Determination of peripherin

Abstract

The invention relates to a method for the qualitative determination of peripherin, comprising the combining and incubating of a diluted sample with a first antibody conjugate comprising a first antibody bound to a solid phase and a second antibody conjugate comprising a biotinylated second antibody, the removal of the liquid phase and the initiation and measuring of a colour reaction. The invention also relates to a quantification of peripherin in a sample by means of a calibration curve, to the creation of a calibration curve and to a kit for peripherin determination.

Description

[0001] P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0002] DETERMINATION OF PERIPHERIN

[0003] Field of invention

[0004] The present invention relates to a method for the qualitative determination of peripherin, comprising combining and incubating a diluted sample with a first antibody conjugate comprising a first antibody bound to a solid phase and a second antibody conjugate comprising a bio-oxidized second antibody, removing the liquid phase, and triggering and measuring a color reaction. The invention further relates to the quantification of peripherin in a sample using a calibration curve, the creation of a calibration curve, and a kit for peripherin determination.

[0005] Background of the invention

[0006] Peripherin is a protein found in the cell matrix of neurons. Together with other so-called intermediate filaments, it ensures the stability of the nerve cell and its processes. Peripherin has not yet been found outside of nerve cells and is therefore a protein specifically found only in nerve cells.

[0007] Peripherin is found in every nerve cell outside the brain, for example, in motor neurons at the level of the spinal cord and in nerve cell processes such as axons. In the autonomic nervous system, peripherin, along with other matrix proteins, serves to stabilize nerve cells and nerve cell processes. A protein called peripherin II is found in the retina of the eye. Here, it has the same function and plays an important role in retinal diseases such as macular degeneration.

[0008] In the event of nerve cell damage, the cell matrix of the nerve cells and nerve cell processes is partially or completely destroyed. In this case, peripherin or its breakdown products are released into the bloodstream via the surrounding tissue. The extent of the release of peripherin and / or its breakdown products depends on the extent of nerve cell damage. This means that the concentration of peripherin and / or its breakdown products in a body fluid allows conclusions to be drawn about the extent of damage to the cellular matrix of the neuron and nerve cell processes. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0009] A similar correlation has already been demonstrated for other proteins that are components of the neuronal matrix. However, since peripherin is only found in nerve cells of the peripheral nervous system and not in those of the central nervous system, the extent and compartment (peripheral or central nervous system) of nerve cell damage can be determined based on the measurement of peripherin and / or its breakdown products in body fluids. Elevated concentrations of peripherin and / or its breakdown products in body fluids occur in diseases involving damage to the cellular matrix of neurons outside the brain, i.e., the nervous system including the peripheral and autonomic nervous systems.

[0010] To date, peripherin and / or its breakdown products in blood and other body fluids have not been satisfactorily detected for medical purposes. Existing detection methods using ELISA techniques are not sensitive enough to accurately and reliably detect peripherin and / or its breakdown products at the normally low concentrations found in blood, cerebrospinal fluid, or other body fluids. Therefore, these techniques are not yet established in routine clinical practice.

[0011] Therefore, there remains a need for a medical device to detect Peripherin and / or its breakdown products in blood or other body fluids.

[0012] It is therefore an object of the present invention to overcome the existing methodological disadvantages in peripheralin measurement. In particular, it is an object of the present invention to provide a sensitive method for the detection of peripheralin and / or its degradation products in blood or other body fluids. It is also an object of the present invention to provide a reliable method for the detection of peripheralin and / or its degradation products in blood or other body fluids. Furthermore, it is an object of the present invention to provide a method for the detection of peripheralin and / or its degradation products in blood or other body fluids that is easy to use, for example, with regard to the stability of the components used. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0013] It is particularly an object of the present invention to provide a method which enables reliable and sensitive detection of peripherin and / or its degradation products in the blood and which is easy to handle.

[0014] It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which can be reliably applied across a wide range of peripherin concentrations. It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which also enables reliable determination of peripherin concentrations in non-neurological diseases. It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which enables reliable determination of peripherin concentrations for both EDTA plasma and serum.It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which enables reliable determinations of peripherin concentrations even at very low sample dilutions. It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which enables reliable determinations of peripherin concentrations even at very high sample dilutions. It is particularly an object of the present invention to provide a method for detecting peripherin and / or its degradation products in blood or other body fluids, which enables reliable determinations of peripherin concentrations even without the use of additional conjugates (for example, helper beads).

[0015] Brief description of the invention

[0016] The present invention relates to a method for the qualitative determination of peripherin. The method comprises: a. providing a sample; b. diluting the sample with a dilution buffer to obtain a diluted sample; c. combining the diluted sample with a first antibody conjugate and a second antibody conjugate in a reaction vessel to obtain a first mixture; d. incubating the first mixture to obtain an antibody-peripherin-antibody conjugate, wherein the peripherin in the antibody-peripherin-antibody conjugate is bound between the first antibody and the second antibody; e. removing the liquid phase and washing the solid phase with a wash buffer; f. initiating a color reaction by adding at least one reporter enzyme and at least one substrate to the solid phase; g. measuring the color reaction.

[0017] The first antibody conjugate comprises a solid-phase-bound first antibody for peripherin recognition. The second antibody conjugate comprises a biotinylated second antibody, also for peripherin recognition. The first antibody is not 8G2, and the second antibody is not mAb A-3. The reporter enzyme has a streptavidin tag and a binding site to the substrate, but no binding sites to the first antibody or peripherin. The substrate has a binding site to the reporter enzyme and a dye tag, but no binding sites to the first antibody, the second antibody, or peripherin.

[0018] The present invention further relates to a method for creating a calibration curve for Peripherin. The method comprises determining Peripherin in five or more samples with known Peripherin concentrations according to the method described above for the qualitative determination of Peripherin and creating a calibration curve based on the Peripherin concentrations of the samples and the measured color reaction.

[0019] The present invention further relates to a method for the quantitative determination of peripherin. The method comprises: a) the qualitative determination of peripherin in a sample according to the method for the qualitative determination of peripherin described above; b) the determination of a calibration curve according to the method for creating a calibration curve for peripherin described above; and c) the quantification of the color reaction measured in step a) by comparison with the calibration curve determined in step b) for the determination of the peripherin concentration in the sample.

[0020] The present invention further relates to a kit for a heterogeneous immunoassay for the determination of peripherin. The kit comprises: i. a first antibody conjugate, wherein the first antibody conjugate comprises a first antibody bound to a solid phase for the detection of peripherin, and wherein the first antibody is not 8G2; ii. a second antibody conjugate, wherein the second antibody conjugate comprises a biotinylated second antibody, also for the detection of peripherin, and wherein the second antibody is not mAb A-3; iii. a reporter enzyme, wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme does not have a binding site to the first antibody and peripherin; and iv.a substrate, wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody and peripherin.

