a-hCG conjugates were
prepared by adding 4 ml anti-a-hCG MAB (with 750 ~g anti-a-hCG
MAB/ml in 5 mM KH2P04-buffer, pH 6.2) to 8 mg of dry carbon
powder (e. g. Degussa Spezialschwarz 100). The suspension with
750 ~g anti-a-hCG MAB/ml 0.2~(w/v) carbon particles was
homogenized on ice for 1 minute by means of a Branson Model 250
Sonifier: Output control 3 ~ 27 Watt, 20 KHz.
A deep black, stable suspension of colloidal carbon particle/
anti-a-hCG MAB-conjugates was formed. In order to remove any
unbound anti-a-hCG MAB, the conjugates formed were washed by
centrifugation (see also method 1).
p_rP~a_rati_on of nitrocellulose strips
Nitrocellulose strips were made with:
a. a linear dilution series of anti-(i-hCG mouse monoclonal
antibodies (MAB) spotted thereon;
b. a slot with rat anti-mouse monoclonal antibodies (negative
test slot) and a slot with anti-~-hCG MAB (positive test
slot) blotted thereon.
a. See Example I.
A linear dilution series of an anti-~-hCG MAB of 1000 ng;
500 ng; 250 ng; 125 ng; 62 ng; 31 ng; 15 ng; 8 ng; 4 ng; 2 ng in
10 mM PBS, pH 7.2 was spotted.
b. A slot with 1000 ng rat anti-mouse monoclonal antibodies
and a slot with 500 ng anti-~-hCG MAB were blotted per row on
nitrocellulose membranes (Schleicher and Schuell, type AE-99
having a pore diameter of 8.0 Vim) by means of a vacuum slotblot
apparatus (PR-600, Hoefer Scientific Instruments). After
blotting the membranes were air-dried for 3 hours.
CA 02149062 2004-11-26
28
The free positions of the nitrocellulose were blocked by
immersing the membranes for 1.5 hours at 37°C in 10 mM PBS, 2%
(w/v) BSA, 0.02% (w/v) NaN3, pH 7.2. The membranes were air-
dried again and then affixed onto an adhesive plastic carrier
material (Costar Serocluster platesealers) and finally cut to
size (10 x 75 mm).
The strips were kept dry in the dark and at room temperature.
Test procedure: Two-step method
In vitro experiment
In stoppable polystyrene tubes (Greiner) of 4.5 ml a series of
seven "anti-~-hCG strips" was pre-incubated with shaking with:
500 ~.1 20 mM Tris-HC1, 600 mM NaCl, 1% (w/v) BSA, 0.2%
(v/v) Tween-20, 0.02% (w/v) NaN3, pH 8.5
500 ~.1 2.5 mM Tris-HC1, pH 8.5 with such an amount of
purified hCG (Sigma) that after combining said
buffers the final concentration of the hCG was 50;
5; 1; 0.5; 0.1: 0.05 or 0 U/ml.
After the pre-incubation the strips were rinsed 3 times
with
10 mM PBS-T, pH 7.2 and once with demineralized water. The
strips were post-incubated with:
500 ~1 20 mM Tris-HC1, 600 mM NaCl, 1% (w/v) casein, 0.2%
(v/v) Tween-20, 0 . 02 % (w/v) NaN3, pH 8 . 5
500 ~tl 2.5 mM Tris-HC1, pH 8.5
1 ml C sol anti-~-hCG conjugate.
With the hCG concentrations (50-0.1 U/ml), the first four spots
occurred after about five minutes. After 1 hour of incubation,
the strip with:
50 U/ml hCG showed 7 spots decreasing in intensity:
5 U/ml hCG showed 5 spots decreasing in intensity;
1 U/ml hCG showed 4 spots decreasing in intensity;
0.5 U/ml hCG showed 4 spots decreasing in intensity;
0.1 U/ml hCG showed 4 spots decreasing in intensity;
0.05 U/ml hCG showed 4 spots all having a low intensity; and
0 U/ml hCG showed no spots at all.
CA 02149062 2004-11-26
29
In vivo experiment
In stoppable polystyrene tubes (Greiner) of 4.5 ml two "anti-~i-
hCG strips" were pre-incubated with shaking with
Control
strip:
250 ~tl 2.5 mM Tris-HC1, pH 8.5
250 ~1 20 mM Tris-HC1, 600 mN NaCl,1% (w/v) BSA, 0.2%
(v/v) Tween-20;M 0.02% (w/v)NaN3, pH 8.5
500 ~.1 urine of a non-p regnant
woman 4
weeks before
conception;
Test
strip:
250 ~tl 2.5 mM Tris-HC1, pH 8.5.
250 ~.1 20 mM Tris-HC1, 600 mM NaCl,1% (w/v) BSA, 0.2%
x~
(v/v) Tween-20, 0.02% (w/v)NaN3, pH 8.5
500 ~.1 urine of the sam e woman n the negative test,
as i
but this time days after conception.
16
After the pre-incubation the strips were rinsed 3 times with
10 mM PBS-T, pH 7.2 and once with demineralized water. The
strips were post-incubated with:
500 ~.1 2.5 mM Tris-HC1, 600 mM NaCI, 1% (w/v) casein,
0.2% (v/v) Tween-20;'~ 0.02% (w/v) NaN3, pH 8.5
500 ~1 2.5 mM Tris-HC1, pH 8.5
1 ml C-sol anti-a-hCG conjugate.
After a few minutes 3 spots occurred on the test strip, whereas
the control strip did not show any spots, even after incubating
overnight.
