Method for depositing metal particles on a marker

An improved method for depositing metal particles on a marker which catalyzes the reduction of metal ions from a physical developer comprising a solution of metal ions, a molar excess of complexant in respect to the metal ions and a reducing agent. Said method being preferably employed for the detection of one or more components of an aggregate formed between a least one specific binding agent and its corresponding bindable substance by labelling at least one component of said aggregate with a marker and contacting said aggregate with a physical developer, whereby under influence of the marker a metal particle is formed which can be detected. Further the invention also relates to solutions and test-kits adapted for carrying out the above mentioned method.

BACKGROUND OF THE INVENTION 
Various methods are presently used for the qualitative and/or quantitative 
determination of specific binding agents and/or their corresponding 
bindable substances. Although these methods differ widely from each other 
in sensitivity, ease of operation and chemical and physical principles 
involved, important similarities are generally recognized. Typical 
examples of the relationship between a specific binding agent and its 
corresponding bindable substance(s) are of the type antigen-antibody, 
antibody-antigen, protein-protein, protein-ligand, receptor-ligand or 
nucleic acid-complementary nucleic acid. Antigen-antibody or immunological 
interactions are by far the most important in this connection, and 
particularly for diagnostic purposes, detection methods based on such 
interactions are the most widely used today. 
Various techniques can be employed to detect and optionally quantify the 
aggregates, by aggregates we mean complexes formed between the specific 
binding agents and bindable substances involved. In certain instances, the 
complexation reaction will lead to a directly visible signal as a result 
of agglutination and/or precipitation of the aggregate itself. This will 
however not always be the case and, in general, the concentration of 
binding agent and bindable substance, needed to produce such result, will 
be far above the practical and useful limits. In order to circumvent this 
lack of sensitivity or to detect otherwise un-detectable aggregates, 
various methods have been developed such as, for example, complement 
fixation, passive haemagglutination, radio-immuno assay (RIA), 
immuno-fluorescence and enzyme-linked immuno sorbent assay (ELISA). In the 
last three methods, the detection of the aggregate is improved by 
labelling the aggregate with an easily detectable marker, which is either 
bound directly to the specific binding agent, to a secondary binding agent 
for which the primary binding agent acts as a bindable substance, or to 
the bindable substance. In the three methods listed, the marker is 
respectively a radioactive atom or group, a fluorescent substance or an 
enzyme. Such methods are described i.a. in Weir's Handbook of Experimental 
Immunology (1967), Blackwell Scientific Publications, Oxford and Edinburgh 
and U.S. Pat. No. 3,654,090 (ELISA). 
During the last years, methods have been introduced wherein aggregates 
formed between specific binding agents and bindable substances are 
detected by labelling the said aggregates directly or indirectly with 
small sized metal particles, particularly gold particles. Depending on the 
circumstances, these particles can be detected, e.g. by direct visual 
examination, by microscopic or spectrophotometric techniques. A 
description of the "immunogold staining (IGS) technique", "the sol 
particle immuno assay (SPIA) technique" of specific applications and 
improvements thereof can be found e.g. in U.S. Pat. Nos. 4,313,734, 
4,446,238 and 4,420,558, in U.S. Ser. No. 622,923, which corresponds to 
the European Patent Publication No. 165,634, in U.S. Ser. No. 660,832, 
which corresponds to the Eur. Pat. Publ. No. 158,746 and in IBRO handbook 
series, Wiley, New York, 1983, pages 347 to 372. 
Metal particles have further been employed for the staining of acceptor 
substances, such as proteins and nucleic acid, which are directly 
immobilized on a solid support. Such a method is for example described in 
U.S. Ser. No. 744,091, which corresponds to the European Patent 
Publication No. 165,633. 
Starting from a relatively unknown method for labelling cell surface 
antigens, metal particles have today become widely used in a variety of 
detection and/or quantitive determination problems. The possibility of 
direct visual examination of metal particles and the advantage that the 
signal generated is permanent and not prone to rapid degradation makes it 
an interesting marker for simple and rapid assays. Moreover metal markers, 
preferably gold markers, seem preferable over radioisotope markers due to 
the very low health hazard related to working with the former. 
In the European Patent Publication No. 158,746 page 10 lines 18 to 32 there 
is described a method to improve the signal of a colloidal gold marker 
significantly by subjecting the colloidal gold particles bound to the 
surface of a blotting medium to a so-called physical developing procedure. 
The art-known physical developers generally consist of a solution 
containing a soluble metal salt, such as silver nitrate, a reducing agent, 
such as hydroquinone and an appropriate buffer system to establish a 
specific pH, preferable less than pH 4. 
Initially the reduction of silver ions to metallic silver is catalyzed at 
the surface of gold particles resulting in a specific deposition of 
metallic silver at the gold particle site. In turn, the thus formed 
metallic silver particles catalyze the reduction, creating an 
auto-catalytic process. The effect of a physical development is that the 
reddish optical gold signal turns into a deep-brown to black silver 
signal, with a much higher intensity. The use of these art-known physical 
developers results in an improved signal, although there are a number of 
drawbacks associated with it. 
One of the major problems is the solubility of the metal salts. Indeed, it 
is well known that metal ions, such as silver ions, form insoluble salts 
with many counter ions. Apart from depleting the available silver ion 
supply, these insoluble salts also form nuclei at which the reduction 
process is catalyzed as well, which results in a seriously augmented noise 
level. Moreover, silver ions may form light sensitive silver salts, such 
as silver bromide and silver chloride, which are readily reduced to 
metallic silver under the influence of light, starting an auto-catalytic 
process. It is therefore absolutely necessary to work with extremely clean 
contacting surfaces, e.g. vessels, analytical grade chemicals and 
ultra-pure water. Usually it is also necessary to introduce multiple 
washing steps between the incubation with the metal-marked specific 
binding agent and the physical development of the marker in order to 
remove unwanted ions present in the incubation medium. All this tends to 
make traditional physically developed metal-based assays more complex, 
expensive and error prone. 