[0021] The method according to the invention solves the problems stated above. In particular, the method according to the invention offers a highly sensitive method for reliably detecting peripherin even in low concentrations in both blood and other body fluids. For this purpose, the peripherin is bound to the defined peripherin-specific antibodies in a sandwich technique and then detected using a colorimetric reaction. The colorimetric reaction can indicate the amount of peripherin in the sample by comparison with a calibration curve.

[0022] The presence and amount of peripherin is a marker to draw conclusions about damage to the nervous system outside the brain.

[0023] Peripherin can also be used as a biomarker for diseases for which there were previously no reliably and sensitively measurable, known biomarkers in blood and / or other bodily fluids. Examples of such diseases include inflammatory diseases. (P77550WO Laboratory Berlin - Charité Vivantes Services GmbH)

[0024] Intestinal diseases, polyneuropathy, motor neuron diseases and

[0025] Adrenal cortex disease.

[0026] In general, the ability to reliably and sensitively determine peripheral levels is advantageous in the following areas:

[0027] - all damage to the spinal cord, (e.g. multiple sclerosis, spinal ischemia, metabolic diseases, spinal tumors, hereditary diseases (for example: spinal muscular atrophy, neurological diseases due to mutations in the SCA genes, metabolic diseases involving the nervous system, spinal paralysis);

[0028] - Damage to the gastrointestinal nervous system, e.g. Hirschsprung's disease, ileus, ulcerative colitis, Crohn's disease, celiac disease, pancreatitis, cystic fibrosis;

[0029] - Damage to the eye: e.g. macular degeneration and macular dystrophy, retinal degeneration and retinitis pigmentosa;

[0030] - Damage to the central nervous system (in the case of a measurement together with a biomarker for damage to the central nervous system, a central damage can be inferred by exclusion procedure, such as in stroke, multiple sclerosis or amyotrophic lateral sclerosis);

[0031] - Cochlear disorders: e.g., acute inner ear diseases, Meniere's disease, verstibular neuronitis;

[0032] - isolated damage to the second motor neuron, e.g. in spinal muscular atrophy or progressive muscular atrophy in amyotrophic lateral sclerosis;

[0033] - Damage to the peripheral nerves, as occurs, for example, in all polyneuropathies regardless of the causative agent. This damage can be demonstrated in both chronic and acute polyneuropathy (e.g., Guillain-Barré syndrome);

[0034] - Adrenal gland disorders involving cell damage, such as Chushing's syndrome or Addison's disease, but especially neoplasms in the adrenal gland, such as adrenocortical adenomas or adrenocortical carcinomas.

[0035] Brief description of the figures using the SIMOA technology as an example:

[0036] Figures 1a-d are schematic representations of a sandwich ELISA. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0037] Figs. 2a-d are schematic representations of the SIMOA technology.

[0038] Fig. 3 is a schematic representation of the determination of a calibration curve.

[0039] Fig. 4 is an example calibration curve.

[0040] Detailed description of the invention

[0041] Within the scope of the present invention, the term “peripherin” describes both peripherin and its degradation products.

[0042] Within the scope of the present invention, the term "qualitative" or "qualitative determination" describes the determination of the presence or absence of a component, e.g., Peripherin, in a sample. "Quantitative" or "quantitative determination" describes the determination of the amount or concentration of a component, e.g., Peripherin, in a sample.

[0043] In the context of the present invention, "dilution" means reducing the concentration of a component (e.g., Peripherin) in a sample.

[0044] Within the scope of the present invention, the term "antibody" refers to peripheralin antibodies. The terms "antibody," "antibody for recognizing peripheralin," and "peripherin antibody" are used analogously.

[0045] The term "size" in relation to particles refers to the largest diameter of a particle. Within the scope of the present invention, the terms "particle size" and "size" are used analogously. Similarly, within the scope of the present invention, the terms "particle" and "bead" are used analogously. Particle sizes can be determined, for example, using ISO 13320:2020. Within the scope of the present invention, particle size refers to the mean particle size according to ISO 9276-2.

[0046] "Approximately" in the context of the present invention means a deviation of less than 10%, e.g., less than 5% or less than 3%, particularly preferably less than 1%. "Approximately spherical" in the context of the present invention, with regard to the shape of particles, means that the largest diameter and the smallest diameter are approximately 1%. [P77550WO Labor Berlin - Charite Vivantes Services GmbH]

[0047] The diameter of a particle does not differ from each other by more than 10% (e.g. less than 5% or less than 3%, particularly preferably less than 1%).

[0048] Within the scope of the present invention, "room temperature" refers to a temperature in the range of 20-25°C.

[0049] “Lysis buffers” are buffered solutions that can break the bonds between proteins in protein aggregates. According to the present invention, the use of lysis buffers is advantageous for increasing the solubility of the samples.

[0050] The present invention relates to a method for the qualitative determination of peripherin, comprising: a. providing a sample; b. diluting the sample with a dilution buffer to obtain a diluted sample; c. combining the diluted sample with a first antibody conjugate and a second antibody conjugate in a reaction vessel to obtain a first mixture, wherein the first antibody conjugate comprises a first antibody bound to a solid phase for the recognition of peripherin, wherein the second antibody conjugate comprises a biotinylated second antibody also for the recognition of peripherin, and wherein the first antibody is not 8G2, and wherein the second antibody is not mAb A-3; d. incubating the first mixture to obtain an antibody-peripherin-antibody conjugate, wherein the peripherin is bound in the antibody-peripherin-antibody conjugate between the first antibody and the second antibody; e.Removal of the liquid phase and washing of the solid phase with a wash buffer; f. Triggering a color reaction by adding at least one reporter enzyme and at least one substrate to the solid phase; P77550WO Laboratory Berlin - Charité Vivantes Services GmbH wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme has no binding site to the first antibody and peripherin, and wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody and peripherin; g. Measuring the color reaction.

[0051] Body fluids can be used as samples in the method according to the invention. Blood and cerebrospinal fluid (CSF) samples are preferred. This means that the method according to the invention is suitable for the detection of peripherin from both blood and CSF samples. The samples are preferably cell-free, in particular cell-free CSF, serum, and any plasma samples. Cell-free CSF samples can be obtained by taking a CSF sample, centrifuging, and separating the liquid phase. Serum can be obtained by taking a blood sample, initiating coagulation (e.g., by adding a coagulation activator or using a blood collection tube containing coagulation activators), centrifuging, and separating the liquid phase. Plasma can be obtained by taking a blood sample, preventing coagulation (e.g., by adding an anticoagulant such as EDTA or using an EDTA or heparin blood collection tube), centrifuging, and separating the liquid phase.

