DE102007037068A1 - Detecting analytes of biological samples, comprises providing reversible binding partner, adding biological sample and binding analyte to reversibly immobilized analyte binder, adding stripping buffer, detecting analyte in stripping buffer - Google Patents

Abstract

Detecting analytes of biological samples, comprises: providing reversible binding partner, (1) which is immobilized on solid phase and to which analyte binder is reversibly bound via reversible binding partner (2); adding biological sample and binding analyte to reversibly immobilized analyte binder in event that biological sample contains analyte; separating biological sample; adding stripping buffer; and detecting analyte in stripping buffer in event that biological sample contains analyte or determining absence of analyte in event that biological sample does not contain analyte. Detecting analytes of biological samples comprises: providing a reversible binding partner, which is a liquid phase, (1) which is immobilized on a solid phase and to which an analyte binder is reversibly bound via a reversible binding partner, which is a solid phase, (2) which is bound to the analyte binder, where the analyte binder is immobilized by binding the reversible binding partners (1) and (2); adding the biological sample and binding the analyte to the reversibly immobilized analyte binder in the event that the biological sample contains the analyte; separating the biological sample; adding a stripping buffer which dissolves the bond between the reversible binding partners (1) and (2), where the bond between the analyte and the analyte binder optionally remains; and detecting the analyte in the stripping buffer in the event that the biological sample contains the analyte or determining the absence of the analyte in the event that the biological sample does not contain the analyte. Independent claims are included for: (1) a device for the execution of a method, comprising a solid phase with a binding partner reversibly immobilized at it (1) to which an analyte binder is reversibly bound via a reversible binding partner (2), which is bound to the analyte binder; where the bond between the reversible binding partners (1) and (2) is dissolved by the addition of a stripping buffer, where the binding of the analyte to the analyte binder optionally remains with the addition of the stripping buffer; and (2) a kit for the execution of the method comprising the solid phase with a binding partner (1) reversibly immobilized at it; a complex comprising analyte-binder that binds to the reversible binding partners (2), where this complex can optionally immobilized through the reversible binding partner (1) and (2) and on the solid phase; stripping buffer, which is dissolved by the bond between the reversible binding partners (1) and (2), where the bond of the analyte optionally remains at the analyte-binder.

Description

• a) Bereitstellung eines an eine Festphase immobilisierten Reversiblen Bindungspartners 1, an den ein Analyt-Binder über einen an den Analyt-Binder gebundenen Reversiblen Bindungspartner 2 reversibel gebunden ist, wobei der Analyt-Binder immobilisiert wird durch Bindung zwischen den Reversiblen Bindungspartnern 1 und 2,
• b) Hinzugabe der biologischen Probe und Bindung des Analyten an den reversibel immobilisierten Analyt-Binder im Fall, dass die biologische Probe den Analyten enthält,
• c) Abtrennung der biologischen Probe,
• d) Zugabe eines Ablösepuffers, der die Bindung zwischen den Reversiblen Bindungspartnern 1 und 2 löst, wobei die Bindung des Analyten an den Analyt-Binder optional bestehen bleibt, und
• e) Detektion des Analyten im Ablösepuffer im Fall, dass die biologische Probe den Analyten enthält beziehungsweise Feststellen der Abwesenheit des Analyten im Fall, dass die biologische Probe den Analyten nicht enthält.

The present invention is a method for detecting analytes from biological samples comprising the following method steps:

• a) providing a reversible binding partner 1 immobilized on a solid phase, to which an analyte binder is reversibly bound via a reversible binding partner 2 bound to the analyte binder, the analyte binder being immobilized by binding between the reversible binding partners 1 and 2,
• b) adding the biological sample and binding the analyte to the reversibly immobilized analyte binder in case the biological sample contains the analyte,
• c) separation of the biological sample,
• d) adding a release buffer which solves the bond between the reversible binding partners 1 and 2, whereby the binding of the analyte to the analyte binder optionally remains, and
• e) detection of the analyte in the release buffer in the event that the biological sample contains the analyte or determination of the absence of the analyte in the event that the biological sample does not contain the analyte.

rapid test, or point-of-care (POC) testing procedures are in various technologies been developed (1). However, very few of them have market maturity obtained.

nowadays established rapid test methods, such. B. the TRIAGE system (biosite, San Diego, USA), but especially the most widespread Methods, immunochromatography, are disadvantageous classical - be it manually or automatically processed - immunoassays a lower analytical sensitivity as well as decreased Precision up, but can be beneficial, anyway in appropriate embodiments, unprocessed whole blood samples to process. (2) - (7)

The Disadvantages are mainly based on the sample matrix dependency the method and the limitation of the usable sample volume.

task The present invention is therefore a u. a. as a quick test to develop the process to be performed, the unprocessed Can process whole blood samples and analytical sensitivity and has the same precision as classic immunoassays is comparable.

object In particular, the use of a reversible binding system according to the invention for the immobilization of an analyte-specific binder, z. B. anti-PCT antibody.

• a) Bereitstellung eines an eine Festphase immobilisierten Reversiblen Bindungspartners 1, an den ein Analyt-Binder über einen an den Analyt-Binder gebundenen Reversiblen Bindungspartner 2 reversibel gebunden ist, wobei der Analyt-Binder immobilisiert wird durch Bindung zwischen den Reversiblen Bindungspartnern 1 und 2,
• b) Hinzugabe der biologischen Probe und Bindung des Analyten an den reversibel immobilisierten Analyt-Binder im Fall, dass die biologische Probe den Analyten enthält,
• c) Abtrennung der biologischen Probe,
• d) Zugabe eines Ablösepuffers, der die Bindung zwischen den Reversiblen Bindungspartnern 1 und 2 löst, wobei die Bindung des Analyten an den Analyt-Binder optional bestehen bleibt, und
• e) Detektion des Analyten im Ablösepuffer im Fall, dass die biologische Probe den Analyten enthält beziehungsweise Feststellen der Abwesenheit des Analyten im Fall, dass die biologische Probe den Analyten nicht enthält.

The present invention thus provides a method for detecting analytes from biological samples comprising the following method steps:

• a) providing a reversible binding partner 1 immobilized on a solid phase, to which an analyte binder is reversibly bound via a reversible binding partner 2 bound to the analyte binder, the analyte binder being immobilized by binding between the reversible binding partners 1 and 2,
• b) adding the biological sample and binding the analyte to the reversibly immobilized analyte binder in case the biological sample contains the analyte,
• c) separation of the biological sample,
• d) adding a release buffer which solves the bond between the reversible binding partners 1 and 2, whereby the binding of the analyte to the analyte binder optionally remains, and
• e) detection of the analyte in the release buffer in the event that the biological sample contains the analyte or determination of the absence of the analyte in the event that the biological sample does not contain the analyte.

the It is clear to a person skilled in the art that between step b) and c) some Incubation period is observed. The incubation period should not shorter than 30 s and no longer than 24 h, especially preferred in the context of a rapid test procedure is an incubation period between 5 and 15 min. The removal process according to step d) may preferably be up to 24 hours, preferably in the frame a rapid test procedure, the detachment process is longer as 15 s and shorter than 15 min, preferably between 5 to 15 minutes.

The binding of the analyte to the analyte binder remains or does not occur during and / or after detachment according to d). Both options are conceivable according to the invention. Preferably remains at and / or after Add the release buffer according to d) the binding between the analyte and the analyte binder. The binding remains preferably at least 90% of the analyte molecules exist. If the bond does not persist, it is preferably restored at a later date. Most preferably, at the time of detection, the binding between analyte and analyte binder, that is, either retained during the stripping process, is restored.

In In a preferred embodiment, the analyte binder is an anti-analyte antibody.

In a preferred embodiment of the invention Procedure, the biological fluid is an unprocessed Whole blood sample.

According to the invention the immobilized reversible binding partner 1 directly or by means of a carrier protein is immobilized. An exemplary Carrier protein is BSA (bovine serum albumin). The expert suitable carrier proteins are known.

According to the invention various binding partners that form strong bonds However, under certain conditions, this bond can give up and thus a "reversible binding system" represent. Of particular interest here are binding systems, where the binding is destabilized by relatively mild conditions conditions, which are the conformation of proteins, in particular antibodies, and their binding with a Do not significantly affect the antigen.

The Application of such reversible binding systems is known in the context purification possibilities of recombinant proteins complex protein mixtures such as cell extracts. By recombinant DNA technology is used in this context for the protein sequence coding cDNA sequence added a sequence, which coded for a so-called "tag", so that expressed a protein extended by one day becomes. Via a binding partner immobilized on a solid phase for the "tag" can then the "tag" protein selectively bound from the protein mixture and then by a specific destabilization of the tag bond be separated again from the solid phase. The protein of interest is then enriched or pure in solution. overviews to common "tags", corresponding immobilized Find binding partners and specific destabilization methods z. At (8) - (10).

Such Binding systems can be part of the current Invention can be used as a binding partner.

• a) positiv und negativ geladene Peptidoligomere,
• b) Ca²⁺-bindendes Peptid/Protein und Antikörper, der das Peptid/Protein mit größerer Affinität bindet, wenn das Peptid/Protein Ca²⁺ gebunden hat,
• c) Oligohistidin (z. B. 6 His) und Ni-NTA,
• d) Biotin und Avidin oder Streptavidin oder Neutravidin.

In the context of the method according to the invention, in particular the binding pair Reversible binding partner 1 and 2 can be selected from one of the following binding pairs:

• a) positively and negatively charged peptide oligomers,
• b) Ca ²⁺ -binding peptide / protein and antibody that binds the peptide / protein with greater affinity when the peptide / protein has bound Ca ²⁺ ,
• c) oligohistidine (eg 6 His) and Ni-NTA,
• d) biotin and avidin or streptavidin or neutravidin.