The major disadvantage of the traditional methods lies within the nature of 
physical developing itself. In the case of a silver-based physical 
developer, for example, the reducing agent reduces all silver ions at a 
certain rate. To obtain optimal sensitivity the amplification process has 
to be aborted by removing the physical developer from the metal 
marker-containing phase before the non-marker-induced reduction, the 
so-called `self-nucleation`, becomes apparent. It is obvious that physical 
developers become more flexible and powerful if the ratio between 
metal-specific reduction speed and speed of self-nucleation can be 
augmented. With the traditionally used physical developers, this ratio can 
hardly be augmented. The only parameter which can be modulated is the 
overall speed of the process; self-nucleation can be postponed only at the 
expense of a slower metal marker amplification. One of the most obvious 
ways to do this is to change the concentration, nature or environment of 
the reducing agent. A frequently used approach is the use of hydroquinone 
at a pH lower than 4. The reducing action of hydroquinone is strongly 
inhibited in an acid environment; the user of the physical developer 
therefore has enough time to stop the metal marker amplification before 
self-nucleation causes too much noise. However, acid additions are in many 
cases not compatible with the nature of the binding between the marked 
specific binding agent and its corresponding bindable substance. Most 
monoclonal antibodies have only a low or average affinity to their 
antigens at said pH. Moreover, no real gain in sensitivity can be 
accomplished because the marker amplification is slowed down to the same 
degree as the self-nucleation is slowed down. 
Thus there is a strong need for improving the sensitivity and practicality 
of metal based detection and/or quantitative determination techniques. 
DESCRIPTION OF THE INVENTION 
The present invention provides a method for depositing metal particles on a 
marker which catalyzes directly or indirectly the reduction of metal ions 
from a physical developer whereby the physical developer used comprises a 
solution of metal ions, a molar excess of complexant in respect to the 
metal ions, a reducing agent and, if desired, a buffer system and one or 
more adjuvants. Said method is preferably employed for qualitatively 
and/or quantitatively determining one or more components of an aggregate 
formed between at least one specific agent and its corresponding bindable 
substance by labelling at least one component of said aggregate with the 
aforementioned marker. Said preferred method can conveniently be carried 
out by immobilizing the specific binding agent or the corresponding 
bindable substance, directly or indirectly, on a solid support, contacting 
the support with a counterpart labelled with a marker which catalyzes the 
reduction of the complexed metal ions of the physical developer, and 
adding the physical developer before or after the separation of the bound 
and free labelled components, whereby during the reaction or after an 
adequate reaction time, the formed metal particles are quantitatively 
and/or qualitatively determined in the test sample and/or in the derived 
fractions to provide a qualitative and/or quantitative indication of the 
component or components to be determined. In some instances it may be 
preferable to contact the support containing the immobilized bindable 
substance with a first binding agent specific to said bindable substance 
to form an aggregate herewith, and subsequently contacting the support 
carrying the thus formed aggregate with a second binding protein, which is 
specific to the said first binding protein, labelled with marker. The thus 
described method is particularly suited for the determination of 
immunochemical components, such as haptens, antigens and antibodies. 
Further, the present invention may also be employed for quantitatively 
and/or qualitatively determining an acceptor substance, such as a protein 
or a nucleic acid, which is directly immobilized on a solid support and 
bound with the aforementioned marker. 
Another aspect of the present invention is to provide versatile solutions 
and test-kits adapted for carrying out the above mentioned methods. 
The present invention remedies the drawbacks associated with the 
traditional developers by adding an excess of complexant to the developer. 
The complexants for use in the method according to the invention comprise 
any agent capable of forming water-soluble complexes with the metal ions 
of the physical developer. The complexation constant is selected so that 
on the one hand sufficient uncomplexed metal ions are present to allow the 
growth of the metal particles on the marker, while on the other hand the 
concentration of uncomplexed metal ions is sufficiently low to avoid the 
aforementioned undesirable side effects. 
Useful complexants in the method according the invention have at least one 
nitrogen donor atom having a lone pair of electrons available on nitrogen, 
such as, for example, amino acids, e.g., glycine, histidine, and 
heterocyclic bases. Preferred heterocyclic bases are optionally 
substituted mono- or bicyclic bases having at least one nitrogen 
accommodating a lone electron pair in a sp.sup.2 hybrid orbital, such as, 
aromatic heterocyclic ring systems containing (i) a five membered ring 
having two ring nitrogen atoms, e.g., imidazole, benzimidazole, pyrazole, 
purine and the like, with imidazole being most preferred, and (ii) 
aromatic heterocyclic ring systems containing a six membered ring having 
one ring nitrogen atom, e.g., pyridine, aminopyridine, nicotinamide, 
quinoline and the like. In connection with formation constants of silver 
complexes with imidazole and pyridine derivatives reference may be made to 
the Bulletin of the Chemical Society of Japan, 42, 3598-3600 (1969). 
The marker for use in the method according to the invention is meant to 
include any particle which can catalyze the reduction of metal ions, 
resulting in a deposition of the corresponding metal particles at the site 
of the said marker. Often the thus disposed metal particles in turn 
catalyze the reduction, creating an autocatalytic process. 
The markers to be used comprise metals, metal compounds or polymers 
optionally coated or impregnated with metals or metal compounds which can 
catalyze directly or indirectly the reduction of metal ions on their 
surface. As examples of such metals there may be named gold, silver, 
thallium, platinum, palladium as well as copper, nickel and the like with 
gold being preferred. As examples of metal compounds there may be named 
their corresponding complexes and sulfides. Polymers coated or impregnated 
with metals or metal compounds have similar properties as the metal or 
metal compounds but size, density and metal content can be optimally 
combined. For use in the preferred method the marker should be selected so 
that specific binding agents or any agent bindable thereby can be attached 
to the marker without loosing their affinity for their binding or bindable 
counterpart. 
Particularly preferred markers for use in the method according to the 
present invention are either (i) colloidal metal particles, optionally a 
sol, containing metals or metal sulfides; or (ii) metal chelates, 
especially those incorporating ethylenediaminotetraacetic acid (EDTA) or 
diethylenetriaminepentaacetic acid (DTPA) groups; or (iii) polymers 
optionally impregnated with metals or metal sulfides, e.g., polymerization 
products of benzidine derivatives, such as, for example diaminobenzidine 
polymers. 
