Process for the immunochemical determination of an analyte

The invention relates to a process for the immunochemical determination of an analyte in a sample by means of a first specific binding partner, where the specific binding partner being immobilized on a support and the extent of the binding of the analyte to the specific binding partner being determined by means of a further specific binding partner which directly or indirectly bears a label, wherein there is additionally added to the process a binding factor, which is not labelled.

The invention relates to a process for the immunochemical determination of 
an analyte in a sample by means of a first specific binding partner, the 
specific binding partner being immobilized on a support and the extent of 
the binding of the analyte to the specific binding partner being 
determined by means of a further specific binding partner which directly 
or indirectly bears a label. 
Customary immunological processes for diagnosing diseases which are 
accompanied by formation of specific antibodies against a disease-causing 
agent, such as viruses, bacteria, allergens, autoantigens or particular 
pharmaceuticals depend on the ability of these antibodies to form 
complexes with antigenic structures of the causative agent. 
In some of these processes, generally designated as heterogeneous 
immunoassay, a sample which is to be examined for the content of, for 
example, specific antibodies (analyte antibodies) is brought into contact 
with antigenic structures of the disease-causing agent, these antigenic 
structures being immobilized on suitable known support materials. Analyte 
antibodies contained in the sample are bound as an immune complex to the 
antigenic structures of the disease-causing agent which are immobilized on 
the support material, and detected. For the detection, detection 
antibodies or other specific receptors, for example protein A, may be used 
which are able to form complexes with the analyte antibody of the sample. 
As a rule, the detection reagent bears a label which permits measurement by 
instrumentation of the quantity of the bound specific antibody. 
Common labels are: radioactive isotopes, enzymes, fluorescent, 
phosphorescent or luminescent substances, substances having stable 
unpaired electrons, erythrocytes, latex particles, magnetic particles and 
metal sols. 
In these processes, both single-step and multi-step detection methods are 
known. Each process step is customarily terminated by a separation process 
(washing step). 
In heterogeneous immunoassays, the technique of the single-step method, 
which is very simple to perform, is not, however, suitable for detecting 
all disease markers. For technical reasons, two-step or else multi-step 
processes must often be used. 
These methods are very specific, but have the disadvantage, however, that 
the disease-causing agents to be detected, or antibodies directed against 
them, which have entered a complex with the immobilized specific receptor 
in the first process step, can partially dissociate again from the complex 
in the subsequent incubation steps, in a reverse reaction known to the 
person skilled in the art, and thereby elude the detection reaction, 
resulting, inter alia, in the sensitivity being markedly reduced. 
The diagnostic efficiency of such multi-step processes is always 
particularly strongly reduced when the rate of the reverse reaction 
between immobilized receptor and agent to be detected is high. This is the 
case, for example, with low-affinity antibodies against disease-causing 
agents or against pharmaceuticals. These problems are also known 
particularly in the case of processes for detecting frequently mutating 
disease-causing agents or disease markers, which show lower interaction 
with the immobilized specific receptor after mutation. There was therefore 
the object of finding reagents which do not possess the indicated 
disadvantages. 
Surprisingly, it was established that the rate of the reverse reaction is 
substantially reduced by addition of a binding factor against structural 
features of the agent to be detected. This binding factor must possess 
more than one site which is capable of binding the agent to be detected 
and must not interfere with the immunochemical detection of the agent. 
The agent to be detected (analyte), in the sense of this invention, can be 
either an antibody which is induced, for example, by a disease-causing 
agent, or an antigen such as, for example, the disease-causing agent 
itself. 
The invention therefore relates to a process for the immunological 
determination of one or more analytes by means of a specific binding 
partner, the specific binding partner being immobilized on a support and 
the extent of the binding of the analyte to the specific binding partner 
being determined by means of a further specific binding partner which 
directly or indirectly bears a label, wherein, in the process, a binding 
factor is additionally used which possesses more than one site which is 
capable of binding the agent to be detected, possesses no affinity for the 
immobilized specific binding partner, and is not labeled.

In this context, not labeled means that it bears either no label or at 
least not that label with which the extent of the binding of the analyte 
to the specific binding partner is determined. 
In the sense of this invention, addition also denotes that the binding 
factor is immobilized on the solid phase in the relevant process steps or, 
in the case of the detecting agents based on matrix chemistry and known to 
the person skilled in the art, is already present on the, phase, having 
been dried into it. 
The processes in which binding factors may be used are known per se in all 
their embodiments to the person skilled in the art. Among these processes 
are single-step and multi-step processes, with, in the case of the latter, 
a washing step being inserted as a rule between the individual steps. 
It is important that the process according to the invention can be employed 
in a suitable form in all immunochemical processes in which, in a first 
step, an immunochemical or comparable binding of an analyte to a 
preferably immobilized, specific binding partner is effected, and, in a 
second, but not necessarily temporally separated, step a direct or 
indirect detection is effected. 