[0052] According to step b., the sample is diluted with a dilution buffer to obtain a diluted sample. Preferably, the sample is diluted in a sample-to-dilution buffer ratio of 1:1 to 1:100, for example 1:2 to 1:50, particularly preferably 1:3 to 1:20.

[0053] Preferably, the dilution buffer is a lysis buffer (e.g., an immunocomplex lysis buffer). The pH of the dilution buffer can be in the range of 7 to 8, for example, 7.2 to 7.8, preferably 7.4 to 7.6.

[0054] According to step c, the diluted sample is combined with a first antibody conjugate (also called capture antibody) and a second antibody conjugate (also called detector antibody) in a reaction vessel to obtain a first mixture. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0055] The components can be combined in any order. Preferably, the diluted sample is first combined with the first antibody conjugate before the second antibody conjugate is added. It is also possible, in principle, to combine the diluted sample with the second antibody conjugate before adding the first, or to combine the two antibody conjugates before adding the diluted sample, or to combine all three components at least partially simultaneously.

[0056] The combination can be carried out while moving, for example by tilting, the reaction vessel.

[0057] Suitable antibodies within the scope of the present invention are, in principle, monoclonal or polyclonal peripheralin antibodies, peripheralin antibody fragments, and other molecules or structures that can recognize and bind peripheralin. Peripherin antibodies are known and commercially available.

[0058] The first antibody conjugate acts as a capture antibody. It comprises a first antibody bound to a solid phase.

[0059] The solid phase is preferably in the form of particles, especially paramagnetic particles. The particles can have a size of 0.5 to 10 pm, e.g. 1 to 5 pm, preferably 2 to 3 pm.

[0060] The particles can have any shape. They are preferably spherical or nearly spherical.

[0061] The particles are preferably labeled with a dye that emits a specific and known color signal when irradiated with light of a particular and known wavelength. Suitable particles include, for example, Simoa 488nm-dyed singleplex beads (Order No. 104006, Quanterix, Billercia, Massachusetts). These emit a specific color signal when irradiated with a 488 nm wavelength laser. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0062] The first antibody is not 8G2. Suitable commercially available first antibodies include, for example, PRPH Monoclonal Antibody 3B3 (Order No. H00005630-M02, Mouse Monoclonal, Non-biotinylated, GST-tag, C-terminal, IgG2b, Immunogen 374-470, Thermo Fisher Scientific, 64293 Darmstadt, Germany) and Polyclonal Peripherin (PRPH) Antibody (Order No. ABX320907-100UL, Rabbit Polyclonal, Non-biotinylated, N-terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands). The antibody MC6 (Sigma Aldrich monoclonal peripherin antibody, catalog-, P5117) is a clone of 8G2.

[0063] Preferably, the first antibodies and the particles are covalently linked. The first antibodies and the particles can also be linked via an amide bond. For example, the first antibodies are bound to a reactive carboxyl group of the particles via a primary amino group.

[0064] In the first mixture, the amount of first antibody conjugate can be at least equal to the amount of peripherin. Preferably, in the first mixture, the amount of first antibody conjugate is higher than the amount of peripherin.

[0065] The second antibody is not mAb A-3. Suitable second antibodies include, for example, Peripherin Antibody 8G2 (Order No. NB300-138B, Mouse Monoclonal, biotinylated, IgG1, Bio-Techne GmbH, 65205 Wiesbaden Nordenstadt, Germany) and PRPH Monoclonal Antibody 3B3 (Peripherin, Neurofilament 4, NEF4, PRPH1 (Biotin) Clone: ​​[3B3] Mouse Monoclonal (Order No. MBS6143769-0.1), biotinylated, GST tag, C-terminal, Immunogen 374-470, BIOZOL Diagnostica Vertrieb GmbH, Leipziger Straße 4, 85386 Eching, Germany). It may be preferable that the second antibody is not MC3 (Santa Cruz Peripherin A-3 antibody catalog# sc-377093).

[0066] Preferably, the first antibody and the second antibody are different.

[0067] The first antibody and the second antibody are particularly preferred to be different, and the second antibody is 8G2.

[0068] Within the scope of the present invention, it may be preferred that the first antibody is not MC6 and the second antibody is not MC3, since this combination does not show the desired effectiveness in the detection of peripherin. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0069] According to step d., the first mixture is incubated to obtain an antibody-peripherin-antibody conjugate. In the antibody-peripherin-antibody conjugate, the peripherin is bound between the first antibody and the second antibody, forming a sandwich structure.

[0070] Preferably, the first mixture is incubated at room temperature. During incubation, the first mixture can either be left undisturbed or kept in motion, for example by stirring, shaking, or swirling.

[0071] Preferably, the incubation time of the first mixture is 1 min to 60 min, e.g. 5 to 50 min or 10 to 45 min or 20 to 40 min, particularly preferably 35 to 45 min.

[0072] Incubation can, in principle, be carried out under various light conditions, for example, in complete darkness, in daylight, in ambient light, or under irradiation. Incubation in complete darkness is preferred.

[0073] According to step e. the liquid phase is removed, and the solid phase is washed with a washing buffer.

[0074] In principle, the liquid phase can be removed by any method known to those skilled in the art. Preferably, the liquid phase is removed by pipetting. Particularly preferably, the removal of the liquid phase is automated using an automated pipette.

[0075] The washing buffer used is preferably suitable for removing components that are not part of the antibody-peripherin-antibody conjugate. Examples include unbound antibodies, unbound components from the sample, particularly components other than peripherin, and unbound components of the dilution buffer. Preferably, the washing buffer is a phosphate buffer. The preferred pH of the washing buffer is in the range of 7.1 to 7.7, particularly preferably in the range of 7.4 to 7.5.

[0076] According to step f., a color reaction is triggered by adding at least one reporter enzyme and at least one substrate to the solid phase. The color reaction is preferably a fluorescence or chemiluminescence reaction, particularly preferably a fluorescence reaction. P77550WO Labor Berlin - Charité Vivantes Services GmbH

[0077] The reporter enzyme has a streptavidin tag and a binding site to the substrate. The streptavidin tag allows the reporter enzyme to bind to the biotin of the second antibody conjugate.

[0078] The reporter enzyme lacks a binding site to the first antibody and to peripherin. Therefore, the reporter enzyme can selectively bind to the second antibody conjugate of the antibody-peripherin-antibody conjugate.