The reducing agents for use in the method according to the present 
invention are meant to include any agent which reduces metal ions, 
preferably silver, gold, platinum, palladium or thallium ions, from a 
physical developer in proximity of an active site. Preferably said 
reducing agents form stable solutions with one or more constituents of the 
protected physical developer. As reducing agent there may particularly be 
mentioned, 1,2-dihydroxybenzene, 1,4-dihydroxybenzene (Hydroquinone), 
4-methylaminophenolsulfate (Metol.RTM.), 4-aminophenol, 
1,4-diaminobenzene, 1,2-diaminobenzene, N-(4-hydroxyphenyl)glycine, 
2,4-diaminophenol, 1-phenyl-3-hydroxypyrazole (Phenidone.RTM.) or mixtures 
thereof. As other constituents (adjuvants) of the protected physical 
developer there may be mentioned, buffers, preservatives, e.g., 
anti-oxidants or organic stabilizers, speed regulators, bactericides and 
the like, such as, for example, sodium sulfite, sodium bisulfite, sodium 
citrate and the like. 
The preparation of colloidal markers, in particular colloidal gold 
particles, their attachment to specific binding agents or any agents 
bindable thereby and the various methodologies of combining them, directly 
or indirectly, with the desired bindable substances are sufficiently 
known. In this connection, reference may be made to U.S. Pat. No. 
4,313,784; U.S. Ser. No. 660,832 which corresponds to the European Patent 
Publication No. 0,158,746; U.S. Ser. No. 744,091 which corresponds to the 
European Patent Publication No. 0,165,633; Immunohistochemistry, Cuello, 
A.C. (ed.), IBRO handbook series, Wiley, New York, 1983, pages 347 to 372 
and Techniques in Immunocytochemistry Vol. 2, pages 217 to 284 (1983). 
In general, the attachment is easily effected by contacting the particles 
with an aqueous medium of appropriate pH wherein the desired binding 
agents, e.g., antibodies, are dissolved. In order to protect the particles 
from non-selective interactions with non-specific proteins of the test 
samples it may be appropriate to add quenching or stabilizing agents such 
as, for example, immunochemically inert polar macromolecules, e.g. bovine 
serum albumin (BSA), polyvinyl pyrrolidone (PVP) and polyethylene glycol 
(PEG). After a suitable period of time the unstabilized particles and free 
or loosely bound binding agents are removed by repeated centrifugation and 
washing. If desired, the particles can also be sized according to a 
procedure described in J. Cell Biol. 90, 533-536. 
Alternatively, the proteins can also be covalently bound to the markers 
following the procedure described in U.S. Pat. No. 3,857,931 using a 
water soluble carbodiimide coupling agent. 
The attachment of metal chelates to binding agents such as antibodies is 
easily carried out following methodologies described in for example, 
Analytical Biochemistry 142, 68-79 (1984) and in Cancer Research 45, 
5694-5699 (1985). In general, reactions for coupling chelating agents such 
as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminotetraacetic 
acid (EDTA) and the like to proteins include diazonium coupling and 
acylation with activated carboxyl groups. The thus obtained 
chelator-conjugated proteins retain their immunoreactivity and can easily 
be charged with the desired metallic element. 
As polymers which could be used as marker for use in the methods of the 
present invention there may be named the polymerization products of 
benzidine derivatives being optionally charged with metals or metal 
compounds. The use and preparation of diaminobenzidine polymers is 
described, for example in J. Histochem. Cytochem. 30, 183-184 (1982) and 
Neuroscience 13, 513-525 (1984). 
The determinations to be made according to the preferred method of this 
invention may be performed homogeneously or heterogeneously. Homogeneous 
determinations are particularly simple to perform but require a measurable 
change of the perceived signal arising from either those markers present 
in the labelled reagent or in the labelled aggregate formed between the 
labelled reagent and the particles to be determined. In those instances 
where no such distinction is possible, heterogenous determinations will 
have to be performed. 
Homogeneous determinations are advantageous due to the fact that it is not 
necessary to physically separate the bound and unbound labelled species, 
thus reducing the number of steps necessary to perform an assay. The 
reaction between the labelled component and the corresponding binding 
counterpart causes the measurable change in the label's participation in 
or modulation of the signal generating moiety necessary to perform a 
homogeneous determination. The distribution of the markers between the 
bound and unbound species may be differentiated by the inability or 
altered ability of the said markers to affect the signal arising therefrom 
after development when present in the bound species. 
A homogenous determination may conveniently be performed according to 
art-known procedures such as, for example, the competitive binding 
technique. The sample containing the analyte is combined with a binding 
counterpart of the analyte, a labelled reagent comprising a marker coupled 
to the analyte or a specific binding analogue thereof, and the physical 
developer necessary to convert the marker to the signal generating moiety 
itself. Alternatively, a sequential determination may be performed whereby 
the sample and the analyte binding counterpart are first combined and 
thereafter the detectant reagent added. 
In many instances it is not possible to perform homogeneous determinations. 
In these cases a heterogeneous determination can be a particularly 
attractive alternative. In general, the heterogeneous determination system 
comprises at least two basic constituents and the physical developer which 
are combined simultaneously or subsequently, i.e. the analyte to be 
detected, a binding counterpart labelled with a marker and the physical 
developer necessary to convert the marker to the signal generating moiety 
itself. If necessary after an appropriate incubation period or periods the 
labelled reagent becomes bound to the corresponding bindable substance to 
be detected whereby the ratio of the bound species to the unbound species 
is a function of the amount of analyte being present. The bound and 
unbound species are physically separated and the amount of label being 
present in one thereof is determined. 
Various means of performing the separation step and of performing the 
binding reactions are known in the art. The said separation may involve 
conventional techniques such as, for example, by employing a solid-phase 
antibody or antigen, a second antibody, or a solid-phase second antibody; 
or by the use of immuno complex precipitation agents, adsorbents, and the 
like. The said binding reactions may for example include the so-called 
competitive binding technique, the sequential saturation technique, the 
sandwich technique, and the like. 
The preferred determinations to be made according to the method of this 
invention are heterogeneous determinations which are generally based on 
the principle that the labelled aggregate formed between the specific 
binding protein and the bindable substances is at some time immobilized in 
such manner that any unreacted particles can be washed off, whereupon the 
immobilized particles are detected "in situ" or, if desired, after 
disengagement in any other phase derived therefrom. 