Some reversible binding systems which are within the scope of the present invention are briefly presented here:
An example of a Ca ²⁺ -binding peptide / protein and an antibody that binds the peptide / protein with greater affinity when the peptide / protein has bound Ca ²⁺ is the FLAG / M1 system, wherein the Ca ²⁺ -binding Peptide / protein is a FLAG peptide and the antibody is an M1 antibody. In another system, the Ca ²⁺ -binding peptide / protein is a protein C peptide and the antibody is an HPC4 antibody.

a) FLAG / M1

An example is thus the binding between the monoclonal antibody M1 (also described as 4E11) and the so-called FLAG peptide. This peptide is able to bind calcium ²⁺ , thereby assuming a specific conformation. Only this conformation is efficiently bound by M1. By adding calcium ^{2 +} -komplexierender agents such as EDTA calcium ²⁺ the peptide is removed from the peptide assumes a different conformation, whereby the M1 antibody to the peptide reduced its affinity dramatically; US Pat 4,851,341 (11).

b) protein C / HPC4

Another analogous example of such a binding pair is a human protein C-derived peptide, which may also adopt a calcium ²⁺ -dependent conformation, and the monoclonal antibody HPC4. (12)

c) Ni ²⁺ -NTA / 6 His

Another example is the Ni ²⁺ -NTA / 6 His system, which can be specifically destabilized by imidazole addition. (13)

d) charged peptide oligomers

One Another example is a binding pair of positive or negative charged peptide oligomers, such as. B. Oligo-Lys or Oligo-Asp, its bonding with each other by increasing the ionic strength can be destabilized. As a binding pair is described a charged peptide oligomer in association with an ion exchange matrix; this bond is also due to the increase in ionic strength to destabilize. (14) - (16)

Next reversible binding systems in which one of the binding partners integrated into a recombinant protein, systems are also known in which one of the binding partners chemically to be immobilized to the Protein is conjugated. For example, here is the biotin / avidin system. Biotin can z. In the form of an NHS ester to primary amine groups of a protein (17). This binding system is also destabilize by adding an excess of biotin, but only under drastic conditions such as elevated temperature and longer incubation time (18). It is however a variant This system described which so-called monomeric avidin used; after saturation "non-reversible" Binding sites with biotin, are the remaining "reversible" Binding sites suitable for binding a biotinylated protein, then under mild conditions using biotin from the binding site can be solved (19).

Peptide "tags" as described above for recombinant proteins also be chemically conjugated to a protein, whereby the so derivatized Protein are then fed to a reversible bond can. In the context of the present invention is the chemical conjugation regarded as a preferred variant of the protein derivatization, since the degree of derivatization is controllable and the chemical conjugation comparatively time-saving and cost-effective.

All previously mentioned examples of reversible binding systems based on a noncovalent interaction of the binding partners. in the Frameworks of the present invention are as reversible binding systems However, to look at such systems, where initially there is a covalent bond, which is then dissolved chemically can be. To name a few examples here is the covalent crosslinking by means of a cleavable hetero-bifunctional crosslinker such as about SPDP (20); by adding a reducing agent, the Disulfide bridge cleaved and dissolved the bond with it become.

object The invention is therefore also the invention Rapid test method, wherein the analyte binder is covalently attached to the reversible Binding partner 2 is bound.

object The present invention is thus in a preferred embodiment the inventive method, wherein the analyte binder is an anti-procalcitonin antibody.

One preferred object of the invention is thus the application of a reversible binding system for the immobilization of a analyte-specific binder, z. B. anti-PCT antibody.

The Application is exemplified below for the reversible Binding system of the charged peptide oligomers (see above) formulated, is to be transferred analogously for the other systems. Oligo-Asp is linked to a carrier protein such as bovine serum albumin (BSA) conjugate, and this conjugate becomes stable on a solid phase such as B. highly binding polystyrene microtiter plates immobilized. Oligo-Lys is conjugated to an anti-PCT antibody. Incubation of the oligo-Lys-anti-PCT antibody with the solid phase leads to immobilization of the antibody by binding between the oligo-lys and oligo-asp portions ionic interaction.

In an alternative embodiment, the anti-PCT antibody may also be indirectly by means of an oli go-Lys-derivatized antibody against the anti-PCT antibody are immobilized (for example, an anti-mouse IgG antibody, when the anti-PCT antibody is a mouse monoclonal antibody).

• – der Möglichkeit, unprozessiertes Vollblut zu verwenden
• – der Möglichkeit, größere Probevolumina zu verwenden, damit relativ große Menge des Analyten zu extrahieren und schließlich eine hohe analytische Assaysensitivität im sich anschließenden Bestimmungsverfahren zu erreichen
• – der Matrixunabhängigkeit für Folgeschritte im Bestimmungsverfahren, die sich vorteilhaft auf Präzision und Richtigkeit des Bestimmungsverfahrens auswirken

The principle of immune extraction is exemplified below for the PCT analysis:
First, an anti-PCT antibody is immobilized on a solid phase via a reversible binding system. A whole blood sample to be examined (peptide oligomers charged for the example and EDTA blood for the example) is added and PCT is immuno-extracted from the sample. In this case, a comparatively large volume of sample can be used, so that a comparatively large amount of PCT can be extracted, which subsequently leads to a high analytical sensitivity of the test. After a short incubation period, the sample is separated (by washing or other suitable separation techniques such as suction or appropriate centrifugation). In the next step, a buffer is added ("dissolution buffer") containing an agent (in the oligo-lys / oligo-asp system eg the strongly negatively charged heparin) which is suitable for binding the antibody to the antibody However, the solid phase does not destabilize the binding between the PCT and the antibody to a large extent, so a solution containing the analyte to be determined (complexed to a specific antibody) in a large amount is always of identical nature for each sample tested because the sample matrix was previously separated, the benefits of this step are:

• - the possibility to use unprocessed whole blood
• The ability to use larger sample volumes to extract relatively large quantities of the analyte and ultimately achieve high analytical assay sensitivity in the subsequent assay
• - the independence of the matrix for subsequent steps in the determination process, which have an advantageous effect on the precision and accuracy of the determination process

Of the Immuno-extracted and solubilized analyte can by different methods are detected. An example is the TRACE Technology, another method (explained below) the immunochromatography.

The TRACE technology used for a sandwich immunoassay two antibodies with different fluorescent labels are marked (for example, cyanine or cryptate). When sandwiching with the analyte in solution, both labels achieve a spatial Near to a specific light emission after appropriate Light excitation leads.

in the exemplary context in the analysis of PCT will be with the Oligo-Lys conjugated anti-PCT antibodies before immobilization first also marked with crypt. Herewith the immune extraction is performed. In the detachment buffer is now next to the release agent, the second for cyanine-labeled anti-PCT antibodies necessary for sandwiching contain. This occurs both by adding the release buffer for detachment of the antigen-antibody complex from the solid phase as well as the sandwich formation, which according to suitable Light excitation is detectable. Replacement and addition of the second antibody for sandwich formation can also be done sequentially.

In In a variant described above, the analyte-specific antibody indirectly via an oligo-Lys derivatized before the immune extraction z. B. anti-mouse IgG antibody immobilized. In this Embodiment, the fluorescence label then to the one or the other antibody.

At the Example of immunochromatography becomes that conjugated with oligo-Lys anti-PCT antibodies before immobilization initially also labeled with biotin. This is the immune extraction carried out. In the detachment buffer is now beside the peel agent, the second for sandwiching necessary, labeled with colloidal gold anti-PCT antibody contain. Replacement and addition of the second antibody for sandwiching can also be done sequentially. The Reaction mixture is subsequently subjected to an immunochromatographic Test strip applied to the as catcher for the formed sandwich a biotin binder such. B. avidin as fine Line at the back of the test strip across the direction of travel the reaction solution is sprayed on.

In a particularly preferred embodiment of the invention Procedure, the analyte binder is marked and remains after addition of the release buffer labeled with a label for the detection of the analyte.

In the embodiment of immunochromatography, the following further variants may be conceivable:
The immunodextraction antibody is additionally biotinylated in addition to the derivatization with binding partner 2. One second antibody for sandwiching is labeled (eg with colloidal gold). The catcher on the test strip would then be a biotin binder such as avidin or streptavidin or neutravidin.

Of the Immune extraction antibody (eg sheep polyclonal) derivatized only with binding partner 2. A second antibody for sandwiching would have to be from another animal species are monoclonal (eg mouse) and labeled (eg with colloidal Gold).

Of the Catcher on the test strip would then be an anti-sheep IgG antibodies. Both variants are also conceivable in the form the immune extraction antibody carries the label and wherein in the first variant then the second antibody is biotinylated (alternatively, here could be the second antibody also be sprayed directly on the strip).

In another preferred embodiment of the method the release buffer contains another for the detection required marked component.

In a particularly preferred embodiment of the method is the further marked required for detection Component a second analyte binder for the implementation a sandwich immunoassay.

Especially Preferably, the detection of the labeled analyte by a Immunoassay using TRACE technology.

In In another preferred embodiment, the detection takes place of the labeled analyte by immunochromatography.

In a particularly preferred variant according to the invention the biological sample is undiluted and in such a way Volume is used that the coated portion of the solid phase completely brought into contact with the biological sample becomes.

Especially preferably, the duration of addition of the sample exceeds until detection is not 30 minutes and the method is thus a rapid test method to watch.

• • eine Festphase mit einem daran immobilisierten Reversiblen Bindungspartners 1, an den ein Analyt-Binder über einen an den Analyt-Binder gebundenen Reversiblen Bindungspartner 2 reversibel gebunden ist,
• • wobei die Bindung zwischen den Reversiblen Bindungspartnern 1 und 2 durch Zugabe eines Ablösepuffers gelöst wird, jedoch die Bindung des Analyten an den Analyt-Binder bei Zugabe des Ablösepuffers optional bestehen bleibt.

Likewise provided by the present invention is an apparatus for carrying out the inventive method comprising:

• A solid phase having a reversible binding partner 1 immobilized thereon, to which an analyte binder is reversibly bound via a reversible binding partner 2 bound to the analyte binder,
• Wherein the bond between the reversible binding partners 1 and 2 is dissolved by the addition of a release buffer, but the binding of the analyte to the analyte binder optionally remains with the addition of the release buffer.