[0079] The substrate has a binding site for the reporter enzyme and a dye tag. The substrate has no binding site for the first antibody, the second antibody, or peripherin.

[0080] Preferably, the color reaction is triggered by the cleavage and release of the dye of the dye tag from the substrate by reaction with the reporter enzyme. The dye tag bound to the substrate preferably exhibits a different color characteristic than the free dye of the dye tag. The color characteristic can be, for example, a color in the visible range, phosphorescence, fluorescence, or chemiluminescence. Preferably, the color characteristic is fluorescence or chemiluminescence, and particularly preferably fluorescence.

[0081] Suitable dyes include, for example, phenoxazine dyes. Phenoxazine dyes are disclosed, for example, in WO 2010 / 003970 A2. Resorufin is particularly preferred.

[0082] The reporter enzyme can be chosen to selectively cleave the substrate upstream of the dye tag, thus releasing the dye of the dye tag. Examples of such enzyme / substrate pairs are galactosidase and β-galactopyranoside.

[0083] For example, streptavidin-beta-galatosidase (SbG) can be used as the preferred reporter enzyme and resorufin-β-galactopyranoside (RGP) as the preferred substrate. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0084] Preferably, the initiation of the color reaction comprises: f1. the addition of the reporter enzyme and mixing with the solid phase; and f2. the addition of the substrate.

[0085] The color reaction is preferably determined using the Simoa technique.

[0086] Simoa stands for "single-molecule array." In the Simoa technique, a sample (here comprising the antibody-peripherin-antibody conjugate) is placed in arrays of a Simoa disk containing numerous microwells, each microwell being large enough for one particle (here, an antibody-peripherin-antibody conjugate). The color reaction can be detected after triggering by determining the number of microwells showing a color reaction.

[0087] The result of the color reaction can be expressed in absolute values ​​(i.e., the total number of microwaves with a color reaction) or as a percentage (i.e., the percentage of microwaves with a color reaction relative to the total number of microwaves). The result of the color reaction is preferably expressed in absolute values.

[0088] The presence of a color reaction means that Peripherin was present in the sample.

[0089] According to the invention, a quantitative determination of the peripherin is carried out by comparing the number of fluorescences of the sample with a calibration curve, which is calculated based on the fluorescent color reaction of samples with known peripherin concentration.

[0090] The present invention therefore also relates to a method for creating a calibration curve for Peripherin comprising i) determining Peripherin in five or more samples as described above, wherein the Peripherin concentration of each of the samples is known; ii) creating a calibration curve based on the Peripherin concentrations of the samples and the measured color reaction.

[0091] To generate the calibration curve, peripherin is first qualitatively determined in five or more samples. The determination is carried out according to the procedure described herein for the qualitative determination of peripherin. The possible and preferred embodiments described in procedure P77550WO Labor Berlin - Charité Vivantes Services GmbH for the qualitative determination of peripherin also apply to the determination of peripherin when generating the calibration curve.

[0092] Preferably, the color reaction is determined using the Simoa technique as described above when determining the peripherin.

[0093] To determine the calibration curve, 5 or more samples are used, for example 5 to 15 samples, preferably 8 to 10 samples.

[0094] The samples can have a peripherin concentration in the range of more than 0 pg / mL up to 1,000,000 pg / mL, preferably in the range of 0.01 pg / mL up to 100,000 pg / mL. A reference sample that does not contain peripherin is also preferably used.

[0095] The sample concentration is preferably distributed within a specific concentration range, for example, logarithmically. Preferably, at least one of the samples has a peripheralin concentration in the range of 1 to 25 pg / mL, e.g., from 5 to 20 pg / mL. Preferably, at least one of the samples has a peripheralin concentration in the range of 500 to 100,000 pg / mL, e.g., from 500 to 5,000 pg / mL.

[0096] A calibration curve can be created by plotting the results on a graph, where the X-axis represents the peripheral nit concentration in the samples and the Y-axis represents the color reaction, e.g. in absolute values ​​or as a percentage, preferably in absolute values.

[0097] Preferably, the calibration curve is based on a fit equation of the 4PL regression.

[0098] formula

[0099] A — D r(%) = D +

[0100] 1 + (?) ß definitely. It says

[0101] Y for AEB (activated enzyme), x for the measured concentration,

[0102] D represents the theoretical value at an infinitely high value.

[0103] A for minimum AEB, c for concentration for the signal between the highest and lowest value and P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0104] B for the slope of the curve.

[0105] For the quantitative determination of Peripherin, a qualitative Peripherin determination described herein is quantified using a calibration curve described herein.

[0106] The present invention therefore also relates to a method for the quantitative determination of Peripherin comprising a) the qualitative determination of Peripherin in a sample as described herein; b) the determination of a calibration curve as described herein; and c) the quantification of the color reaction measured in step a) by comparison with the calibration curve determined in step b).

[0107] The possible and preferred embodiments described in the procedure for the qualitative determination of Peripherin and the procedure for creating a calibration curve also apply to the quantitative determination of Peripherin.

[0108] The color reaction is preferably determined in the qualitative determination of peripherin and in the creation of the calibration curve using the Simoa technique.

[0109] Preferably, the measured color reaction is a fluorescence or chemiluminescence. Preferably a fluorescence.

[0110] The present invention also relates to a kit for a heterogeneous immunoassay for the determination of peripherin, comprising: i. a first antibody conjugate, wherein the first antibody conjugate comprises a first antibody bound to a solid phase for the detection of peripherin, and wherein the first antibody is not 8G2; ii. a second antibody conjugate, wherein the second antibody conjugate comprises a biotinylated second antibody also for the detection of peripherin, and wherein the second antibody is not mAb A-3; iii. a reporter enzyme, wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme has no binding site to the first antibody and peripherin; and iv. P77550WO Labor Berlin - Charite Vivantes Services GmbHa substrate, wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody and peripherin.

[0111] The possible and preferred embodiments of the kit components described in the procedure for the qualitative determination of Peripherin also apply to the kit described here.

[0112] Preferably, the kit also includes a calibration component. A calibration curve can be generated using the calibration component. The calibration component comprises a known quantity of Peripherin. The Peripherin can be in solid or dissolved form. The calibration component is preferably a calibration fluid in which the Peripherin is dissolved in a liquid medium, e.g., an aqueous buffer. If the Peripherin is in solid form, the kit preferably also includes a liquid medium, e.g., an aqueous buffer, for dissolving the Peripherin.

[0113] If the kit includes a calibration component, it preferably also includes instructions for creating a dilution series of the calibration component.