In a particularly preferred embodiment, the binding substance to be 
detected, which may be contained in a crude test specimen or in a purified 
or partly purified fraction derived therefrom, is immobilized on an 
appropriate immobilizing support prior to its complexing with the labelled 
binding agent, specific to said bindable substance. 
The immobilization of the bindable substance may be carried out following 
the usual techniques, e.g., by spotting an aliquot of the test specimen on 
the immobilizing support or by immersing the latter in the test sample and 
subsequently drying and optionally washing off non-immobilized material. 
This is the so-called direct technique. As immobilizing supports for this 
technique use can be made of various materials, in general polymeric 
materials like, nitrocellulose, diazobenzyloxymethyl (DBH)- and 
diazophenylthioether (DPT) modified cellulose paper, paper, paper or 
cellulose acetate activated with cyanogen bromide, agarose, nylon, 
plastics and the like, which may take any form which is convenient for the 
determination process, e.g. sheets, beats, welled plates, dip-sticks and 
the like. 
The support is then brought into contact with a labelled binding agent 
under conditions which allow aggregate formation between the binding agent 
and the corresponding bindable substances. Consequently, at the sites 
where the bindable substance is immobilized, markers will be immobilized 
in turn in amounts proportional to the concentration of the immobilized 
bindable substance. 
In a variant of this method, the immobilized bindable substance is first 
allowed to react with a first binding agent which is specific therefor and 
subsequently the thus immobilized phase is brought into contact with the 
markers attached to a second binding agent which is specific for said 
first binding agent. 
Because of the lack of selectivity and specificity of the immobilizing 
process as described above, the direct method is usually employed with 
relatively pure or purified test samples or fractions. For more complex 
samples, the direct method will often be less suitable, as the 
non-specific immobilization of a large excess of non-desired material will 
interfere with the sensitivity and specificity of the determination. 
To avoid this problem, which is important with regard to routine analyses, 
an indirect or so-called sandwich technique can be used. In this 
technique, a purified or enriched primary specific binding agent is 
immobilized on a solid support. The latter is contacted with the test 
sample under conditions which allow the complexing of the corresponding 
bindable substances, which consequently become immobilized themselves. 
After removal of the test sample and washing of the support, the latter is 
contacted with a suspension of markers coated with secondary specific 
binding agents which are able to bind to uncomplexed sites of the 
immobilized bindable substance. 
The most straightforward case of embodiment to which the invention is 
applicable is a flow-through environment consisting of bindable substance 
which is immobilized, directly or indirectly, to a solid phase, and a 
liquid phase mobile relative to the solid phase. Depending on the 
direction of the liquid phase flow versus the solid phase, this solid 
phase can be liquid-permeable or -unpermeable. For example, a permeable 
membrane can be used as solid phase, allowing for a perpendicular flow of 
the liquid phase through that membrane. On the other hand, an unpermeable 
solid phase can be used in combination with a lateral liquid flow. 
During the first step of the embodiment, the liquid phase containing the 
marked specific binding agent is brought into contact with the bindable 
substance immobilized on the solid phase. The movement of this liquid 
phase relative to the solid phase may be continuous or discontinuous and 
must be such that the contact time between both phases allows for binding 
between the immobilized bindable substance and the marked specific binding 
agent to take place. However, this binding process not necessarily needs 
to reach its saturation point. The pressure drop between source and 
destination of the liquid phase, creating its flow, may be built in 
several ways. In the case of a permeable membrane as solid phase, a 
perpendicular flow may be created by bringing one side of this membrane 
into contact with a fluid-absorbing material and by applying the liquid 
phase on the other side. In the case of a non-permeable solid phase, a 
lateral flow of the liquid phase may be created by a pump. 
For the second step a protected physical developer according to this 
invention is applied as the liquid phase. Said protected physical 
developer comprises complexed metal ions as well as a reducing agent. In 
order to maximize the ratio between marker-specific reduction speed and 
the speed of self-nucleation, both components are kept apart until 
immediatly before use. However, it should be emphasized that in comparison 
with the prior-art, relatively stable formulations of the physical 
developer can be made by mixing two stable and liquid components. In some 
instances it may be preferable to apply both liquid components, one 
comprising metal ions, a molar excess of complexant in respect to the 
metal ions and the other comprising a reducing agent, subsequently and 
thus forming the protected physical developer "in situ". In some 
instances, stable and liquid components may be prepared from their 
corresponding dry consituents by adding an appropriate amount of water. In 
view of the above considerations the physical developer can easily be 
optimized towards the marker used, the sensitivity required and the 
contact time between the solid phase and the liquid physical developer. 
In the preferred flow-through embodiment, the mixing of the two stable 
liquid components and the application of the resulting protected physical 
developer should be combined into a single action. The application of the 
flowing protected physical developer has a dual effect. Initially, the 
liquid will wash away from the solid phase all remaining marked specific 
binding agents which were not or only loosely bound during the first step. 
Because the physical development of the marker is a gradually progressive 
process, this material will be washed from the solid phase before any 
signal becomes apparent. The remaining, bound marked specific binding 
agents will generate a visible signal during the further contact with the 
protected physical developer. The flow and volume of the developer applied 
to the solid phase can be chosen to ensure that the contact time is long 
enough for an optimal detection of the immobilized marked compounds and 
short enough to avoid non-specific reduction at the solid phase caused by 
self-nucleation. Typically, these times can be modulated from a few 
seconds to several minutes. For most protected physical developers there 
is no need for an additional treatment or fixing step to keep this signal 
permanent. 
The advantages of this new embodiment are obvious since it offers all the 
advantages of the physically developed colloidal metal marker systems 
described earlier, but without the also described disadvantages inherent 
to the classical physical developers. 