• • eine Festphase mit einem immobilisierten Reversiblen Bindungspartner 1
• • Komplex umfassend Analyt-Binder gebunden an den Reversiblen Bindungspartner 2, wobei dieser Komplex optional bereits durch Bindung der Reversiblen Bindungspartner 1 und 2 auf der Festphase immobilisiert vorliegen kann
• • Ablösepuffers, der die Bindung zwischen den Reversiblen Bindungspartnern 1 und 2 löst, wobei jedoch die Bindung des Analyten an den Analyt-Binder optional bestehen bleibt.

Likewise provided by the present invention is a kit for carrying out the process according to the invention comprising:

• • a solid phase with an immobilized reversible binding partner 1
• Complex comprising analyte-binder bound to the reversible binding partner 2, which complex may optionally already be immobilized by binding of the reversible binding partner 1 and 2 on the solid phase
• Release buffer, which solves the binding between the Reversible binding partners 1 and 2, but the binding of the analyte to the analyte binder optionally remains.

In a particularly preferred embodiment is in the Kit analyte binders an anti-PCT antibody.

• • positiv und negativ geladene Peptidoligomere,
• • Ca²⁺-bindendes Peptid/Protein und Antikörper, der das Peptid/Protein mit größerer Affinität bindet, wenn das Peptid/Protein Ca²⁺ gebunden hat,
• • Oligohistidin (z. B. 6 His) und Ni-NTA,
• • Biotin und Avidin oder Streptavidin oder Neutravidin.

The most preferred kit contains as selected binding pair Reversible binding partner 1 and 2 selected from one of the following binding pairs:

• Positive and negatively charged peptide oligomers,
• Ca ²⁺ -binding peptide / protein and antibody that binds the peptide / protein with greater affinity when the peptide / protein has bound Ca ²⁺ ,
• Oligohistidine (eg 6 His) and Ni-NTA,
• • biotin and avidin or streptavidin or neutravidin.

The The following examples are intended to explain the invention in more detail without restricting the subject matter of the invention:

Examples

Immune Extraction from PCT thoroughbred

When Basis for the processing of further steps was first checked whether and how effective an analyte is, here by example PCT examined from unprocessed EDTA whole blood by means of a immobilized on a solid phase analyte-specific antibody extract. After a short incubation (5 min) and separation the whole blood sample was bound PCT with a second, chemiluminescent-labeled anti-PCT antibodies detected. For comparison, PCT-containing Serum, diluted in buffer, incubated on the same solid phase, however, longer (2 hours) and the bound PCT became separated the diluted sample as demonstrated above.

in the Specifically, the experiment was carried out as follows:

to Determination of immune extraction were the components from the BRAHMS PCT sensitiv LIA Kit (BRAHMS Aktiengesellschaft, Hennigsdorf, Germany) Germany). The luminescently labeled anti-PCT tracer antibody A was dissolved in tracer reconstitution buffer B according to the instructions. A standard series was in EDTA whole blood of a healthy blood donor to the following concentrations: c = 0.007; 0.014; 0.036; 0.171; 0.383; 1.73; 7.99 ng / ml. In the with anti PCT antibodies coated tubes were each 300 ul whole blood standards pipetted and incubated for 5 min at room temperature. After that were tubes with 5 x 1 ml wash solution (8mM Tris, 60mM NaCl, 0.2% Tween 20, pH 7.5). Subsequently 200 μl of luminescence-labeled anti-PCT antibody pipetted and incubated for 2 h at room temperature; the tubes were washed again with 5 × 1 ml of washing solution. The chemiluminescence bound to the tubes was determined in a luminometer (BERTHOLD, BAD WILDBAD, GERMANY, LB952T; Basic reagents BRAHMS AG).

The reference method (determination of PCT from diluted serum) was carried out on the basis of the BRAHMS PCT sensitive LIA kit (BRAHMS Aktiengesellschaft, Hennigsdorf, Germany) as follows: A standard series was dissolved in zero serum from the test kit at the following concentrations:
c = 0; 0.008; 0.031; 0.042; 0.126; 0.251; 1.21; 5.61; 28.0 ng / ml. 50 μl serum standards and 250 μl tracer reconstitution buffer B were pipetted into the tubes coated with anti PCT antibodies and incubated for 2 hours at room temperature. Thereafter, the tubes were washed with 5 x 1 ml wash solution (8 mM Tris, 60 mM NaCl, 0.2% Tween 20, pH 7.5). Subsequently, 200 μl of luminescence-labeled anti-PCT antibody were pipetted and incubated for 2 h at room temperature; the tubes were washed again with 5 x 1 ml of washing solution. The chemiluminescence bound to the coated tubes was measured in a luminometer (BERTHOLD, BAD WILDBAD, GERMANY, LB952T, basic reagents BRAHMS AG) ( 1 )

Representation of reversible to be bound to a solid phase antibodies

Several of the reversible binding systems described above were examined for their suitability to reversibly bind an antibody, in this case using an anti-PCT antibody, to a solid phase. For this purpose, the antibody was derivatized differently (see Table 1) and labeled with a chemiluminescence label. It was thus investigated how these antibodies bind to solid phases corresponding to the respective derivative and can be removed again. The summary of the results in Table 1 shows that all tested reversible binding systems except the biotin / avidin system can be specifically and rapidly destabilized and, with limitations for the 6 His / Ni-NTA system, are unaffected by blood matrix. Derivative on the antibody (binding partner 1) Solid phase (binding partner 2) peeling agent Proportion of the antibody which can be unspecifically removed by blood (10 min incubation) based on the previously bound amount Proportion of the antibody which can be removed by detaching agent (10 min incubation) based on the previously bound amount Proportion of the antibody which can be released by the detachment agent (30 min incubation) based on the previously bound amount biotin avidin 25 mM biotin 0.7% 1.9% 2.5% M1 antibody FLAG peptide-BSA 10mM EDTA 7.8% 58.7% 74.8% 6 His Ni-NTA 250 mM imidazole 38.6% 89.4% 98.8% HPC4 antibody Protein C-peptide-BSA 10mM EDTA 3.5% 38.3% 69.2% Oligo-Asp Oligo-Lys BSA 0.01% heparin 5.3% 80.0% 93.2% Tab. 1

It was further observed that the efficiency of binding and more specific Replacement can be influenced by the respective degree of derivatization (the molar ratio of binding partner 1 per antibody), the concentration of immobilized on the solid phase binding partner 2, by whether binding partner 1 conjugates to the antibody and binding partner 2 was immobilized, or binding partner 2 conjugated to the antibody and immobilized binding partner 1 and, in the case of the Oligo-Asp / Oligo-Lys system, the length both oligomers. Therefore fall within the scope of this invention such Variants although also under the concept of reversible binding systems and the claims exclude such variants expressly included, but these are exemplary here variants described as preferred embodiments to watch. Polylysine solid phases also proved to be an alternative to oligo-Lys-BSA as suitable.

in the Specifically, the experiment was carried out as follows:

Binding partner 1 (liquid phase)

1. Preparation of MACN Chemiluminescent Labeled antibody

A polyclonal anti-calcitonin sheep antibody was treated as follows:
Three marks were applied; these were rebuffered differently after incubation (see below).

For chemiluminescent labeling of the antibody, 1 ml of the antibody solution (c = 3.19 mg / ml) was mixed with 40 μl of 1 M potassium phosphate, pH 7.8, and with 2.7 μl of MACN-acridinium-NHS ester (c = 1 mg / ml). ml; InVent GmbH, Hennigsdorf, Germany) (molar labeling ratio of antibody: MACN 10: 1). Then it was incubated for 30 min at room temperature. Subsequently, the labeling mixtures were rebuffered via NAP-10 gel filtration columns (Pharmacia, Upsalla, Sweden) in 1.5 ml of eluent and freed from low-molecular constituents: Mark 1 PBS pH 7.4 for biotinylation see 2.1. Mark 2 100 mM sodium phosphate, 5 mM EDTA, pH 6.9 for SPDP activation see 2.2. Mark 3 PBS, 10mM EDTA, pH 8.0 for SMCC activation see 2.3.

Of the Protein content was determined photometrically, the labeled antibody were stored portioned at -20 ° C.

2. Derivatizations of the MACN-chemiluminescence-labeled antibody

2.1. biotinylation

Of the MACN antibody was coupled with EZ-Link NHS-chromogenic-biotin (Pierce, Rockford, IL, USA, Art. No. 21325) in molar ratio biotinylated from 1:10.

1 mg of MACN antibody was probed with 5.41 μl of 1.233 mM Biotin solution (freshly dissolved in DMSO) and Incubated for 60 min at room temperature. The reaction was treated with 100 μl 50 mM glycine stopped for 10 min at room temperature and over a NAPS column (Pharmacia, Upsalla, Sweden).

to Separation of last residues from non-antibody bound Biotin was subjected to gel filtration HPLC (column: Bio-Sil Sec 400, Biorad, Munich, Germany). The Sample was applied and at a flow rate of 0.8 ml / min with PBS, pH 7.4. With a flow photometer the wavelengths 280 nm and 354 nm were measured. Of the Marking degree μM biotin / μM antibody was at the peak 0.48. The antibody-containing fractions (retention time 11-12 min) were pooled.

Of the Protein content was determined photometrically, the conjugate was portioned stored at -20 ° C.

2.2. Conjugation with anti FLAG-tag M1 antibody, or anti ProtC-tag HPC4 Anti body

For the conjugation with anti FLAG-tag M1 (Sigma, Deisenhofen, Germany, Art. No. F 3040), or anti ProtC-tag HPC4 (Roche, Nutley, NJ, USA, USA, Art. No. 11814516001) Antibody these and the MACN antibody were labeled with SPDP (N-succinimidyl 3- (2-pyridyldithio) propionate, Pierce, Rockford, IL, USA, Art. No. 21857) in the molar ratio of 1: 7 activated (see "Instructions SPDP Reagents", Pierce, Rockford, IL, USA).

ever 2 mg of antibody were mixed with 4.67 μl each of 20 mM SPDP (freshly dissolved in ethanol) and 30 min at room temperature incubated. Only the activated M1 and HPC4 antibodies were with 200 mM DTT (dithiothreitol dissolved in 100 mM sodium phosphate, 5 mM EDTA, pH 6.9) to a final concentration of 10 mM DTT and reduced for 15 min at room temperature.