[0114] Preferably, the kit also includes a dilution buffer. Preferably, the dilution buffer is a lysis buffer (e.g., an immunocomplex lysis buffer). The pH of the dilution buffer can be in the range of 7 to 8, for example, 7.2 to 7.8, preferably 7.4 to 7.6.

[0115] The first antibody conjugate acts as a capture antibody. It comprises a first antibody bound to a solid phase.

[0116] The solid phase is preferably in the form of particles, especially paramagnetic particles. The particles can have a size of 0.5 to 10 pm, e.g. 1 to 5 pm, preferably 2 to 3 pm.

[0117] The first antibody is not 8G2. Suitable, commercially available first antibodies include, for example, PRPH Monoclonal Antibody 3B3 (Order No. H00005630-M02, Mouse P77550WO, Laboratory Berlin - Charité Vivantes Services GmbH).

[0118] Monoclonal, Non-biotinylated, GST-tag, C-terminal, IgG2b, Immunogen 374-470, Thermo Fisher Scientific, 64293 Darmstadt, Germany), Polyclonal Peripherin (PRPH) Antibody, (Order No. ABX320907-100UL, Rabbit Polyclonal, Non-biotinylated, N-terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands).

[0119] Preferably, the first antibodies and the particles are covalently linked. The first antibodies and the particles can also be linked via an amide bond. For example, the first antibodies are bound to a reactive carboxyl group of the particles via a primary amino group.

[0120] In the first mixture, the amount of first antibody conjugate can be at least equal to the amount of peripherin. Preferably, in the first mixture, the amount of first antibody conjugate is higher than the amount of peripherin.

[0121] The second antibody is not mAb A-3. Suitable second antibodies include, for example, Peripherin Antibody 8G2 (Order No. NB300-138B, Mouse Monoclonal, biotinylated, IgG1, Bio-Techne GmbH, 65205 Wiesbaden Nordenstadt, Germany) and PRPH Monoclonal Antibody 3B3 (Peripherin, Neurofilament 4, NEF4, PRPH1 (Biotin) Clone: ​​[3B3] Mouse Monoclonal (Order No. MBS6143769-0.1), biotinylated, GST tag, C-terminal, Immunogen 374-470, BIOZOL Diagnostica Vertrieb GmbH, Leipziger Straße 4, 85386 Eching, Germany).

[0122] Preferably, the first antibody and the second antibody are different.

[0123] The first antibody and the second antibody are particularly preferred to be different, and the second antibody is 8G2.

[0124] The reporter enzyme has a streptavidin tag and a binding site to the substrate. The streptavidin tag allows the reporter enzyme to bind to the biotin of the second antibody conjugate.

[0125] The reporter enzyme lacks a binding site to the first antibody and to peripherin. Therefore, the reporter enzyme can selectively bind to the second antibody conjugate of the antibody-peripherin-antibody conjugate. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0126] The substrate has a binding site for the reporter enzyme and a dye tag. The substrate has no binding site for the first antibody, the second antibody, or peripherin.

[0127] P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0128] Exemplary embodiments

[0129] A. Method for the qualitative determination of peripherin comprising: a. providing a sample; b. diluting the sample with a dilution buffer to obtain a diluted sample; c. combining the diluted sample with a first antibody conjugate and a second antibody conjugate in a reaction vessel to obtain a first mixture, wherein the first antibody conjugate comprises a first antibody bound to a solid phase for the recognition of peripherin, wherein the second antibody conjugate comprises a biotinylated second antibody also for the recognition of peripherin, and wherein the first antibody is not 8G2, and wherein the second antibody is not mAb A-3; d. incubating the first mixture to obtain an antibody-peripherin-antibody conjugate, wherein the peripherin is bound in the antibody-peripherin-antibody conjugate between the first antibody and the second antibody; e.f. Removal of the liquid phase and washing of the solid phase with a wash buffer; f. Initiation of a color reaction by adding at least one reporter enzyme and at least one substrate to the solid phase; wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme has no binding site to the first antibody and peripherin; wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody, and peripherin; g. Measurement of the color reaction.

[0130] B. The method according to embodiment A or B, wherein the first antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3, Polyclonal Peripherin (PRPH) Antibody (Order No. ABX320907-100UL, Rabbit P77550WO Labor Berlin - Charité Vivantes Services GmbH)

[0131] Polyclonal, Non-biotinylated, N-Terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands), and wherein the second antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3 and Peripherin Antibody 8G2.

[0132] C. The method according to one of the preceding embodiments, wherein the first antibody and the second antibody are different, and wherein the second antibody is 8G2.

[0133] D. The method according to one of the preceding embodiments, wherein the sample is a blood sample, preferably a cell-free cerebrospinal fluid sample, particularly preferably human serum or human plasma.

[0134] E. The method according to one of the preceding embodiments, wherein the sample is diluted in a sample to dilution buffer ratio of 1:1 to 1:100.

[0135] F. The method according to one of the preceding embodiments, wherein the dilution buffer is a lysis buffer.

[0136] G. The method according to one of the preceding embodiments, wherein in the first mixture the amount of first antibody conjugate is at least equal to the amount of peripheralin.

[0137] H. The method according to one of the preceding embodiments, wherein the first antibody conjugate comprises paramagnetic particles to which the first antibodies are bound.

[0138] I. The method according to embodiment H, wherein the particles have a size of 0.5 to 10 pm.

[0139] J. The method according to one of the preceding embodiments, wherein the first antibody and the second antibody are different.

[0140] K. The method according to one of the preceding embodiments, wherein the first mixture is incubated at room temperature. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0141] L. The method according to one of the preceding embodiments, wherein the incubation time of the first mixture is 1 min to 60 min (preferably 5 to 50 min, e.g. 10 to 45 min or 20 to 40 min, particularly preferably 35 to 45 min).

[0142] M. The method according to one of the preceding embodiments, wherein the liquid phase is removed by pipetting.

[0143] N. The method according to one of the preceding embodiments, wherein the washing buffer is a phosphate buffer.

[0144] O. The method according to one of the preceding embodiments, wherein initiating the color reaction comprises: f1. Addition of the reporter enzyme and mixing with the solid phase; and f2. Addition of the substrate.

[0145] P. The method according to one of the preceding embodiments, wherein the color reaction is determined using the Simoa technique.

[0146] Q. Method for creating a calibration curve for Peripherin comprising i) determining Peripherin in five or more samples according to one of embodiments A to P, wherein the Peripherin concentration of each of the samples is known; ii) creating a calibration curve based on the Peripherin concentrations of the samples and the measured color reaction.