Without thereby postulating a particular mode of action of the binding 
factors, it appears to be advantageous if the analyte, which may be a 
peptide or a protein, possesses, besides the binding sites for the 
specific solid phase binding partner and detection binding partner, at 
least one further epitope. 
Preferably, the binding factors are employed in the processes known to the 
person skilled in the art as sandwich ELISA, an enzyme, preferably with a 
chromogenic or fluorogenic substrate or a chemiluminescent label, 
preferably being used as the abeling system. However, the embodiment of 
the detection method does not have a primary influence on the possible 
uses of the binding factors. 
Microtiter plates, magnetic particles, latex particles or test elements 
based on matrix chemistry, such as, for example, fibers or test modules 
containing membranes, are preferably used as solid phases. 
It is also known to the person skilled in the art that immunochemical 
processes as described above may also be employed for the simultaneous 
determination of different analytes, such as, for example, HIV 1/2 or HIV 
1+2/HCV. Such embodiments are also included here. 
Processes are advantageous in which the binding factor is added in the 
reaction step in which the analyte binds to the second, preferably 
labeled, specific binding partner. 
The use of the binding factor has a particularly advantageous effect in 
multi-step processes, the binding factor preferably being added after the 
first washing step. In addition, the invention relates to a reagent for 
use in the abovementioned process which contains a binding factor. 
Furthermore, the invention relates to the abovementioned process in which 
the binding factor is an antibody conjugate. 
Binding factors, in the sense of the invention, are specific binding 
partners which possess more than one binding site with bioaffinity for the 
agent to be detected. A binding factor or components of this binding 
factor may be constituted by conjugates of the antibodies as well as the 
antibodies themselves. In the sense of this invention, antibodies are 
monoclonal or polyclonal antibodies as well as the known immuno-reactive 
fragments. 
Lectins, or conjugates of a plurality of lectins, or conjugates of lectins 
with agent-specific antibodies or their fragments, are likewise suitable. 
The binding factor can be added at any point in the respective reaction 
step of the determination; preferably, however, the addition is effected 
after the binding of the analyte to the solid phase. 
Binding factors which are particularly preferred are antibodies against the 
specific immunoglobulin class of the antibody to be detected. 
In the case of the antigen detection, the antibody used as a binding factor 
is preferably one which does not recognize the same epitope as the 
solid-phase antibody or the conjugate antibody. 
A process is preferred in which at least one washing step is used. 
Preferably, the binding factors are not homologous to the analyte 
antibodies. If the analyte antibody is a human antibody, mouse or rabbit 
antibodies or antibody conjugates, or conjugates of antibody fragments, 
are very particularly preferred. 
Methods which are familiar to the person skilled in the art for preparing 
such conjugates, while preserving their bioaffinity function, are, for 
example, linking by means of chemical reagents or by means of interaction 
based on bioaffinity. 
Hybrid molecules can also be produced by chemical synthesis, by the 
hybridoma technique, or by methods of gene technology. If a plurality of 
relevant agents (e.g. antibodies of the immunoglobulin classes G and M) 
against one or more defined disease-causing agents, e.g. against HIV 1 and 
HIV 2, are being detected, the binding factor can, for example, interact 
on a bioaffmity basis simultaneously with two or more agents. 
The reagent according to the invention can be used in a multiplicity of 
processes within human and veterinary diagnostics. Examples which may be 
listed are two-step or multi-step tests for detecting antibodies of 
different immunoglobulin classes against structural features of viruses 
(e.g. viruses of the hepatitis A, B or C type as well as various HIV 
types), of bacterial and parasitic pathogens, and of allergic disorders. 
Additional examples are the detection of disease-causing agents such as 
viruses (e.g. hepatitis B virus), bacteria, parasites or allergens as well 
as of markers of diseases(e.g. tumor markers) in one-step or multi-step 
detection processes. 
The invention is illustrated by the following example, without thereby 
being limited to it: 
EXAMPLE 
a) Preparation of a reagent according to the invention 
1 ml of 0.2 M LiBO3/20% dioxane is added to 4 mg of monoclonal anti-human 
IgG antibody (Fc-specific) in 1 ml of PBS, pH 7.2, and the mixture is 
treated with a 15-fold molar excess of 
N-.gamma.-maleimidobutyrtyl-succinimide (GMBS) and incubated at room 
temperature for 1 h. The heterobifunctional reagent which remains 
unreacted is separated off by gel chromatography (Sephadex G-25) with 0.1 
molar Na phosphate buffer +5 mmol/ml nitrilotriacetic acid (NTA), pH 6.0. 
4 mg of monoclonal anti-human IgG antibody (Fc-specific) in 4 ml of 10 
mmol/l sodium phosphate and 100 mmol/l NaCL pH 7.4, is incubated with a 
24-fold molar excess of N-succinimidyl-3-(2-pyridyl-dithio)propionate 
(SPDP) at room temperature for 30 min., and then subsequently reduced with 
dithiothreitol (100-fold molar excess in relation to SPDP) at room 
temperature for 15 min. After reduction is complete, the low-molecular 
component is removed by gel filtration on Sephadex G-25 (0.1 M Na 
phosphate, 5 mmol/l NTA, pH 6.0). 