The reduced M1, or HPC4 antibodies were each about purified a NAP10 column (eluent: 100 mM sodium phosphate, 5 mM EDTA, pH 6.9) and the protein content was determined photometrically.

The reduced products were each activated with the unreduced SPDP 1: 1 molar ratio of MACN antibody Night conjugated at 4 ° C. Subsequently, the Reaction each with 50 ul 100 mM cysteine stopped for 10 min.

Of the Protein content was determined photometrically, the conjugates were portioned stored at -20 ° C.

2.3. Conjugation with His-tag, or oligo-Asp peptide

to Coupling of His-tag Peptide "PRG12" (Amino Acid Sequence RGSHHHHHGGC, JPT GmbH, Berlin, Germany, M = 1359.8), or the oligo-Asp peptide "D14C" (amino acid sequence DDDDDDDDDDDDDDC, JPT GmbH, Berlin, Germany, M = 1731.43) was the MACN antibody with SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Pierce, Rockford, IL, USA, Art. No. 22360) in a molar ratio of 1:50 activated (see "Instructions SMCC and Sulfo-SMCC", Pierce, Rockford, IL, USA). 2 mg MACN antibodies were with 6.68 μl of 100 mM SMCC (freshly dissolved in DMSO) and incubated for 30 min at room temperature. The product was over a NAP10 column (Pharmacia, Upsalla, Sweden) (Eluent: PBS, 10 mM EDTA, pH 8) and the protein content photometrically certainly.

Subsequently Immediately conjugation with the His-tag, or oligo-Asp Peptide (dissolved in distilled water) in molar ratio from 1: 100. After incubation for 60 min at room temperature, the Reaction with 100 μl of 100 mM cysteine stopped for 10 min, again each over a NAP25 column (Pharmacia, Upsalla, Sweden) purified (mobile phase: PBS, pH 7.4) and the protein content photometrically certainly.

Both Conjugates were stored aliquoted at -20 ° C.

Binding partner 2 (solid phase)

1.1. Conjugation of BSA with FLAGtag / ProtC-tag

to Coupling of Flag-tag Peptide "PDC12" (Amino Acid Sequence DYKDDDDKGGGC, JPT GmbH, Berlin, Germany, M = 1286.46), or ProtC-tag peptide PEG15 (amino acid sequence EDQVDPRLIDGKGGC, JPT GmbH, Berlin, Germany, M = 1601.7) was protease-free Bovine serum albumin (Sigma, Deisenhofen, Germany) with SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Pierce, Rockford, IL, USA, Art. No. 22360) in a molar ratio 1: 2 activated (see "Instructions SMCC and Sulfo-SMCC", Pierce, Rockford, IL, USA).

2500 μl 0.5% BSA (dissolved in PBS, 10 mM EDTA, pH 8) was mixed with 3.78 μl 100 mM SMCC (freshly dissolved in DMSO) and 30 min incubated at room temperature. The product was about a NAP25 column (Pharmacia, Upsalla, Sweden) (eluent: PBS, 10 mM EDTA, pH 8) and the protein content was determined photometrically.

Subsequently Conjugation with the FLAG-tag or ProtC-tag took place immediately Peptide (dissolved in distilled water) in molar ratio from 1: 100. After incubation for 60 min at room temperature the conjugates each with 100 ul 100 mM cysteine stopped for 10 min and again each on a NAP25 column (Pharmacia, Upsalla, Sweden) (eluent: PBS, pH 7.4); the protein content was determined photometrically.

The BSA + FLAG tag and BSA + ProtC tag conjugate was aliquoted at -20 ° C stored.

1.2. Conjugation of BSA with oligo-Lys peptide

to Coupling of Oligo-Lys Peptide "K14C" (Amino Acid Sequence KKKKKKKKKKKKKC, JPT GmbH, Berlin, Germany, M = 1914,27) was protease-free bovine serum albumin (Sigma, Deisenhofen, Germany) with SMCC in the molar ratio of 1:25 activated.

1000 μl 0.5% BSA (dissolved in PBS, 10 mM EDTA, pH 8) was mixed with 18.9 μl 100 mM SMCC (freshly dissolved in DMSO) and 30 min incubated at room temperature. The product was about a NAP10 column (Pharmacia, Upsalla, Sweden) (eluent: PBS, 10 mM EDTA, pH 8) and the protein content was determined photometrically.

Subsequently Conjugation with the oligo-Lys peptide (dissolved in dest. Water) in a molar ratio of 1: 100. To Incubation for 60 min at room temperature was followed by reaction 100 μl 100 mM cysteine stopped for 10 min, again over a NAP25 column (Pharmacia, Upsalla, Sweden) (Eluent: PBS, pH 7.4) and the protein content determined photometrically.

The BSA + oligo-Lys conjugate was aliquoted at -20 ° C stored.

2. Coupling of the conjugates, or avidin

irradiated Star tube (Greiner, Frickenhausen, Germany) were used with the conjugates from 1.1. and 1.2., or with avidin (Pierce, Rockford, IL, USA, Art. No. 21121, dissolved in dest. Water) coated as follows:

The Components were added to a concentration in 10mM Tris, 100mM NaCl, pH 7.8 diluted to 6.67 μg / ml. Per tube 300 μl of the solutions were pipetted and 20 h incubated at 22 ° C. The solutions were filtered off with suction. Then each tube was filled with 300 μl of 0.5% Bovines Serum albumin, 2% Karion FP 2 h saturated and again aspirated. Subsequently, the tubes were in dried in a vacuum dryer and stored at 4 ° C.

3. Ni-NTA solid phase

It were Ni-NTA microtiter plates (Qiagen, Hilden, Germany Art. No. 1006387).

4. Assay for the detection of reversibly bound antibodies

4.1 Binding of MACN antibodies conjugates

The MACN antibody conjugates were at concentration of 1.5 μg / ml with dilution buffer (see Table 2). Each 300 μl were in several parallel Approaches to the corresponding solid phase pipetted and over Incubated at room temperature overnight. The tubes were with 5 x 1 ml, the Ni-NTA microtiter plate with 5 x 300 μl Washing buffer (see Table 2).

In each of the approaches was the amount of bound MACN antibody Conjugate determines:

The amount of MACN-antibody conjugate bound to the tubes was used in a luminometer (BERTHOLD, BAD WILDBAD, GERMANY, LB952T; Basic reagents BRAHMS AG). The on the microtiter plate bound amount of MACN antibody conjugate was in one Microplate Luminometer (BERTHOLD, BAD WILDBAD, GERMANY, MPL2; Basic reagents BRAHMS AG).

4.2 Measurement of nonspecific detachment the MACN antibody conjugates with whole blood

In further approaches of bound MACN antibodies Conjugates were each pipetted 300 μl of whole blood (see Table 2) and incubated for 10 min. The tubes were 5x1 ml, the Ni-NTA microtiter plate with 5 × 300 μl washing buffer (see Table 2). The remaining on the tube Amount of MACN antibody conjugate was in a luminometer (BERTHOLD Company, BAD WILDBAD, GERMANY, LB952T; Basic Reagents BRAHMS AG).

The amount of MACN-antibody conjugate remaining on the microtiter plate was used in a Microplate Luminometer (BERTHOLD, BAD WILDBAD, GERMANY, MPL2; Basic reagents BRAHMS AG).

4.3 Measurement of the specific detachment the MACN antibody conjugates with separation buffer

In further approaches of bound MACN antibodies Conjugates were each 300 ul Ablösungspuffer (see Table 2) pipetted and incubated for 10 or 30 min. The tubes with 5 x 1 ml, the Ni-NTA microtiter plate with 5 x 300 μl Washing buffer washed (see Table 2). The on the tube remaining amount of MACN-antibody conjugate was in a luminometer (BERTHOLD, BAD WILDBAD, GERMANY, LB952T; Basic reagents BRAHMS AG).

The amount of MACN-antibody conjugate remaining on the microtiter plate was measured in a Microplate luminometer (BERTHOLD, BAD WILDBAD, GERMANY, MPL2, basic reagents BRAHMS AG). solid phase MACN antibody + Dilution buffer wash buffer thoroughbred detachment buffer avidin biotin PBS pH 7.4 0.5% BSA 8 mM Tris pH 7.5 60 mM NaCl, 0.2% Tween 20 EDTA whole blood PBS pH 7.4, 0.5% BSA, 25 mM Biotin Ni-NTA His-tag peptide PBS pH 7.4, 0.5% BSA, 25 mM imidazole Oligo-Lys BSA Oligo-Asp peptide PBS pH 7.4, 0.5% BSA, 2.6 M KF, 0.01% heparin FLAG tag BSA anti FLAG tag M1 50 mM HEPES pH 7.2 150 mM NaCl, 0.02% Tween, 0.5% BSA, 5 mM CaCl ₂ 50 mM HEPES pH 7.2 150 mM NaCl, 0.02% Tween, 5 mM CaCl ₂ Heparin whole blood 50mM HEPES pH 7.2 150mM NaCl, 0.02% Tween, 0.5% BSA, 5mM EDTA ProtC-tag BSA anti ProtC-tag HPC4 Table 2

Further investigation of the Oligo-Asp / Oligo-Lys system

Ablösungsagentien

As described above, was an efficient resolution of binding by adding 0.01% heparin (see Table 1 and related Description). As alternative separation agents were also examined NaCl and KF. A comparable with 0.01% heparin Effect on the dissolution of the bond was with NaCl or KF concentrations> 0.8 Reached. Numerous other variations in the buffer composition are conceivable, which lead to dissolution of the bond (other salts low molecular weight, other charged oligo- or polymers, other pH). All of these variations are within the meaning of this invention to be understood as detachment agents. As particularly preferred However, such variations should be regarded as efficient on the one hand destabilize the binding of the oligo-Asp / oligo-lys system, on the other hand but the binding between antibody and analyte as possible leave undisturbed. Heparin in a concentration 0.01% represents such a preferred variant, as is known Many immunoassays can use heparin plasma as a sample matrix and the concentration of heparin in this plasma typically in the range of 0.01%.