[0147] R. The method according to embodiment Q, wherein the color reaction is determined using the Simoa technique.

[0148] S. The method according to one of embodiments Q or R, wherein at least one of the samples has a peripheralin concentration in the range of 1 to 25 pg / mL. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0149] T. The method according to one of embodiments Q to S, wherein at least one of the samples has a peripheralin concentration in the range of 1,000 to 100,000 pg / mL.

[0150] U. Method for the quantitative determination of Peripherin comprising a) qualitative determination of Peripherin in a sample according to one of embodiments A to P; b) determination of a calibration curve according to one of embodiments Q to T; c) quantification of the color reaction measured in step a) by comparison with the calibration curve determined in step b).

[0151] V. The method according to embodiment U, wherein the measured color reaction is a fluorescence or chemiluminescence.

[0152] W. The method according to embodiment V, wherein the color reaction and the calibration curve are determined using Simoa technique.

[0153] X. Kit for a heterogeneous immunoassay for the determination of peripherin comprising i. a first antibody conjugate, wherein the first antibody conjugate comprises a solid-phase-bound first antibody for the detection of peripherin, and wherein the first antibody is not 8G2; ii. a second antibody conjugate, wherein the second antibody conjugate comprises a biotinylated second antibody, also for the detection of peripherin, and wherein the second antibody is not mAb A-3; iii. a reporter enzyme, wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme has no binding site to the first antibody and peripherin; and iv. a substrate, wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody, and peripherin.

[0154] Y. The kit according to embodiment X, further comprising a dilution buffer (preferably a lysis buffer, particularly preferably an immunocomplex lysis buffer). P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0155] Z. The kit according to one of embodiments X or Y, further comprising a calibration component, wherein the calibration component contains Peripherin in a known quantity.

[0156] AA. The kit according to one of embodiments X to Z, wherein the first antibody and the second antibody are different.

[0157] BB. The kit according to one of embodiments X to AA, wherein the first antibody conjugate comprises paramagnetic particles to which the first antibodies are bound.

[0158] CC. The kit according to one of the embodiments X to BB, wherein the particles have a size of 0.5 to 10 pm.

[0159] DD. The kit according to one of the embodiments AA to CC, wherein the first antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3, Polyclonal Peripherin (PRPH) Antibody (Order No. ABX320907-100UL, Rabbit Polyclonal, Non-biotinylated, N-Terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands), and wherein the second antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3 and Peripherin Antibody 8G2.

[0160] EE. The kit according to one of embodiments X to DD, wherein the first antibody and the second antibody are different, and wherein the second antibody is 8G2.

[0161] P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0162] Examples

[0163] materials

[0164] Table 1: Materials P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0165] SIMOA technology

[0166] Assays based on SIMOA (single molecule array) technology from Quanterix are known for their sensitive protein detection with low detection limits. This P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0167] Technology makes it possible to detect proteins at the single molecule level.

[0168] The technique is based on a sandwich ELISA (enzyme-linked immunosorbent assay), which is shown schematically in FIGS. 1a to 1d. Two different antibodies are used, each specific for the respective target protein (130). A first specific antibody (capture antibody, 120), linked to a solid phase (beads, 110), binds the target protein (130) from the matrix. A second antibody (detection antibody, 140), also specifically recognizing the target protein (peripherin, 130), binds to the target protein (130) bound to the solid phase (110) via the first antibody (120). The second antibody (140) is tagged with a biotin tag (160). To trigger a color signal, a reporter enzyme (150) tagged with a streptavidin tag is first bound to the biotin tag (160) of the second antibody (140).Subsequently, a substrate (170) is added, which has a binding site (172) to the reporter enzyme (150) and a dye tag (174). The reporter enzyme can then cleave the substrate between the binding site (172) and the dye tag (174). This releases the dye (180), which can be detected.

[0169] SIMOA technology is a particularly sensitive technology, schematically illustrated in FIG. 2. The reaction sample contains unloaded first antibodies (100) bound to beads (110), i.e., first antibodies to which no target proteins (130) are bound, and loaded first antibodies (101) bound to beads (110), i.e., first antibodies to which the target protein (130) and the second antibody (140) with tag (160) are sandwiched together. As shown in FIG. 2a, the reaction sample is placed on a microtiter plate (240) with wells (230), each well (230) being able to hold one unloaded first antibody (100) bound to a bead or one loaded first antibody (101) bound to a bead. As shown in FIG. 2b, an oil (250) is added to seal the wells (230) over the microtiter plate (240).An oil-sealed microtiter plate (240) is obtained, the wells (230) of which are at least partially filled with unloaded first antibodies (100) bound to a bead (110) or with loaded first antibodies (101) bound to a bead (110) (see FIG. 2c). FIG. 2d shows that the wells (230) containing loaded first antibodies (220) can be identified by triggering a color reaction. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH.

[0170] The number of wells in which a color reaction is detected correlates with the number of bound target proteins (130), so that the concentration of the target proteins (130) in the sample can be inferred.

[0171] Creating a dilution series for a calibration curve

[0172] The calibration curve was created using a fetal calf serum (FCS) sample. The serum sample was centrifuged for 5 minutes at a centrifugal force of 10,000 x g. The liquid phase was then extracted by pipetting.

[0173] The liquid phase was first diluted with a dilution buffer (immune complex lysis buffer 1 L) (1 part FCS serum, 3 parts dilution buffer) to obtain a diluted FCS medium. A Peripherin solution with a concentration of 1 pg / mL in dilution buffer was prepared.

[0174] Subsequently, a pre-dilution and a starting dilution were prepared and stored on ice. The compositions are as follows:

[0175] Table 2: Composition of pre-dilution, starting dilution and

[0176] dilution medium

[0177] A dilution series was prepared on a SIMOA 96-well plate (see FIG. 3): i. 400 pL of the initial dilution were pipetted into well B2. ii. 200 pL of the dilution medium and 200 pL of the initial dilution from well B2 were pipetted into well A2 and mixed well. iii. 200 pL of the dilution medium and 200 pL from well A2 were pipetted into well H1 and mixed well. iv. 200 pL of the dilution medium and 200 pL from well H1 were pipetted into well G1 and mixed well. v. 200 pL of the dilution medium and 200 pL from well G1 were pipetted into well F1 and mixed well. vi. 200 pL of the dilution medium and 200 pL from well F1 were pipetted into well E1 and mixed thoroughly. vii. 200 pL of the dilution medium and 200 pL from well E1 were pipetted into well D1 and mixed thoroughly. viii.200 pL of the dilution medium and 200 pL from well D1 were pipetted into well C1 and mixed thoroughly. 200 pL of the dilution medium and 200 pL from well C1 were pipetted into well B1 and mixed thoroughly. 200 pL of dilution medium were pipetted into well A1.