The present invention remedies the drawbacks associated with the classical 
developers by adding a molar excess of complexant to the developer 
preferably a several fold excess, for example, a two to a twenty fold 
excess. Apart from eliminating the necessity of very clean contacting 
surface, e.g. vessels, analytical grade chemicals, ultra-pure water and 
multiple pre-wash steps it also enables a severalfold increase in the 
ratio between marker-specific reduction speed, and speed of 
self-nucleation. Because the equilibrium is strongly shifted towards the 
complexed form, a more strongly reducing environment is allowable to 
mobilize the free metal ions necessary for the physical development. Under 
these circumstances the catalytic effect of the marker is more pronounced, 
resulting in an accelerated marker-specific reduction speed, without 
effecting the speed of self-nucleation. The increased ratio between marker 
specific reduction speed and the speed of self-nucleation can be exploited 
in several ways. In contrast with the prior art the sensitivity can be 
increased by keeping the marker longer into contact with the physical 
developer, or the speed of the marker specific development can be 
increased without loosing the flexibility offered by a safe period of time 
between the moment of optimum marker development and the moment where 
self-nucleation starts to give an increased background. In some cases a 
combination of both increased overall speed and sensitivity can be 
implemented. Said overall speed can easily be modulated by changing the 
concentration, nature or environment of the reducing agent. The utmost 
speed can be obtained using very small markers, preferably less than 5 nm, 
e.g., about 1 to 4 nm particles. In case gold particles are developed 
according to the present invention, the speed of the system can perfectly 
be modulated from very fast (10-20 sec) to slow (30 min or more). 
Obviously, none of the methods of the prior art can achieve such 
possibilities. Common developing systems only enable average (6-10 min) to 
low speed (15-30 min) systems. Further it should be noted that small 
markers not only accelerate marker-specific reduction speed but also 
accelerate the diffusion rate of the labelled reagents, and enable more 
efficient attachment of the markers to the suitable binding agents, 
resulting in an increased stability of the labelled reagents in 
suspension. Another point which should be emphasized for comparison with 
the prior art concerns the possibility to work in a neutral environment 
(pH 7) during development which results in an increased stability of the 
bound(s) formed between the binding pairs. 
The detection of the formed metal particles in a certain phase of the 
reaction mixture may take place using numerous techniques which are in 
themselves known. Said techniques are based upon the amount and/or the 
physical properties of the metal particles formed, preferably on the 
scattering and adsorption of the metal particles. As examples of these 
techniques there may be cited the spectrophotometric techniques such as 
densitometry, which will be preferred when quantitative determinations are 
desired. However, in view of the high sensitivity obtained the particles 
can easily be observed visually, optionally using a microscope. 
The specific binding agents which can be employed in the preferred method 
according to the invention can be of various nature but will in many 
instances be antibodies to specified antigens or haptens. As an example of 
specific binding substances other than antibodies there can be mentioned 
phages, which are optionally chemically or genetically adapted to bind 
molecular or cellular materials, lectins, which specifically bind 
glycoproteins, Staphylococcus aureus protein A which specifically binds 
immunoglobulins of various animal species and DNA or RNA probes for gene 
identification. In general any other moleculare interaction of sufficient 
specificity and affinity can be employed. Antibodies may be polyclonal or 
monoclonal. 
The binding reaction of the specific binding agent(s) and bindable 
substance(s) will in almost all cases be allowed to proceed under mild 
conditions. The reaction mixture will in general be an aqueous medium with 
any desirable organic co-solvents being present in minor amounts. The 
temperature of the reaction will be maintained at a constant level in 
normal circumstances throughout the incubation period. Temperatures will 
generally be between 0.degree. and 50.degree. C., more usually between 
20.degree. and 40.degree. C. Preferably the reaction will proceed at room 
temperature. The pH of the reaction mixture will vary between 5 and 10, 
more usually between 6 and 9. The concentration of various reagents will 
depend on the level of analyte expected in the test medium, with such 
level usually being between 10.sup.-3 and 10.sup.-12 M. Selection of the 
parameters is primarily based on empirically derived optimization balanced 
against the preferences and needs of the technician who will ultimately 
perform assays on a routine basis. None of the parameters therefore is of 
a critical nature to the present invention, rather they are all within the 
ordinary ranges used in the art. 
In view of their general nature, the methods according to the invention 
have an extremely wide field of application. In principle they can be 
applied to the qualitative and/or quantitative determination of any 
substance which can be labelled with the aforementioned marker. For 
example, such substances comprise but are not limited to cell surface and 
tissue antigens, biological substances excreted by or derived from living 
organisms, particularly biological substances occurring in biological 
fluids such as saliva, lymph, blood and its derived fractions such as, 
plasma and serum, urine, cerebrospinal fluid, amnion fluid, and the like. 
Substances which can be detected include, proteins, polypeptides, 
peptides, like enzymes, hormones, structural proteins, nucleic acids, 
vitamins, polysaccharides, toxins, alkaloids, glycoproteins, haptens, 
metabolites, pharmacological agents, pesticides, pollutants, steroids, and 
any other molecule for which a specific binding counterpart exists in 
biological systems or can be synthesized. 
Representative protein analytes include the classes of protamines, 
mucoproteins, glycoproteins, globulins, albumins, scleroproteins, 
phosphoproteins, histones, lipoproteins, chromoproteins, and 
nucleoproteins. Examples of specific proteins are prealbumin, 
.alpha..sub.1 -lipoprotein, human serum albumin, .alpha..sub.1 -acid 
glycoprotein, .alpha..sub.1 -antitrypsin .alpha..sub.1 -glycoprotein, 
transcortin, thyroxine binding globulin, haptoglobin, hemoglobin, 
myoglobin, ceruloplasmin, .alpha..sub.2 -lipoprotein, .alpha..sub.2 
-macroglobulin, .beta.-lipoprotein, erythropoietin, transferin, hemopexin, 
fibrinogen, the immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, IgG 
being preferred and their fragments, e.g., F.sub.c, F.sub.ab and 
F(ab).sup.2 complement factors, prolactin, blood clotting factors such as 
fibrinogen, thrombin and so forth, insulin, melanotropin, somatotropin, 
thyrotropin, follicle stimulating hormone, luteinizing hormone, 
gonadotropin, thyroid stimulating hormone, placental lactogen, intrinsic 
factor, transcobalamin, serum enzymes such as alkaline phosphatase, lactic 
dehydrogenase, amylase, lipase, phosphatases, cholinesterase, glutamic 
oxaloacetic transaminase, glutamic pyruvic transaminase, and uropepsin, 
endorphins, enkephalins, protamine, tissue antigens, bacterial antigens, 
and viral antigens such as hepatitis associated antigens (e.g., HB.sub.s 
Ag, HB.sub.c Ag and HB.sub.e Ag). 