Specificity d. Binding of the analyte to the solid phase

Gives a biological sample to a solid phase, which is coated with oligo-lys-BSA / oligo-asp analyte binder, one could fear that the analyte of interest from the sample not only bind to the analyte-specific antibody, but may also be unspecific about ionic interaction with possibly free lysine portions of the solid phase. If that were the case, the correctness would be in the subsequent analyte detection (after the detachment step) impaired. Whether and to what extent such nonspecific analyte binding has been studied by using various synthetic Peptides which are largely identical to known natural ones Analytes are and therefore represent chemiluminescence-labeled were diluted into a plasma matrix and placed in tubes incubated with oligo-Lys-BSA and attached thereto Oligo-Asp-anti PCT antibody was bound. The detected Proportion of nonspecifically bound peptides was always below 7% of the offered peptide. Thus, non-specific analyte binding is involved the solid phase is not a problem.

in the Specifically, the experiment was carried out as follows:

1. Preparation of MACN chemiluminescence-labeled peptides

The following peptides (all from JPT GmbH, Berlin, Germany) were labeled with MACN:
"PPL41" (amino acid sequence PEVPPWTGEVSPAQRDGGALGGGGRGPWDSSDRSALLKSKL derived of NT-proANP), "PSW44" (amino acid sequence SSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAHW derived from proendothelin), "PAY33" (amino acid sequence ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY derived from Copeptin), "peptide 45-92" (amino acid sequence ELRMSSSYPTGLADVKAGPAQTLIRPQDMKGASRSPEDSSPDAARIRV derived from proAdre nomedullin)

to Chemilumineszenzmarkierung were the peptides in the molar ratio of 1: 1 with MACN-acridinium-NHS ester (InVent GmbH, Hennigsdorf, Germany) for 1 hour at room temperature. Subsequently the reaction was stopped with 1 M Tris pH 7.8 for 10 min.

to Separation of unincorporated MACN became the tag approaches a reversed phase C18 column (μBondapak, Waters WAT027342, No. 0202352581). The samples were applied and at a flow rate of 1.0 ml / min by means of a rising Water / acetonitrile (+ 0.1% TFA) gradient chromatographed. With a wavelength photometer became the wavelengths 214 nm, 280 nm and 368 nm measured and detected the peaks.

The labeled peptides were portioned at -20 ° C stored.

1.1. Preparation BSA + oligo-Lys conjugate: see above

1.2. Producing anti calcitonin sheep Antibody + oligo-Asp conjugate

to Coupling of Oligo-Asp Peptide "D14C" (Amino Acid Sequence DDDDDDDDDDDDDC, (JPT GmbH, Berlin, Germany, M = 1731.43) became a polyclonal anti calcitonin sheep antibody ("Anti-PCT antibody 1") with SMCC in ratio 1:25 activated (see "Instructions SMCC and Sulfo-SMCC", Pierce).

3 mg of antibody (in 4.5 ml of PBS, 10 mM EDTA, pH 8) with 5.0 μl of 100 mM SMCC (freshly dissolved in DMSO) and incubated for 30 min at room temperature. The product was over purified two NAP25 columns (eluent: PBS, 10 mM EDTA, pH 8) and the protein content is determined photometrically.

Subsequently the conjugation with the oligo-Asp peptide (dissolved in dest. Water) in a molar ratio of 1: 100. To Incubation for 60 min at room temperature was followed by reaction 100 μl 100 mM cysteine stopped for 10 min, unconjugated Peptide separated via NAP25 columns (eluent: PBS, pH 7.4) and the protein content is determined photometrically.

The anti calcitonin sheep antibody + oligo-asp conjugate was aliquoted stored at -20 ° C.

2. Coupling of the conjugates to tubes

2.1. Coupling: BSA + Oligo-Lys conjugate

Irradiated star tubes (Greiner, Frickenhausen, Germany) were coated with BSA-oligo-Lys conjugate as follows:
The conjugate was diluted in 10 mM Tris, 100 mM NaCl, pH 7.8 to a concentration of 6.67 μg / ml. 300 μl of this solution were pipetted into each tube and incubated at 22 ° C. for 20 h. The solution was sucked off. Then, each tube was saturated with 300 ul of 0.5% Bovine Serum Albumin, 2% Karion FP for 2 h and again aspirated. Subsequently, the binding of the anti-calcitonin sheep antibody + oligo-Asp conjugate was carried out:

2.2. Binding: anti calcitonin sheep antibody + oligo-Asp conjugate

The Conjugate was in PBS, 0.5% protease-free BSA pH 7.4 to a concentration of 667 ng / ml. In each tube were Pipet 300 μl of this solution and add in for 1 hour Incubated at 22 ° C. The solution was sucked off. Then each tube was filled with 300 μl 0.5% Bovine Serum Albumin, 2% Karion FP 2 h again saturated and then aspirated.

Subsequently the tubes were dried in a vacuum dryer and stored at 4 ° C.

3. Determination of non-specific binding:

The MACN peptides were diluted in a plasma matrix. ever 300 .mu.l were in the with the oligo-Lys-BSA and the oligo-Asp-anti Pipetted PCT antibody coated tubes and incubated for 30 min at room temperature. The tubes were washed with 3 x 1 ml PBS pH 7.4, 0.5% protease-free BSA. The amount bound to the tubes as well as the total activity used of MACN peptide was measured in a luminometer (BERTHOLD, BAD WILDBAD, GERMANY, LB952T; Basic reagents BRAHMS AG).

The percentage unspecific binding relative to the total activity was: PSW44 3.0% PPL41 3.1% PAY33 6.5% Peptide 45-92 2.9%

Comparison using the example of PCT: Classic Immunochromatography from whole blood vs. Immune Extraction of PCT from Whole Blood, Replacement and analyte detection by means of immunochromatography or the TRACE technology

PCT (Procalcitonin) was selected as a relevant model analyte. It is a marker for bacterial infections, especially serious infections leading to a systemic inflammatory response of the host (sepsis) (21). Sandwich immunoassays for Detection of PCT Use Antibodies Against Calcitonin or katacalcin portion of the peptide (22), (23).

Immunochromatography represents the most common rapid test methodology for the immunological detection of analytes from whole blood in today's routine application and can therefore be referred to as "classical immunochromatography." This method was therefore chosen here as a reference method in order to obtain advantages of the principle according to the invention of immuno-extraction and detachment ( "Immune transfer") with connected analyte detection. Two different detection methods were investigated: immunochromatography on the one hand, and time resolved amplified cryptate emission (TRACE) on the other hand. All three methods were used to determine PCT concentrations from PCT-containing whole blood samples in 10-fold determination. In 2 the resulting precision profiles are contrasted with the methods. It can be clearly seen that both methods, which are the subject of this invention, ie immune transfer in connection with a detection method (be it immunochromatography or TRACE technology), significantly more precise and sensitive results than the reference method (classical immunochromatography directly from whole blood). The details for carrying out the experiments are explained below. Under A is described as a method according to the invention immune transfer with connected analyte detection using TRACE technology; under B is described as a method according to the invention immune transfer with connected analyte detection by means of immunochromatography; under C is described as a reference method classical immunochromatography. A comparison of the precision profiles of variants A, B and C is in 2 shown.

A. Immune Extraction of PCT from Whole Blood and analyte detection using the TRACE technology

When Solid phase were microtiter plates of high-binding polystyrene used, which were coated with oligo-Lys-BSA, what a first anti-PCT antibody bound previously with oligo-Asp and Cryptate had been derivatized. On it, PCT-containing whole blood sample was briefly incubated and PCT from the sample to the solid phase immune extracted. After washing the solid phase, release buffer was added and incubated briefly. One aliquot was transformed into another, low binding transferred black microtiter plate and with a Mixed solution, a second, labeled with cyanine anti-PCT antibody. After a short incubation was the sandwich formation after excitation with laser light by means of TRACE Technology detected.

in the Specifically, the experiment was carried out as follows:

A.1. Preparation of the conjugates for the solid phase

A.1.1. Preparation BSA + oligo-Lys conjugate: s. above

A.1.2. Producing anti calcitonin sheep Antibody Crypt MonoMP + Oligo-Asp Conjugate

For the label with cryptate monophosphate ("KMonoMP", company Cezanne, Nimes, France) became a polyclonal anti-calcitonin sheep antibody ("Anti-PCT antibody 1") with SPDP (N-succinimidyl 3- (2-pyridyldithio) propionate, Pierce, Rockford, IL, USA, Art. No. 21857) in molar ratio of 1: 4 (see "Instructions SPDP Reagents", Pierce, Rockford, IL, USA).

5 mg of antibody (dissolved in 1.5 ml of 100 mM sodium phosphate, 5 mM EDTA, pH 6.9) were mixed with 41.6 μl of 3.2 mM SPDP (fresh dissolved in ethanol) and 30 min at room temperature incubated.

Of the activated antibody was treated with 81.2 μl of 200 mM DTT (dithiothreitol dissolved in 100 mM sodium phosphate, 5 mM EDTA, pH 6.9) for 15 min at room temperature. The product was purified over two NAP10 columns (eluent: 100 mM sodium phosphate, 5 mM EDTA, pH 6.9) and the protein content determined photometrically.