Tegt t~rocedL_re- pne-RtP~ me hod
Two nitrocellulose strips (Schleicher and Schuell, type AE99)
with slots of RAM and anti-~-hCG MAB were pre-wetted on the
place of application with 10 ~,1 10 mM PBS-T, 1% (w/v) BSA, 0.02%
(w/v) NaN3, pH 7.2 (= running liquid) after which 10 ~tl C-sol
anti-a-hCG conjugate was immediately applied on the same place
of application.
Subsequently, the strips were immersed with the side on which
the place of application was situated in glass vessels
CA 02149062 2004-11-26
containing 2 ml running liquid with 40 U/ml hCG (positive test),
2 ml running liquid with 5 mU/ml hCG (positive test), and 2 ml
running liquid without hCG (negative test), respectively. When
the liquid front had passed the entire length of the strip (this
5 took about 3-5 minutes), the positive test strips showed a black
coloring of both the RAM slot and the anti-~-hCG slot, while on
the negative test strip only the RAM slot was colored.
xa g,le 44 - One-step immunochromatographic test strip for
10 determining human Chorionic Gonadotropin (hCG) by means of a
(colloidal) carbon anti-hCG conjugate.
Preparation of carbon marticles anti-~hGC con~~~aate
A monoclonal antibody (MAb) directed against the a-subunit of
15 hCG was dissolved in 5 mM phosphate buffer, pH 6.7 and incubated
with a colloidal carbon (Printex 150T) suspension (2 mg/ml; made
by method A) in the same buffer to a final protein concentration
of 0.75 mg/ml. After incubation and washing (see Example 3), the
final pellet was resuspended in 5 mM Tris-HC1, 1~ (w/v) BSA,
20 0.02 (w/v) NaN3, pH 8.5, to the original volume. The conjugate
was stored in a glass tube at 4°C.
Pre~,aration of nitrocellulose strj,~
Nitrocellulose strips (Schleicher and Schuell, type AE99) were
25 line-sprayed with anti-~-hCG and RAM (Control) MABs (see Example
3) by means of a Linomat IV (CAMAG) at 1 ~1 (1 ~,g) per 0.5 cm.
The strips were blocked with 0.1 M borate, 1$ (w/v) BSA, 5~
(w/v) trehalose, 0.05$ (v/v) Tween-20, 0.02 (w/v) NaN3, pH 8.9.
30 One-stes euneriment
The strips were placed in devices with a sample window and a
test-result window. Urine of a non-pregnant woman was buffered
(final concentration 0.5 M Tris/HC1, 0.05 (v/v) Tween-20;M pH
8.5) and spiked with hCG in a serial dilution of 5 to 300 mIU/
ml. Spiked human urine (50 ~,1) was applied in the sample window
on a reservoir filter. Results appeared in about one minute. The
sensitivity of this test format was 10 mIU hCG/ml.
CA 02149062 2004-11-26
31
~ple 5 - Carbon sol particle immunoassay for human serum
albumin (HSA).
Preparation of carbon particles anti-aHSA conjugate
A monoclonal antibody (0.75 mg/ml final concentration) directed
against HSA was coupled onto colloidal carbon particles (Degussa
Spezialschwarz 100, 0.2% (w/v)) in 2.5 mM Tris-HC1, pH 8. The
suspension was gently stirred for 3 h at room temperature. For
assessing whether adsorption was successful, a flocculation test
was carried out as described in Example 2. The stable conjugate
was washed three times according to Example 1. The final pellet
was resuspended in buffer to the original volume. The conjugate
was stored at 4°C.
Preparation of nitrocellulose strips
HSA was spotted onto nitrocellulose strips (Schleicher and
Schuell, BA-85/23, pore diameter 0.45 Eun) in eight serial
dilutions (1000 to 7.8 ng) in 10 mM PBS, pH 7.4. Dried strips
were blocked in 10 mM PBS, 2% (w/v) BSA, 0.02% (w/v) NaN3, pH
7.4 for 90 min at 37°C. Strips were plastic-backed with
Serocluster Plate-sealers (Costar, Cambridge, UK).
On~~-stem ext~eriment
HSA was detected by a SPIA test format. The strips were placed
in 4.5 ml plastic tubes (Greiner) and were incubated (from
min to 16 h) in a suspension containing 250 ~,1 buffer (i.e.
20 mM Tris-HC1, 600 mM NaCl, 1% (w/v) BSA, 0.2% (v/v) Tween-20,
0.02% (w/v) NaN3, pH 8.5), 700 ~1 distilled water and 50 ~,1 of
the colloidal carbon-monoclonal antibody conjugate. Results were
30 evaluated by visual examination and by computer image analysis.
An amount of 7 ng HSA spotted onto a strip could be detected
visually. The quantification of the average grey level of each
spot was performed by computer image analysis. The grey level
scaling was expressed as a function of the logarithm of the
amount of HSA spotted. With the colloidal carbon conjugate used,
average grey levels of 7 to 500 ng HSA spots could be
distinguished. The shape of the curve plotted was similar to
those obtained with comparable ELISA's.
CA 02149062 2004-11-26
32
In a competitive test format, nitrocellulose strips onto which
62 ng of HSA had been spotted were incubated each in the same
buffer containing increasing amounts of free HSA (0.25 to
6.75 fig) mixed with the colloidal carbon - anti-HSA monoclonal
antibody conjugate in a total volume of 1 ml containing 250 ~1
20 mM Tris-HCl, 600 mM NaCl, 1% (w/v) BSA, 0.2% (v/v) Tween-20,M
0.02% (w/v) NaN3, pH 8.5. Inhibition was assessed visually and
by computer image analysis. By visual examination 0.25 ~g of
free HSA was already judged as an inhibitory amount.
Digitalization of the results gave a good correlation (r=0.996)
between the grey level scaling and the logarithm of the amount
of free HSA added.