The SH-activated anti-human IgG is incubated with the maleimide-activated 
anti-human IgG at room temperature for 2 hours and the reaction is 
subsequently stopped with 1/10 vol. of 0.1 M N-ethyl-maleimide. The 
conjugate is purified by gel chromatography (ACA 34, LKB) using 50 mmol/l 
TRIS/HCl, pH 7.4, and subsequently concentrated down to 1-3 mg/ml. 
b) Preparation of an HIV 1 (env)-peptide-peroxidase conjugate 
10 mg of HIV 1 (gp 41) peptide (IAF BioChem, Canada) are dissolved in 1 ml 
of glacial acetic acid/water (50:50, v/v). After neutralization with 5 N 
sodium hydroxide solution, a 10-fold molar excess of GMBS is added to the 
mixture, which is then incubated at room temperature for 1 h. The GMBS 
which remains unreacted is separated off by gel filtration (Sephadex G-25) 
using 0.1 M sodium phosphate/5 mmol/1 NTA, pH 6.0. 10 mg of horseradish 
peroxidase are incubated in 5 ml of 10 mmol/l , sodium phosphate and 100 
mmol/l NaCl, pH 8.0, with a 100-fold molar excess of 2-iminothiolane at 
room temperature for 1 h. Subsequently, free modifying reagent is removed 
by gel chromatography (Sephadex G-25) using 0.1 M sodium phosphate/5 
mmol/l NTA, pH 6.0. 
Both eluates (SH-activated peroxidase and maleimide-modified HIV 1 peptide) 
are combined and incubated at room temperature overnight. After the 
reaction has been stopped with 1/10 vol of 0.1 M N-ethylmaleimide, the 
conjugate is freed of non-reacted HIV 1 peptide by gel chromatography 
(Sephadex G-25). After concentration (2 mg/ml), the peptide-peroxidase 
conjugate is stored at -20.degree. C. 
c) 2-Step enzyme immunoassay for detecting HIV 1 antibodies 
An enzyme immunoassay for detecting anti-HIV 1 antibodies is carried out as 
follows: 
25 .mu.l of sample buffer (0.3 M Tris/HCl and 1% albumin, 2% Tween 20, pH 
7.2) are incubated with 100 .mu.l of human serum at 37.degree. C. for 30 
min. in the wells of a test plate (.RTM.Enzygnost Anti HIV 1+2, 
Behringwerke AG, Marburg, FRG) coated wit HIV 1 and HIV 2 peptides. After 
washing 4 times with 50 mmol/l PBS, 0.1% Tween 20, 100 .mu.l of the HIV 1 
peptide-peroxidase conjugate (1:1000 in 0.1M Tris/HCl, 1% albumin and 2% 
pluronic/F 64, pH 8.1) prepared according to Example 1 b) are pipetted in. 
The 30-minute incubation (+37.degree. C.) is terminated with 4 further 
washing steps. The bound peroxidase activity, which correlates directly 
with the number of bound HIV 1-specific antibodies, is determined by 
addition of H.sub.2 O.sub.2 tetramethylbenzidine (Behringwerke AG, 
Marburg, FRG). 
d) Use of the reagent according to the invention 
Anti-HIV 1 positive sera and anti-HIV negative sera are examined in the 
enzyme immunoassay according to c). In the one case, 10 .mu.l of 50 mmol/l 
Tris/HCl, pH 7.4 (reference system), and in the other, 10 .mu.l of the 
reagent prepared according to a) according to the invention (system 
according to the invention) are added to 10 ml of conjugate solution. 
The results (extinction units) of the investigations are to be found in the 
table, and comparative titrations of HIV 1 antibodies of positive sera in 
FIG. 1 and FIG. 2. 
TABLE 
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System according 
Reference system 
to the invention 
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Control serum, negative 
0.019 0.018 
Control serum, positive 
0.964 1.681 
0.985 1.756 
Anti-HIV neg. sera 
0.015 0.019 
0.017 0.021 
0.032 0.028 
0.019 0.024 
0.030 0.018 
Anti-HIV neg. plasmas 
0.014 0.016 
0.017 0.016 
0.028 0.017 
0.016 0.016 
0.016 0.015 
Anti-HIV 1 pos. plasma 
&gt;2.500 &gt;2.500 
1:500 &gt;2.500 &gt;2.500 
1:1000 1.424 2.440 
1:2000 0.694 1.054 
1:4000 0.317 0.478 
1:8000 0.161 0.216 
1:16000 0.083 0.107 
1:32000 0.052 0.064 
1:64000 0.032 0.039 
Cut off value 0.119 0.118 
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