Subsequently Immediate conjugation of the eluate with lyophilized KMonoMP in a molar ratio of 1: 8. After incubation for 20 h at 4 ° C, the product was re-crossed over three NAP10 columns purified (eluent: PBS, 10 mM EDTA pH 8) and the protein content determined photometrically.

to Coupling of Oligo-Asp Peptide "D14C" (Amino Acid Sequence DDDDDDDDDDDDDC, JPT GmbH, Berlin, Germany, M = 1731.43) the product was activated with 1:25 SMCC (See "Instructions SMCC and Sulfo-SMCC", Pierce, Rockford, IL, USA) .: 3 mg of product (eluted with 4.5 ml PBS, 10 mM EDTA, pH 8) were dissolved with 5.0 μl of 100 mM SMCC (fresh in DMSO) and incubated for 30 min at room temperature. The product was purified on two NAP25 columns (eluent: PBS, 10 mM EDTA, pH 8) and the protein content was determined photometrically.

Subsequently the conjugation with the oligo-Asp peptide (dissolved in dest. Water) in a molar ratio of 1: 100. To Incubation for 60 min at room temperature was followed by reaction 100 μl 100 mM cysteine stopped for 10 min, unconjugated Peptide separated via NAP25 columns (eluent: PBS, pH 7.4) and the protein content is determined photometrically.

The anti calcitonin sheep antibody-K MonoMP + oligo-Asp conjugate was stored aliquoted at -20 ° C.

A.2. Coupling of the conjugates to microtiter plates

A.2.1. Coupling: BSA + Oligo-Lys conjugate

Irradiated microtiter plates (Greiner, Frickenhausen, Germany) were coated with BSA-oligo-Lys conjugate as follows:
The conjugate was diluted in 10 mM Tris, 100 mM NaCl, pH 7.8 to a concentration of 6.67 μg / ml. 300 μl of this solution were pipetted into each well and incubated at 22 ° C. for 20 h. The solution was sucked off. Each well was then saturated with 300 μl 0.5% Bovine Serum Albumin, 2% Karion FP for 2 h and aspirated again. Subsequently, the binding of the anti-calcitonin sheep antibody-cryptate MonoMP + oligo-Asp conjugate was carried out:

A.2.2. Binding: anti calcitonin sheep Antibody Crypt MonoMP + Oligo-Asp Conjugate

The Conjugate was in PBS, 0.5% protease-free BSA pH 7.4 to a concentration of 667 ng / ml. In each cavity were Pipet 300 μl of this solution and add in for 1 hour Incubated at 22 ° C. The solution was sucked off. Then Each well was filled with 300 μl of 0.5% Bovine Serum Albumin, 2% Karion FP 2 h saturated and again aspirated.

Subsequently The microtiter plates were dried in a vacuum dryer and stored at 4 ° C.

A.3. Preparation of an anti-catacalcin Mouse antibody-Cy5 conjugate for the liquid phase

One monoclonal anti-catacalcin antibody ("anti-PCT Anit body 2 ") was in the presence of 45% (2-hydroxyl-propyl) -β-cyclodextrin (Aldrich, Seelze, Germany, Art. No. 33,260-7) with Cy5-NHS (Amersham, Art. No. PA15100) in molar ratio marked by 1:20 (see Product Booklet "Amersham CyDye mono-reactive NHS ester "). 213 μl antibody (c = 10 mg / ml in PBS pH 7.4) were mixed with 750 μl of 60% cyclodextrin (dissolved in 100 mM carbonate buffer pH 9) and mixed with 37.3 μl 7.7 mM Cy5-NHS (dissolved in DMSO). After incubation of one hour at room temperature, the batch became stopped with 50 μl of 50 mM glycine for 10 min a NAP10 column (eluent: PBS, pH 7.4) prepurified.

to Separation of last residues not bound to antibodies Cy5-NHS was subjected to gel filtration HPLC (column: Bio-Sil Sec 125, Biorad, Munich, Germany). The Sample was applied and at a flow rate of 1 ml / min chromatographed with PBS, pH 7.4. With a flow photometer the wavelengths 280 nm and 648 nm were measured. The Absorbance ratio 648 nm / 280 nm as a measure of the degree of labeling of the antibody was 1.48 at the peak. The antibody-containing fractions (retention time 8-10 min) were pooled.

The anti Katacalcin mouse antibody Cy5 conjugate was portioned and stored at -20 ° C.

A.4. Immune transfer and TRACE assay

Of the The assay described below was for each sample in 10-fold determination performed. In the cavities that with BSA + oligo-lys and anti calcitonin sheep antibody-cryptate MonoMP + D14C conjugate coated microtiter plates were 300 μl EDTA whole blood samples supplemented with recombinant PCT (InVivo GmbH, Hennigsdorf, Germany), pipetted and 15 min at room temperature incubated (the PCT concentrations of the samples were determined by measurement with the assay BRAHMS PCT LIA sensitiv (BRAHMS AG, Hennigsdorf, Germany) determined from plasma). Thereafter, 2 × with 300 ul PBS, pH 7.4, then 2 × with 300 μl washing solution (8mM Tris, 60mM NaCl, 0.2% Tween 20, pH 7.5). Subsequently was mixed with 300 μl of dissolution buffer (PBS pH 7.4, 0.6 M KF, 0.5% BSA, 0.01% heparin) for 10 min. From the detached immune complex 100 μl were suitable for the TRACE measurement Microtiter plates (Costar Half Area Plates, 96 well, black, polystyrene, Sigma / Aldrich, Seelze, Germany, Cat. No. CLS 3694-100EA) and with 50 μl of anti-catacalcin mouse antibody Cy5-NHS Conjugate (c = 20 μg / ml in PBS pH 7.4, 0.6 M KF, 0.5% BSA, 0.01% heparin) for 19 min at room temperature. The measurement The TRACE signals (620 nm and 665 nm) were performed using the high-performance Time-Resolved Fluorescence Microtiter Plate Reader RUBYstar (Company BMG LABTECH GmbH, Offenburg, Germany).

The following device settings were made on the RUBYstar: Number of flashes 20 Integration delay [μs] 50 Integration time [μs] 400 Interval time [μs] 10 Number of intervals 45 Ratio multiplicator 10000

For each sample, the ratio OD _665nm / OD _620nm , Delta F was calculated as follows: Delta F = (Ratio pos - Ratio neg ) / Ratio neg

Ratio _{neg means} the ratio of a PCT-negative sample (ie PCT not containing or not detectable sample) and Ratio _pos the ratio of a sample to be tested (ie, a potentially PCT-containing sample). From the 10-fold determinations of the individual samples, the respective mean value was formed. These values served as standard concentrations at which the concentrations of all individual samples were determined using the MultiCalc (Spline Fit) software. For each 10-fold determination, the coefficient of variation was determined.

B. Immune Extraction of PCT from Whole Blood and analyte detection by immunochromatography

The solid phase used was polystyrene tubes coated with oligo-Lys-BSA bound by a first anti-PCT antibody previously derivatized with oligo-Asp and biotin. Dar PCT-containing whole blood sample was briefly incubated, thereby immunopotenting PCT from the sample to the solid phase. After washing the solid phase, release buffer containing a second colloidal gold-labeled anti-PCT antibody was added and incubated briefly. The solution was finally immunochromatographed on a test strip sprayed with streptavidin as a test band. The biotinylated first anti-PCT antibody and thus also bound PCT and the second gold-labeled anti-PCT antibody bound to it bind to the streptavidin. The detection of the label was carried out by reflectometry.

in the Specifically, the experiment was carried out as follows:

B.1. Preparation of the conjugates for the solid phase

B.1.1. Preparation BSA + oligo-Lys conjugate: see above

B.1.2. Producing anti-calcitonin sheep Antibody biotinylates + Oligo-Asp conjugate

One polyclonal anti-calcitonin sheep antibody ("anti-PCT Antibody 1 ") was incubated with EZ-Link sulfo-NHS-LC-LC-biotin (Pierce, Rockford, IL, USA, Art. No. 21338) in molar ratio of 1:10 (see "Instructions EZ-Link Sulfo-NHS-LC-LC-Biotin", Pierce, Rockford, IL, USA): 1.6 mg of antibody (in PBS pH 7.4) were mixed with 10 μl of 10 mM EZ-Link sulfo-NHS-LC-LC-biotin (freshly dissolved in distilled water) and 30 min at Room temperature incubated.

The Product was purified on a NAP5 column (eluent: PBS, 10 mM EDTA pH 8.0) and the protein content was determined photometrically.

to Coupling of Oligo-Asp Peptide "D14C" (Amino Acid Sequence DDDDDDDDDDDDDC, (JPT GmbH, Berlin, Germany, M = 1731.43) the product was activated with 1:50 SMCC (See "Instructions SMCC and Sulfo-SMCC", Pierce, Rockford, IL, USA).

1.3 mg of product (eluted with 1.0 ml of PBS, 10 mM EDTA, pH 8) were washed with Add 4.33 μl of 100 mM SMCC (freshly dissolved in DMSO) and incubated for 30 min at room temperature. The product was over purified a NAP10 column (eluent: PBS, 10 mM EDTA, pH 8) and the protein content is determined photometrically.

Subsequently the conjugation with the oligo-Asp peptide (dissolved in dest. Water) in a molar ratio of 1: 100. To Incubation for 60 min at room temperature was followed by reaction 100 μl 100 mM cysteine stopped for 10 min, unconjugated Separated peptide via a NAP25 column (eluent: PBS, pH 7.4) and the protein content is determined photometrically.

The anti-calcitonin sheep antibody biotinylated + oligo-Asp Conjugate was aliquoted at -20 ° C stored.