[0178] The preparation of the dilution series for the calibration curve is shown schematically in Figure 3a. In row 1 of the microtiter plate, a dilution series is prepared as described above. A1 contains the pure dilution medium as a reference. H1 contains the starting dilution, i.e., the highest concentration of Peripherin. The decreasing amount of Peripherin from H1 to B1 is shown schematically by the increasing brightness of the fill.

[0179] Each sample A1 to B2 is analyzed using the SIMOA technique as described. The result for the sample from well B1 is shown schematically in Figure 3b.

[0180] Production of a first antibody conjugate

[0181] The first antibody conjugate was prepared using the Simoa Homebrew Assay Starter Kit (Order No. 101351, Quanterix, Billercia, Massachusetts). The entire subsequent procedure was performed on ice.

[0182] The beads were washed three times with Bead Wash Buffer (Quanterix, (Order No. 101335), Quanterix, Billercia, Massachusetts), and then three times with Bead Conjugation Buffer (Quanterix, (Catalog 101357), Quanterix, Billercia, Massachusetts). After washing, 291 pL of Bead Conjugation Buffer was added to the beads.

[0183] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was diluted to 10 mg / mL with Bead Conjugation Buffer. 9 pL of the diluted EDC were added to the beads in 291 pL Bead P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0184] Conjugation buffer was added and the beads were mixed in suspension for 30 ± 2 min at 2-8°C to activate them.

[0185] 80 pg of peripherin-capture antibody was made up to 500 pL with bead conjugation buffer. Using an Amicon filter from the Simoa Homebrew Assay Starter Kit, the antibodies were washed and centrifuged five times with bead conjugation buffer (three times at a centrifugal force of 14,000 x g for 5 min and twice at a centrifugal force of 1,000 x g for 2 min). 300 pL of bead conjugation buffer with an antibody concentration of 0.2 mg / mL was obtained.

[0186] The activated beads were washed again with 300 pL of bead wash buffer. After adding the 300 pL antibody mixture to the beads, they were incubated again at 2-8°C with shaking for 120 ± 2 minutes.

[0187] After incubation, the suspension was washed twice with Bead Wash Buffer before the bead-antibody mixture was resuspended in 300 pL Bead Blocking Buffer (Quanterix, Order No. 101356, Quanterix, Billercia, Massachusetts). Following a second incubation at room temperature using a shaker plate for 45 ± 2 min, the final washing steps were performed with Bead Wash Buffer and Bead Diluent (Quanterix, Order No. 101355, Quanterix, Billercia, Massachusetts) before the beads were resuspended in 300 pL Bead Diluent as the first antibody conjugate.

[0188] Determination of peripheral

[0189] The SIMOA technique is used to determine peripheralin levels. The procedure is automated under the following conditions:

[0190] Equipment and materials

[0191] SIMOA HD-1 / HD-X Analyzer (Quanterix Corporation, Billerica, Massachusetts)

[0192] SIMOA pipette tips (Quanterix Corporation, Billerica, Massachusetts)

[0193] SIMOA test tubes “cuvettes” (Quanterix Corporation, Billerica, Massachusetts) SIMOA Discs (Quanterix Corporation, Billerica, Massachusetts)

[0194] SIMOA 96-well microtiter plates (Quanterix Corporation, Billerica, Massachusetts) Evaporation protection films for 96-well microtiter plates

[0195] SIMOA 15-ml Bottles (Quanterix Corporation, Billerica, Massachusetts) P77550WO Labor Berlin - Charite Vivantes Services GmbH

[0196] material

[0197] SIMOA Bead Diluent Buffer

[0198] SIMOA SbG Diluent

[0199] SIMOA SbG Concentrate

[0200] SIMOA Detector / Sample Diluent

[0201] SIMOA RPG Reagent

[0202] Immune complex lysis buffer

[0203] Peripheral glucocorticoids (8G2)

[0204] Bead-bound peripheral lin antibodies (3B3)

[0205] Fetal Calf Serum (FCS)

[0206] A list of materials with more detailed manufacturer information is also provided at the beginning of the examples.

[0207] Rehearsal preparation

[0208] Using an Excel spreadsheet provided by Quanterix to calculate the volumes, the 15-ml bottles are filled with the respective reagents:

[0209] - Peripheral antibody (8G2) with SIMOA detector / sample diluent,

[0210] - SbG concentrate with SIMOA SbG Diluent,

[0211] - Bead-bound peripheral lin antibodies (3B3) with SIMOA Bead Diluent Buffer.

[0212] The 15-ml bottles containing the respective diluted reagents are stored on ice.

[0213] A human serum sample is centrifuged for 5 minutes at a centrifugal force of 10,000 x g. The liquid phase is withdrawn by pipetting. The liquid phase is diluted with the dilution buffer (immune complex lysis buffer 1 L) (1 part serum, 3 parts dilution buffer) to obtain a diluted serum sample. The diluted sample is stored on ice until measurement.

[0214] 100 pL of the diluted sample are required per measurement per well.

[0215] implementation

[0216] The measurement is performed on a SIMOA HD-1 / HD-X analyzer (Quanterix Corporation, Billerica, Massachusetts) with integrated software. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0217] The software allows you to configure the following specific settings for performing a user-defined assay for peripheral rin determination. The following headings refer to the names of the respective tabs in the software:

[0218] “Reagents”

[0219] Table 3: first antibody conjugate

[0220] Table 4: second antibody conjugate P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0221] Table 5: SbG

[0222] Table 6: Calibrator P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0223] “Overview”

[0224] Table 7: General settings of the assay P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0225] "Plexes"

[0226] Table 8: Settings for determining the calibration curve

[0227] Table 9: Measured quantities

[0228] Table 10: Acquisition channel

[0229] Table 11: further settings P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0230] P77550WO Laboratory Berlin - Charité Vivantes Services GmbH

[0231] Reference symbol list

[0232] 100 first antibody conjugate

[0233] 101 Antibody-peripherin-antibody conjugate with reporter enzyme and substrate

[0234] 110 fixed phase

[0235] 120 first antibody

[0236] 130 Target protein (e.g., peripherin)