Representative hapten analytes include the general classes of drugs, 
metabolites, hormones, pesticides, pollutants, vitamins, and the like 
organic compounds. Haptenic hormones include thyroxine and 
triiodothyronine. Vitamins include vitamins A, B, e.g. B.sub.12, C, D, E 
and K, folic acid and thiamine. Drugs include antibiotics such as 
aminoglycosides, e.g., gentamicin, tobramycin, amidacin, sisomicin, 
kanamycin, and netilmicin, penicillin, tetracycline, terramycin, 
chloromycetin, and actinomycetin; nucleosides and nucleotides such as 
adenosine diphosphate (ADP) adenosine triphosphate (ATP), flavin 
mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD) and its 
phosphate derivative (NADP), thymidine, guanosine and adenosine; 
prostaglandins; steroids such as the oestrogens, e.g., oestriol and 
oestradiol, steroids; and others such as phenobarbital, phenytoin, 
pirimidone, ethosuximide, carbamazepine, valproate, theophylline, 
caffeine, propranolol, procainamide, quinidine, amitryptiline, cortisol, 
desipramine, disopyramide, doxepin, doxorubicin, nortryptiline, 
methotrexate, imipramine, lidocaine, N-acetyl-procainamide, amphetamines, 
catecholamines, and antihistamines. Further cardiac glycosides, and 
derivatives of benzodiazepine, benzimidazole, piperidine, piperazine, 
imidazole, triazole, pyridazine, 1,2,4-triazinedione or 
2,3,5,6-tetrahydro-imidazo[2,1-b]thiazoles, or amides, hydratropic acid 
derivatives or trialkylamines. 
Benzimidazole haptens comprise thiabendazole, fuberidazole, ciclobendazole, 
oxibendazole, parbendazole, cambendazole, mebendazole, fenbendazole, 
flubendazole, albendazole, oxfendazole, nocodazole and astemizole. 
Piperidine haptens comprise diphenoxylate, phenoperidine, haloperidol, 
haloperidol decanoate, bromperidol decanoate, bromperidol, moperone, 
trifluperidol, pipamperone, piritramide, fentanyl, benperidol, droperidol, 
benzitramide, benzetimide, domperidone, sufentanil, carfentanil, 
alfentanil, dexetimide, milenperone, difenoxin, fluspirilene, penfluridol, 
pimozide, lorcainide, loperamide, astemizole, ketanserine, levocabastine, 
cisapride, altanserin, ritanserin, 
3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2,7-dimethyl-4H-pyrido-[1,2 
-a]pyrimidin-4-one, 
3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-methyl-4H-py 
rido[1,2-a]pyrimidin-4-one and 
3-[2-[4-[[3-(2-furanylmethyl)-3H-imidazo[4,5-b]pyridin-2-yl]amino]-1-piper 
idinyl]ethyl]-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one. 
Piperazine haptens include azaperone, fluanisone, lidoflazine, flunarizine, 
mianserine, oxatomide, mioflazine, clocinizine and cinnarizine. 
Examples of imidazole haptens are metronidazole, ornidazole, ipronidazole, 
tinidazole, isoconazole, nimorazole, miconazole, burimamide, metiamide, 
metomidate, enilconazole or imazalil, etomidate, econazole, clotrimazole, 
carnidazole, cimetidine, doconazole, sulconazole, parconazole, orconazole, 
butoconazole, triadiminole, tioconazole, valconazole, fluotrimazole, 
ketoconazole, oxiconazole, lombazole, bifonazole, oxmetidine, 
fenticonazole, tubulazole and 
(Z)-1-[2-chloro-2-(2,4-dichlorophenyl)ethenyl]-1H-imidazole. 
Triazole haptens comprise virazole, azaconazole, etaconazole, 
propiconazole, penconazole, itraconazole and terconazole. 
Pyridazine haptens comprise for example, 
3-chloro-6-[3,6-dihydro-4-(3-methylphenyl)-1(2H)-pyridinyl]pyridazine, 
3-methoxy-6-[4-(3-methylphenyl)-1-piperazinyl]pyridazine and the compounds 
of Publ. Eur. Pat. Appl. No. 0,156,433. 
1,2,4-Triazinediones comprise for example, 
2-chloro-.alpha.-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2 
(3H)-yl)benzeneacetonitrile, 
2,6-dichloro-.alpha.-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triaz 
in-2(3H)-yl)benzeneacetonitrile and the compounds of Publ. Eur. Pat. Appl. 
No. 0,170,316. 
Trialkylamines are, for example, diisopromine, prozapine. 
2,3,5,6-Tetrahydro-imidazo[2,1-b]thiazoles comprise, for example, 
tetramisole or levamisole. 
Amides comprise for example, closantel, ambucetamide, isopropamide, uzepide 
metiodide, dextromoramide. A hydratropic acid hapten is, for example, 
suprofen. 
The purposes of the determinations can be multiple. In certain applications 
they will be used merely as a scientific tool, to visualize particular 
substances, e.g. on histological coupes, on chromatograms, 
electrophoretograms, blots, etc. For example, when applying different 
labels to different specific proteins or other bindable substances on a 
chromatogram, electrophoretogram, protein blot and the like, a reference 
pattern is obtained which can advantageously be used to localize other 
proteins or other substances. Apart from its scientific utility, the 
method of the invention will find utility in a wide variety of diagnostic 
tests such as, for example, the following: the detection and 
characterisation of subpopulations of T-lymphocytes; pregnancy tests based 
on the presence of certain hormones (chorionic gonadotropin) in the urine, 
diagnostic tests for various infections diseases of i.a. fungal, bacterial 
and in particular viral origin, such as, for example, hepatitis B, 
auto-immune-diseases e.g. Lupus erythromatosus and immune deficiency 
diseases, e.g. AIDS, gonorrhoea, rubella, poliomyelitis, and the like; 
diagnostics for metabolic, endocrinological and various endogenous 
diseases, including diagnostics for the detection of congenital 
malfunctions of embryos based on the presence of specific proteins in the 
amnion fluid. 