B.2. Coupling of the conjugate to tubes

B.2.1. Coupling: BSA + Oligo-Lys conjugate

irradiated Star tube (Greiner, Frickenhausen, Germany) were coated with BSA-oligo-Lys conjugate as follows:

The Conjugate was added to a concentration in 10mM Tris, 100mM NaCl, pH 7.8 diluted to 6.67 μg / ml. In every tube 300 μl of this solution were pipetted and 20 h incubated at 22 ° C. The solution was sucked off. Then each tube was filled with 300 μl of 0.5% Bovines Serum albumin, 2% Karion FP 2 h saturated and again aspirated. Subsequently, the binding of the anti-calcitonin Sheep Antibodies Biotinylated + Oligo-Asp Conjugates:

B.2.2. Binding: anti-calcitonin sheep Antibody biotinylates + Oligo-Asp conjugate

The Conjugate was in PBS, 0.5% protease-free BSA pH 7.4 to a concentration of 667 ng / ml. In each tube were Pipet 300 μl of this solution and add in for 1 hour Incubated at 22 ° C. The solution was sucked off. Then each tube was filled with 300 μl 0.5% Bovine Serum Albumin, 2% Karion FP 2 h again saturated and then aspirated.

Subsequently the tubes were dried in a vacuum dryer and stored at 4 ° C.

B.3. Preparation of gold-labeled anti-catacalcin Antibody:

This component was purchased from 8sens.biognostic GmbH, Berlin, Germany. Monoclonal anti-catacalcinin antibody was coupled to colloidal gold (8sens.biognostic GmbH, Berlin, Germany, according to Frens: Frens, G., Nature Physical Science, 1973, 241 20-22) as described in Hermanson ( http://www.amazon.de/Bioconjugate-Techniques-Greg-T-Hermanson/dp/0123423368 ).

B.4. Preparation of the test strips:

These Component was manufactured by company 8sens.biognostic GmbH, Berlin, Germany, acquired. Nitrocellulose membranes from MDI (Advanced Microdevices, Ambala, India) with 4 μg / cm streptavidin (Pierce, Rockford, IL, USA) (test line) and with 2.5 μg / cm anti-mouse IgG Antibodies (Scantibodies, Santee, CA, USA) (Control Line) coated. For applying the capture molecules a microdispenser from Biodot, Irvine, CA, USA was used. Subsequently, the drying of the membrane over Night at 50 ° C. As Saugilad came AP22 of the company Millipore, Billerica, MA, USA.

B.5. Immune transfer and immunochromatography:

Of the The assay described below was for each sample in 10-fold determination performed. In the with BSA + Oligo-Lys and anti-calcitonin sheep antibody biotinylated + D14C Conjugate coated tubes were 300 μl EDTA whole blood samples supplemented with recombinant PCT (InVivo GmbH, Hennigsdorf, Germany) and 15 min at room temperature incubated (the PCT concentrations of the samples were determined by measurement with the assay BRAHMS PCT LIA sensitiv (BRAHMS AG, Hennigsdorf, Germany) determined from plasma). Thereafter, 2 × with 300 ul PBS, pH 7.4, then 2 × with 300 μl wash solution (8 mM Tris, 60 mM NaCl, 0.2% Tween 20, pH 7.5). Subsequently was washed with 150 μl of dissolution buffer (PBS pH 7.4, 5% BSA, 0.5% Tween 20, 0.01% heparin), which also labeled the gold anti-catacalcin monoclonal antibody, supra Shake incubated for 10 min. From the detached one Immune complex was 150 μl in wells of low binding microtiter plates. Subsequently immediate immunochromatography with the test strips, by putting these into the cavities. After 30 min was the staining of the test band with a Reflektrometer the company LRE Medical GmbH, Munich, Germany.

Out the 10-fold determinations of the individual samples became the respective Mean value formed. These values served as standard concentrations, where the concentrations of all individual samples using software MultiCalc (Spline Fit). To every 10-fold Determination, the coefficient of variation was determined.

C. Detection of PCT from whole blood by means of classical immunochromatography

It Test strips were prepared which were attached to the sample application zone a membrane that was capable of blood cells from a withhold the whole blood sample to be applied. Between this membrane and the test strip had a porous pad attached ("Sample pad"), the dried one contained one first, biotinylated anti-PCT antibody, on the other hand a second colloidal gold labeled anti-PCT antibody. As a test band streptavidin was sprayed on the strip. The test strips were inserted in plastic cassettes. PCT-containing whole blood sample was immunochromatographed. To the streptavidin binds the biotinylated first anti-PCT antibody and hence PCT bound to it and the related PCT second, gold-labeled anti-PCT antibody. The detection of the label was reflektrometrisch.

in the Specifically, the experiment was carried out as follows:

C.1. Preparation of the conjugates for the solid phase

C.1.1. Preparation BSA + oligo-Lys conjugate: see above

C.1.2. Producing anti-calcitonin sheep Antibody biotinylated + Oligo-Asp conjugate: see above

C.2. Preparation of gold-labeled anti-catacalcin Antibody: see above

C.3. Preparation of the test strips:

These Component was manufactured by company 8sens.biognostic GmbH, Berlin, Germany, acquired. Nitrocellulose membranes from MDI (Advanced Microdevices, Ambala, India) with 4 μg / cm streptavidin (Pierce, Rockford, IL, USA) (test line) and with 2.5 μg / cm anti-mouse IgG Antibodies (Scantibodies, Santee, CA, USA) (Control Line) coated. For applying the capture molecules a microdispenser from Biodot, Irvine, CA, USA was used. Subsequently, the drying of the membrane over Night at 50 ° C. As Saugpad came AP22 of the company Millipore, Billerica, MA, USA. For the production of Thoroughbred tests became a thoroughbred setup of the company 8sens.biognostic used. In the sample application area were on 2 separate pads on the one hand, the anti-Katacalcin antibody coupled to colloidal gold and second, the biotinylated + oligo-Asp-derivatized anti-calcitonin Immobilized antibody. The cassette became a standard Housing used by the company 8sens.biognostic.

C.4. Classical immunochromatography

The Immunochromatography described below was used for each sample is performed in 10-fold determination. On the Test strips were supplemented with 120 μl of EDTA whole blood samples with recombinant PCT (InVivo GmbH, Hennigsdorf, Germany) pipetted (The PCT concentrations of the samples were determined by measurement with the Assay BRAHMS PCT LIA sensitive (BRAHMS AG, Hennigsdorf, Germany) determined from plasma). After 30 minutes, the staining of the Test band with a reflectometer from LRE Medical GmbH, Munich, Germany, certainly.

Out the 10-fold determinations of the individual samples became the respective Mean value formed. These values served as standard concentrations, where the concentrations of all individual samples using software MultiCalc (Spline Fit). To every 10-fold Determination, the coefficient of variation was determined.

Description d. pictures

1 shows that the analyte (PCT) can be immune-extracted from whole blood after only 5 min incubation with similar efficiency to the reference method after 2 h incubation.

2 : Shown are the coefficients of variation for the multiple determination of whole blood samples with different PCT concentrations as determined by the three following methods (A: immune transfer with TRACE technology, B: immune transfer with immunochromatography, C: reference classical immunochromatography). Both methods containing immune transfer are methods of the invention; classical immunochromatography is the reference method.

Bibliography

• (1) MJ Pugia, CP Price Point-of-Care Testing, 2nd ed., Edited by Price, John, Hicks, American Association for Clinical Chemistry ISBN 1-59425-012-X, 2004; Pages 13-30
• (2) Saadeddin S, Habbab M, Siddieg H, Fayomi M, Dafterdar R. Reliability of the rapid bedside whole-blood quantitative cardiac troponin. T Assay in the diagnosis of myocardial injury in patients with acute coronary syndrome Med Sci Monit. 2004 Mar; 10 (3): MT43-6. Epub 2004 Mar 1
• (3) o. V. Evaluation of a bedside whole blood rapid troponin T assay in the emergency department. Rapid Evaluation by Assay of Cardiac Troponin T (REACTT) Investigator's Study Group Acad Emerg Med. 1997 Nov; 4 (11): 1018-24
• (4) Zugck C, Nelles M, Katus HA, Collinson PO, Gaze DC, Dikkeschei B, Gurr E, Hayen W, Haass M, Hechler C, van Hoof V, Guerti K, Van Waes C, Printzen G, Klopprogge K, Schulz I, Zerback R. Multicentre evaluation of a new point-of-care test for the determination of NT-proBNP in Whole Blood Clin Chem Lab Med. 2006; 44 (10): 1269-77
• (5) Alan HB A Platform for Quantitative Point-of-Care Cardiac Marker Determinations from Roche Diagnostics
• (6) Yeo KT, Wu AH, Apple FS, Kroll MH, Christenson RH, Lewandrowski KB, Sedor FA, Butch AW Multicenter evaluation of the Roche NT-proBNP assay and comparison to the Biosite Triage BNP assay Clin Chim Acta. 2003 Dec; 338 (1-2): 107-15
• (7) Meisner M, Brunkhorst FM, Reith HB, Schmidt J, Lestin HG, Reinhart K. Clinical experiences with a novel semi-quantitative solid phase immunoassay for rapid measurement of procalcitonin Clin Chem Lab Med. 2000 Oct; 38 (10): 989-95
• (8th) Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003 Jan; 60 (5): 523-33. Epub 2002 Nov 7
• (9) RC Stevens Fusion tags used in recombinant protein expression and purification Design of high-throughput methods of protein production for structural biology Structure, 8, R177-R185 (2000) ,
• (10) Stevens RC. Design of high-throughput methods of protein production for structural biology Structure. 2000 Sep 15; 8 (9): R177-85
• (11) Hopp TP, Gallis B, Prickett KS Metal-binding properties of a calcium-dependent monoclonal antibody. Mole of immunol. 1996 May-Jun; 33 (7-8): 601-8
• (12) Stearns DJ, Kurosawa S, Sims PJ, Esmon NL, Esmon CT The interaction of a Ca2 + -dependent monoclonal antibody with the protein. Evidence for obligatory Ca2 + binding to both antigen and antibody J Biol Chem. 1988 Jan 15; 263 (2): 826-32
• (13) QIAexpress Detection and Assay Handbook 10/2002, QIAGEN, HILDEN, GERMANY
• (14) Sassenfeld, HM and Brewer, SJ (1984) Bio / Technology, 2, 76-80 ,
• (15) Dalboge, H., Dahl, H.-HM, Pedersen, J., Hansen, JW and Christensen, T. (1987) Bio / Technology, 5, 161-164
• (16) Zhao, JY, Ford, CF, Glatz, CE, Rougvie, MA and Gendel, SM (1990) J. Biotechnol., 14, 273-283
• (17) Avidin-Biotin Chemistry: A Handbook
• (18) Invitrogen (library)
• (19) Pierce Immobilized Monomeric Avidin Kit www.piercenet.com
• (20) Pierce Two thiol cleavable, heterobifunctional, amine and sulfhydryl-reactive crosslinking agents www.piercenet.com
• (21) Meisner M. Pathobiochemistry and clinical use of procalcitonin. Clin Chim Acta. 2002 Sep; 323 (1-2): 17-29
• (22) Meisner M, Brunkhorst FM, Reith HB, Schmidt J, Lestin HG, Reinhart K. Clinical experiences with a novel semi-quantitative solid phase immunoassay for rapid measurement of procalcitonin Clin Chem Lab Med. 2000 Oct; 38 (10): 989-95
• (23) Morgenthaler NG, Struck J, Fischer-Schulz C, Bergmann A. Sensitive immunoluminometric assay for the detection of procalcitonin. Clin Chem. 2002 May; 48 (5): 788-90