[0237] 140 second antibody

[0238] 150 reporter enzyme

[0239] 160 Biotin Day

[0240] 170 substrate

[0241] 172 Binding site to reporter enzyme

[0242] 174 dye day

[0243] 180 dye

[0244] 230 Well

[0245] 240 microtiter plate

[0246] 250 oil

Claims

1. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH Claims 1. Method for the qualitative determination of peripherin comprising: a. providing a sample; b. diluting the sample with a dilution buffer to obtain a diluted sample; c. combining the diluted sample with a first antibody conjugate and a second antibody conjugate in a reaction vessel to obtain a first mixture, wherein the first antibody conjugate comprises a first antibody bound to a solid phase for the recognition of peripherin, wherein the second antibody conjugate comprises a biotinylated second antibody also for the recognition of peripherin, and wherein the first antibody is not 8G2, and wherein the second antibody is not mAb A-3; d. incubating the first mixture to obtain an antibody-peripherin-antibody conjugate, wherein the peripherin is bound in the antibody-peripherin-antibody conjugate between the first antibody and the second antibody; e.f. Removal of the liquid phase and washing of the solid phase with a wash buffer; f. Initiation of a color reaction by adding at least one reporter enzyme and at least one substrate to the solid phase; wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme has no binding site to the first antibody and peripherin; wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate has no binding site to the first antibody, the second antibody, and peripherin; g. Measurement of the color reaction.

2. The method according to claim 1, wherein the first antibody is selected from the group consisting of PRPH monoclonal antibody 3B3, polyclonal 38 P77550WO Laboratory Berlin - Charité Vivantes Services GmbH Peripherin (PRPH) Antibody (Order No. ABX320907-100UL, Rabbit Polyclonal, Non-biotinylated, N-Terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands), wherein the second antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3 and Peripherin Antibody 8G2.

3. The method according to one of the preceding claims, wherein the first antibody and the second antibody are different, and wherein the second antibody is 8G2.

4. The method according to one of the preceding claims, wherein the sample is a blood sample, preferably a cell-free cerebrospinal fluid sample, particularly preferably human serum or human plasma.

5. The method according to one of the preceding claims, wherein the sample is diluted in a sample to dilution buffer ratio of 1:1 to 1:

100.

6. The method according to one of the preceding claims, wherein the dilution buffer is a lysis buffer, preferably an immunocomplex lysis buffer.

7. The method according to one of the preceding claims, wherein in the first mixture the amount of first antibody conjugate corresponds at least to the amount of peripherin.

8. The method according to one of the preceding claims, wherein the first antibody conjugate comprises paramagnetic particles to which the first antibodies are bound.

9. The method according to claim 6, wherein the particles have a size of 0.5 to 10 pm.

10. The method according to one of the preceding claims, wherein the first antibody and the second antibody are different. 39 P77550WO Laboratory Berlin - Charité Vivantes Services GmbH 11. The method according to one of the preceding claims, wherein the first mixture is incubated at room temperature.

12. The method according to one of the preceding claims, wherein the incubation time of the first mixture is 1 min to 60 min, preferably 5 to 50 min, particularly preferably 35 to 45 min.

13. The method according to one of the preceding claims, wherein the liquid phase is removed by pipetting.

14. The method according to one of the preceding claims, wherein the washing buffer is a phosphate buffer.

15. The method according to any one of the preceding claims, wherein triggering the color reaction comprises: f1. Addition of the reporter enzyme and mixing with the solid phase; and f2. Addition of the substrate.

16. The method according to one of the preceding claims, wherein the color reaction is determined using the Simoa technique.

17. Method for creating a calibration curve for Peripherin comprising i) determining Peripherin in five or more samples according to any one of claims 1 to 16, wherein the Peripherin concentration of each of the samples is known; ii) creating a calibration curve based on the Peripherin concentrations of the samples and the measured color reaction.

18. The method according to claim 17, wherein the color reaction is determined using the Simoa technique.

19. The method according to one of claims 17 or 18, wherein at least one of the samples has a peripheralin concentration in the range of 1 to 25 pg / mL. 40 P77550WO Laboratory Berlin - Charité Vivantes Services GmbH 20. The method according to any one of claims 17 to 19, wherein at least one of the samples has a peripheralin concentration in the range of 1,000 to 100,000 pg / mL.

21. Method for the quantitative determination of peripherin comprising a) qualitative determination of peripherin in a sample according to any one of claims 1 to 16; b) determination of a calibration curve according to any one of claims 17 to 20; c) quantification of the color reaction measured in step a) by comparison with the calibration curve determined in step b).

22. The method according to claim 21, wherein the measured color reaction is a fluorescence or chemiluminescence.

23. The method according to one of claims 21 or 22, wherein the color reaction and the calibration curve are determined using Simoa technique.

24. Kit for a heterogeneous immunoassay for the determination of peripherin comprising: i. a first antibody conjugate, wherein the first antibody conjugate comprises a solid-phase-bound first antibody for the detection of peripherin, and wherein the first antibody is not 8G2; ii. a second antibody conjugate, wherein the second antibody conjugate comprises a biotinylated second antibody, also for the detection of peripherin, and wherein the second antibody is not mAb A-3; iii. a reporter enzyme, wherein the reporter enzyme has a streptavidin tag and a binding site to the substrate, and wherein the reporter enzyme does not have a binding site to the first antibody and peripherin; and iv. a substrate, wherein the substrate has a binding site to the reporter enzyme and a dye tag, and wherein the substrate does not have a binding site to the first antibody, the second antibody, and peripherin.

25. The kit according to claim 24, further comprising a dilution buffer. P77550WO Laboratory Berlin - Charité Vivantes Services GmbH 26. The kit according to claim 25, wherein the dilution buffer is a lysis buffer, preferably an immunocomplex lysis buffer.

27. The kit according to one of claims 24 to 26, further comprising a calibration component, wherein the calibration component contains Peripherin in a known amount.

28. The kit according to any one of claims 24 to 27, wherein the first antibody and the second antibody are different.

29. The kit according to any one of claims 24 to 28, wherein the first antibody conjugate comprises paramagnetic particles to which the first antibodies are bound.

30. The kit according to any one of claims 24 to 29, wherein the particles have a size of 0.5 to 10 pm.

31. The kit according to any one of claims 24 to 30, wherein the first antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3, Polyclonal Peripherin (PRPH) Antibody (Order No. ABX320907-100UL, Rabbit Polyclonal, Non-biotinylated, N-Terminal, IgG, Immunogen 1-260, abbexa, Leiden Bio Science Park, Bargelaan 200, 2333 CW Leiden, Netherlands), and wherein the second antibody is selected from the group consisting of PRPH Monoclonal Antibody 3B3 and Peripherin Antibody 8G2.

32. The kit according to any one of claims 24 to 31, wherein the first antibody and the second antibody are different, and wherein the second antibody is 8G2.

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