Hence it can be employed in virtually all circumstances for which 
immunological techniques are conceived at present. In addition the present 
invention may also be employed for the determination and/or detection of 
acceptor substances, such as proteins or nucleic acids, which are directly 
immobilized in or on a solid support and bound with a colloidal marker 
following procedures described in the European Patent Publication No. 
0,165,633 and Analytical Biochemistry 145, 315-321 (1985) which are 
incorporated herein as reference. Said method comprises the subsequent 
steps of contacting a protein or nucleic acid support for a given time, 
with a sufficient concentration of colloidal markers suspended in a 
medium, preferably containing a detergent that does not interface with 
protein or nucleic acid binding, like for example 0.1% of the non-ionic 
detergent Tween 20, and appropriately pH adjusted, and adding a physical 
developer whereby during the reaction or after an adequate reaction time 
the formed metal particles are quantitatively and/or qualitatively 
determined. The physical developer according to this invention improves 
the sensitivity of this method without the drawbacks associated with the 
traditional developers. 
The methods according to the invention offer a framework which can be used 
for a wide variety of routine and experimental applications. Due to their 
nature and ease of handling, the methods lend themselves particularly for 
simple and rapid qualitative or semi-quantitative assays. These can be 
oriented towards use by experienced laboratory technicians as well as by 
non-technically trained medical personnel or laymen. The methods can also 
be easily automated which is an important factor when large numbers of 
identical determinations must be carried out, e.g., in blood banks and 
specialized clinical laboratories. 
The invention further comprises reagent systems which comprise all of the 
essential chemical elements required to conduct a desired assay method 
encompassed by the present invention. The reagent system can be presented 
in a commercially packaged form, as a composition or admixture where the 
compatability of the reagents will allow it, in a test device 
configuration, or as a test kit, i.e. a packaged combination of one or 
more containers holding the necessary reagents. Included in the reagent 
system may be the reagents appropriate for the binding reaction system 
desired, requiring a labelled reagent and the solutions making up the 
protected physical developer necessary to produce the signal-generating 
reaction. Such binding reaction reagents can include, in addition to the 
labelled reagent, a binding counterpart to the analyte, and so forth. Of 
course, the reagent system can include other reagents as are known in the 
art and which may be desirable from a commercial and user standpoint, such 
as buffers, diluents, standards, and so forth. 
More preferably the invention comprises test kits for depositing metal 
particles on a marker which catalyzes the reduction of metal particles on 
a marker which comprise, besides other reagents, a protected physical 
developer, which is preferably prepared by mixing two equal volumes untill 
immediately before use.

EXAMPLE 1 
1.1 Preparation of Colloidal Gold-labelled Anti-human Immunoglobulin (Ig) 
Antibodies 
A colloidal gold sol with a mean diameter of 20 nm AuroSol G20.RTM. was 
purchased from Janssen Life Sciences Products, B-2340 Beerse, Belgium. 
Affinity-purified goat-anti-human Ig antibodies were dialyzed overnight at 
4.degree. C. against a 5 mM carbonate buffer of pH 9.8. The acidity of the 
gold sol was brought to pH 9.0 with potassium hydroxide. 100 ml of this 
gold sol was stirred in a beaker at room temperature. 1 mg of the dialyzed 
antibodies was added. 2 Minutes later, 1 g bovine serum albumin (BSA) was 
added, predissolved in 10 ml 1 .mu.M potassium hydroxide. Another 2 
minutes later, the gold-antibody-BSA mixture was centrifuged at 4.degree. 
C. for 1 h at 15000 xg. After removal of the supernatant, the pellet was 
resuspended in 100 ml 20 mM Tris-HCl buffer at pH 8.2 containing 1% (w/v) 
BSA and 150 mM sodium chloride. This suspension was centrifuged again and 
the pellet resuspended in the same buffer to a volume at which the optical 
density at 520 nm was about 5.0. 
1.2 Preparation of the Protected Physical Developer 
Two liquid components of this developer were prepared separately. Solution 
A was made by dissolving 12 g histidine, 0.4 g silver nitrate and 4 g 
citric acid in 100 ml of distilled water. Solution B was made by 
dissolving 2.88 g sodium citrate, 2 g sodium sulfite, 6 g 
tris(hydroxymethyl)aminomethane, 0.5 g p-methylaminophenol sulfate and 0.2 
g p-aminophenol.hydrochloride in 100 ml of distilled water. 
1.3 Preparation of the Adsorptive Solid Phase 
Squares of white nitrocellulose paper (12.times.12 mm) were mounted on top 
of and in close contact with stacks of filter paper (40.times.60 mm, 10 mm 
high). This amount of filter paper was able to absorb a few ml of water. 
Each nitrocellulose/filter paper stack was put in a tight cardboard box 
with a hole (10 mm diameter) at the site of the nitrocellulose membrane. 
This hole was lined with a plastic cylinder (10 mm diameter, 7 mm high). 
The design was such that pouring a liquid into this cylinder created a 
flow through the nitrocellulose membrane into the absorbing filter paper. 
1.4 Performance of a Human Ig Detection in Buffer 
Samples with various concentration (10 to 200 .mu.g/ml) of human Ig were 
prepared in 50 mM Tris-HCl buffer of pH 8.2 containing 0.005% BSA. 5 .mu.l 
of such a human Ig solution was applied at the center of the nitrocellulose 
membrane, leaving a wet spot of about 5 mm diameter which dried within a 
few seconds (antigen immobilization step). 100 .mu.l of a 50 mM phosphate 
buffer of pH 7.5 containing 0.5% BSA and 0.5% Tween.RTM.20 was added to 
the membrane (free protein binding site saturation step). This amount of 
fluid covered the entire nitrocellulose membrane surface accessible 
through the cylinder and took about 15 seconds to flow through the 
membrane. 100 .mu.l of the colloidal gold-marked anti-human Ig antibodies 
were added. After this volume was sucked through the membrane (another 15 
seconds), 100 .mu.l protected physical developer solution A was mixed with 
100 .mu.l solution B in a small tube. Immediately afterwards, this 
activated developer was added to the membrane. 30 seconds later, the 
developer was entirely sucked through the membrane and the experiment 
finished. 