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 4851341 [0018]

Cited non-patent literature

• - http://www.amazon.de/Bioconjugate-Techniques-Greg-T-Hermanson/dp/0123423368 [0140]
• - MJ Pugia, CP Price Point-of-Care Testing, 2nd Ed., Edited by Price, John, Hicks, American Association for Clinical Chemistry ISBN 1-59425-012-X, 2004; Pages 13-30 [0150]
• - Saadeddin S, Habbab M, Siddieg H, Fayomi M, Dafterdar R. Reliability of the rapid bedside whole-blood quantitative cardiac troponin. T Assay in the diagnosis of myocardial injury in patients with acute coronary syndrome Med Sci Monit. 2004 Mar; 10 (3): MT43-6. Epub 2004 Mar 1 [0150]
• - o. V. Evaluation of a bedside whole blood rapid troponin T assay in the emergency department. Rapid Evaluation by Assay of Cardiac Troponin T (REACTT) Investigator's Study Group Acad Emerg Med. 1997 Nov; 4 (11): 1018-24 [0150]
• - Zugck C, Nelles M, Katus HA, Collinson PO, Gaze DC, Dikkeschei B, Gurr E, Hayen W, Haass M, Hechler C, van Hoof V, Guerti K, Van Waes C, Printzen G, Klopprogge K, Schulz I , Zerback R. Multicentre evaluation of a new point-of-care test for the determination of NT-proBNP in whole blood Clin Chem Lab Med. 2006; 44 (10): 1269-77 [0150]
• - Alan HB A Platform for Quantitative Point-of-Care Cardiac Marker Determinations from Roche Diagnostics [0150]
• - Yeo KT, Wu AH, Apple FS, Kroll MH, Christenson RH, Lewandrowski KB, Sedor FA, Butch AW Multicenter evaluation of the Roche NT-proBNP assay and comparison to the Biosite Triage BNP assay Clin Chim Acta. 2003 Dec; 338 (1-2): 107-15 [0150]
• - Meisner M, Brunkhorst FM, Reith HB, Schmidt J, Lestin HG, Reinhart K. Clinical experiences with a novel semi-quantitative solid phase immunoassay for rapid measurement of procalcitonin Clin Chem Lab Med. 2000 Oct; 38 (10): 989-95 [0150]
• - Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003 Jan; 60 (5): 523-33. Epub 2002 Nov 7 [0150]
• - RC Stevens Fusion tags used in recombinant protein expression and purification Design of high-throughput methods of protein production for structural biology Structure, 8, R177-R185 (2000) [0150]
• - Stevens RC. Design of high-throughput methods of protein production for structural biology Structure. 2000 Sep 15; 8 (9): R177-85 [0150]
• - Hopp TP, Gallis B, Prickett KS Metal-binding properties of a calcium-dependent monoclonal antibody. Mole of immunol. 1996 May-Jun; 33 (7-8): 601-8 [0150]
• - Stearns DJ, Kurosawa S, Sims PJ, Esmon NL, Esmon CT The interaction of a Ca2 + -dependent monoclonal antibody with the protein. Evidence for obligatory Ca2 + binding to both antigen and antibody J Biol Chem. 1988 Jan 15; 263 (2): 826-32 [0150]
• - QIAexpress Detection and Assay Handbook 10/2002, QIAGEN, HILDEN, GERMANY [0150]
• Sassenfeld, HM and Brewer, SJ (1984) Bio / Technology, 2, 76-80 [0150]
• - Dalboge, H., Dahl, H.-HM, Pedersen, J., Hansen, JW and Christensen, T. (1987) Bio / Technology, 5, 161-164 [0150]
• Zhao, JY, Ford, CF, Glatz, CE, Rougvie, MA and Gendel, SM (1990) J. Biotechnol., 14, 273-283 [0150]
• - Avidin-Biotin Chemistry: A Handbook [0150]
• - Invitrogen (library) [0150]
• - Pierce Immobilized Monomeric Avidin Kit www.piercenet.com [0150]
• Pierce Two thiol cleavable, heterobifunctional, amine and sulfhydryl-reactive crosslinking agents www.piercenet.com [0150]
• - Meisner M. Pathobiochemistry and clinical use of procalcitonin. Clin Chim Acta. 2002 Sep; 323 (1-2): 17-29 [0150]
• - Meisner M, Brunkhorst FM, Reith HB, Schmidt J, Lestin HG, Reinhart K. Clinical experiences with a novel semi-quantitative solid phase immunoassay for rapid measurement of procalcitonin Clin Chem Lab Med. 2000 Oct; 38 (10): 989-95 [0150]
• - Morgenthaler NG, Struck J, Fischer-Schulz C, Bergmann A. Sensitive immunoluminometric assay for the detection of procalcitonin. Clin Chem. 2002 May; 48 (5): 788-90 [0150]

Claims (18)

Method for detecting analytes from biological Samples comprising the following method steps: a) Provision a immobilized to a solid phase Reversible binding partner 1, to which an analyte binder via an analyte binder bound reversible binding partner 2 is reversibly bound, wherein the analyte-binder is immobilized by binding between the reversible binding partners 1 and 2, b) Addition of the biological sample and binding of the analyte to the reversibly immobilized Analyte binder in case the biological sample contains the analyte, c) Separation of the biological sample d) addition of a release buffer, the bond between the reversible binding partners 1 and 2 dissolves, with binding of the analyte to the analyte binder optional persists, and e) Detection of the analyte in Release buffer in case the biological sample is the analyte contains or ascertains the absence of the Analytes in the case that the biological sample does not contain the analyte. The method of claim 1, wherein the biological Liquid is an unprocessed whole blood sample. Method according to one of the claims 1 or 2, wherein the immobilized reversible binding partner 1 immobilized directly or by means of a carrier protein is. Method according to one of the claims 1 to 3, wherein the analyte binder is covalently attached to the reversible binding partner 2 is bound. Method according to one of the claims 1 to 4, wherein the analyte binder is an anti-procalcitonin antibody is. Method according to one of claims 1 to 5, wherein the binding pair Reversible binding partner 1 and 2 is selected from one of the following binding pairs: a) positively and negatively charged peptide oligomers, b) Ca ²⁺ -binding peptide / protein and antibody containing the peptide / Protein with greater affinity binds when the peptide / protein has bound Ca ²⁺ , c) oligohistidine (eg 6 His) and Ni-NTA, d) biotin and avidin or streptavidin or neutravidin. A method according to any one of claims 1 to 6, wherein the Ca ²⁺ -binding peptide / protein is a FLAG peptide and the antibody is an M1 antibody or the Ca ²⁺ -binding peptide / protein is a protein C peptide and the antibody is an HPC4 antibody is. Method according to one of the claims 1 to 7, wherein the analyte binder is labeled and also after addition of the release buffer remains marked with a label for the detection of the analyte. Method according to one of the claims 1 to 8, wherein the release buffer another for the detection contains required labeled component. The method of claim 10, wherein the further required for the detection marked component second analyte binder for carrying out a Sandwich immunoassays is. Method according to one of the claims 1 to 10, wherein the detection of the labeled analyte by a Immunoassay is done using TRACE technology. Method according to one of the claims 1 to 11, wherein the detection of the labeled analyte by immunochromatography he follows. Method according to one of the claims 1 to 12, wherein the biological sample is undiluted and in is used such a volume that the coated portion the solid phase completely in contact with the biological Sample is brought. Method according to one of claims 1 to 13, wherein the duration of addition of the sample to the De does not exceed 30 minutes and the method should be regarded as a rapid test procedure. Apparatus for carrying out a method according to the Claims 1 to 14 comprising: a) a solid phase with a Reversible binding partner 1 immobilized thereon, to the an analyte binder via a bound to the analyte binder Reversible binding partner 2 is reversibly bound, b) where the bond between the reversible binding partners 1 and 2 by Addition of a release buffer is dissolved, however the binding of the analyte to the analyte binder upon addition of the release buffer optionally remains. Kit for carrying out a method according to one of claims 1 to 14 comprising: a) a solid phase with an immobilized reversible binding partner 1, b) Complex comprising analyte binder bound to the reversible binding partner 2, this complex optionally already by binding the reversible Binding partners 1 and 2 are immobilized on the solid phase can c) Release buffer, which fixes the bond between the Reversible binding partners 1 and 2 dissolves, but where the binding of the analyte to the analyte binder is optional remains. The kit of claim 16, wherein the analyte binder an anti-PCT antibody. Kit according to claim 16 or 17, wherein the binding pair Reversible binding partner 1 and 2 is selected from one of the following binding pairs: a) positively and negatively charged peptide oligomers, b) Ca ²⁺ -binding peptide / protein and antibody containing the peptide / protein greater affinity binds when the peptide / protein has bound Ca ²⁺ , c) oligohistidine (eg 6 His) and Ni-NTA, d) biotin and avidin or streptavidin or neutravidin.