1.5 Results 
The presence of human Ig on the membrane surface resulted in a black dot 
(reduced silver) at the site where the sample had been applied. Around 
this dot the membrane stayed white, indicating the very low level of 
background staining. When 5 .mu.l of 50 .mu.g/ml human Ig was added (250 
ng), the developed signal was still clearly visible. Since not all Ig gets 
adsorbed this way, this corresponds to a sensitivity of better than 12 
ng/mm.sup.2. Starting from the immobilized antigen on the saturated 
membrane, the assay time took less than a minute. 
EXAMPLE 2 
2.1 Preparation of DTPA-coupled Human Immunoglobulins (huIg) 
120 mg DTPA were heated at 50.degree. C. for 90 minutes under stirring 
conditions in a mixture of 2 ml acetonitrile and 170 .mu.l triethylamine. 
After cooling to room temperature, 17.65 mg N-hydroxysuccinimide and 24 
.mu.l diisopropylcarbodiimide were added and the whole was stirred for 
another 90 minutes at room temperature. 1 ml of the resulting DTPA 
conjugate was then mixed with 5 ml of a 0.1M sodium hydrogen carbonate 
solution at pH 7.0 containing 5 mg/ml huIg and 0.2 mM EDTA. This mixture 
was kept at room temperature for 60 minutes with a short shaking session 
every 20 minutes. After this incubation, the sample was rapidly cooled in 
at -20.degree. C. for a few minutes and then kept at 4.degree. C. 
Afterwards, the huIg and DTPA-huIg molecules were separated from the 
unreacted DTPA-conjugate by gel filtration on a Sephadex.RTM. G100 column 
with a 0.1M sodium acetate solution at pH 5.0. 
2.2 Preparation of Silver-marked DTPA-huIg 
1 ml of DTPA-huIg (optical density at 280 nm=1.0) was brought to pH 10.7 
with 10 mM potassium hydroxide and drops of a 0.1M silver nitrate solution 
were added until the first signs of precipitation became apparent. A 
control sample was prepared by adding silver nitrate to DTPA-huIg at pH 
5.4, conditions known to be unfavorable for the binding of silver ions by 
DTPA. These mixtures were incubated at room temperature for 24 hours 
before separation of the Ag-DTPA-huIg and DTPA-huIg molecules from the 
free silver ions by gel filtration on a Sephadex.RTM. G25 column with 
phosphate-buffered saline (50 mM phosphate buffer at pH 7.5 containing a 
physiological concentration of sodium chloride). 
2.3 Preparation of the Protected Physical Developer 
Two liquid components of this developer were prepared separately. Solution 
A was made by dissolving 8 g imidazole, 0.18 g silver nitrate, 1.4 g 
sodium citrate (.2H.sub.2 O) and 3.6 g citric acid (H.sub.2 O) in 100 ml 
of distilled water. Solution B was made by dissolving 3.3 g sodium citrate 
(.2H.sub.2 O), 1.5 g citric acid (H.sub.2 O), 1.6 g hydroquinone and 0.75 g 
sodium sulfite in 100 ml of distilled water. 
2.4 Visualization of Ag-DTPA-huIg 
Strips of white nitrocellulose paper (5.times.30 mm) were used as an 
immobilizing matrix. Two 1 .mu.l drops of Ag-DTPA-huIg solution (optical 
density at 280 nm=1.0) and two 1 .mu.l drops of DTPA-huIg control solution 
(see 2.2) were applied to each nitrocellulose strip as four different 
spots. The unsaturated protein binding sites of these strips were blocked 
by incubating the strips for 5 to 10 minutes in a 50 mM phosphate buffer 
at pH 7.5 containing 0.5% BSA (bovine serum albumin) and 0.1% 
Tween.RTM.20. The actual development was performed by mixing 1 ml of 
developer solution A with 1 ml of developer solution B in a 10 ml test 
tube and incubating the spotted and saturated strip in it. Development 
sessions were held at room temperature for 30 minutes as well as for 24 
hours. After development, the strips were briefly rinsed with distilled 
water and air-dried. 
2.5 Results 
After a 30 minutes development, the Ag-DTPA-huIg spots were colored grey 
whereas the DTPA-huIg control spots and the background were not colored at 
all. After a 24 hours development, the Ag-DTPA-huIg spots were colored much 
more intense without any coloration of the control spots or the background. 
This example shows the specificity of visualizing this marker with a 
physical developer. Similar experiments showed that Ag-DTPA-huIg molecules 
have the same immunobinding behaviour as huIg molecules themselves. 
3.0 Visualization of Diaminobenzidine Polymers 
Strips of nitrocellulose were spotted with mouse IgG (1 .mu.l spot, 
starting at 250 .mu.g Ig/.mu.l). After blocking for 30 minutes at 
37.degree. C. with 5% BSA the strips were incubated with goat anti-mouse 
IgG peroxidase labeled for 60 minutes. After 3 washings each 3 minutes 
with 0.05M sodium phosphate buffer pH 7.5 containing 5 g/l BSA--0.5 ml/l 
Tween, the strips were washed 3 times with water. 
A substrate solution was prepared by dissolving 100 mg diaminobenzidine in 
4 ml of a phosphate buffer (5.75 g Na.sub.2 HPO.sub.4 +1.48 g NaH.sub.2 
PO.sub.4 -2 H.sub.2 O/l water). 0.5 ml of this solution was mixed before 
use with 25 ml of the same phosphate buffer and 75 .mu.l 3% H.sub.2 
O.sub.2. The strips were developed for 9 minutes. After washing, the 
strips were treated for 6 minutes with 0.03% HAuCl.sub.4 solution. After 3 
washings 3 minutes each, the strips were sooked in a 0.142% solution of 
Na.sub.2 S. After 3 washing 3 minutes each in water, the silver 
enhancement was done by mixing 0.5 ml solution A and 0.5 ml solution B as 
described in "2.3" with 1 ml Triton X.sub.100 solution (0.25% in H.sub.2 
O). 
3.1 Results 
After 8 minutes of enhancement a strong dark (black) signal was obtained. 
This example clearly shows that the protected physical developer according 
the present invention is much more simple to use than the classical 
developers. In order to obtain similar results with classical developers 
one need several additional steps and one has to take the extreme 
precautions known with the use of classical developers i.e. ultrapure 
water glassware and the like.