RU2780565C2 - Method for determination of members of family of s100 calcium-binding proteins by means of immunoturbidimetry - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to turbidimetric immunoassay. A method for in vitro measurement of the presence of calprotectin in a biological sample from a patient, using turbidimetric immunoassay enhanced with latex particles, is disclosed, including stages of collection of predetermined amount of the specified biological sample; extraction of the specified biological sample in predetermined amount of an aqueous organic buffer, and homogenization of a matrix of the specified biological sample, followed by the removal of any material in the form of particles to obtain a sample solution with the presence of solubilized heterodimeric calprotectin; mixing of certain amount of the resulting solution with certain amount of a reagent containing nanoparticles having immobilized two or more monoclonal antibodies or their fragments specifically binding calprotectin to obtain a particle-bound antibody-antigen reaction for calprotectin, which is present as heterodimer S100A8 and S100A9; incubation of the mixture; obtaining optical properties of the mixture, and determination of a signal indicative for calprotectin content based on optical properties of the mixture; establishment of connection of the specified content to the calibrated control, and assessment of a clinical condition of the specified patient based on the measured presence of calprotectin in the specified biological sample. A test kit for implementation of the above-described method is also disclosed.

EFFECT: group of inventions provides the advantage that used reagents create an environment, which is physiologically similar to the environment, into which granulocytes release their physiological calprotectin.

12 cl, 6 dwg, 5 tbl, 4 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[001] The present invention relates to a turbidimetric method and test system for the quantitative determination of members of the S100 family of calcium-binding proteins in physiological fluids and feces. The test kit contains antibodies, buffers, and a kit of parts for use in the diagnosis of acute and chronic inflammatory diseases and conditions.

BACKGROUND OF THE INVENTION

[002] The term calprotectin refers to two granulocyte proteins with relative molecular weights of 36500 Daltons and isoelectric points at pH 6.3 and 6.5 (US 4833074; Fagerhol et al., Scand. J. Haematol. 24, 393-398 (1980 Fagerhol et al., Bull Europ Physiopath Resp. 16 ( suppl ), 273-281 (1980) Alternate names include the low molecular weight calcium binding proteins S100A8 and myeloid protein 14 (MRP14), migration inhibitory factor-related proteins 8/14 (MRP8–MRP14), S100a/b, calgranulin A/B, cystic fibrosis antigen (CFA), human leukocyte protein , leukocyte protein L1 complex, 60BB antigen , and antigen 27E10 Calprotectin is a proven marker of neutrophil activation because its concentration in body fluids and matrices is indicative of leukocyte turnover and tissue infiltration, thus it has become a widely used biomarker in clinical and chemistry for infections, acute and chronic inflammatory disorders and other diseases. Measurement of calprotectin can be used to distinguish bacterial infection from other causes, as well as to diagnose non-infectious systemic inflammation (Striz, I & Trebichavsky, Calprotectin – a pleiotropic molecule in acute and chronic inflammation, Physiological research/Academia Scientiarum Bohemoslovaca (2004) 53 ( 3): 245–53; Nilsen T et al, A new turbidimetric immunoassay for serum calprotectin for fully automatized clinical analysers , Journal of Inflammation (2015) 12:45; Johne B et al, Functional and clinical aspects of the myelomonocyte protein calprotectin . Mol Pathol 1997;50:113–23; Nielsen T et al, Serum calprotectin levels in elderly males and females without bacterial or viral infections , Clin Biochem 2014;47(12):1065–8). Calprotectin has also been proposed as a marker of cardiovascular disease (CVD) prior to the onset of CVD symptoms in asymptomatic subjects (cf. EP 1 573 335 B1).

[003] The simultaneous presence of various mono- and multimers of S100, in addition to the heterodimer, however, complicates the immunological determination of calprotectin in biological samples (blood, serum, synovial fluid, etc.) and matrices (faeces). The Ca ²⁺ and Zn ²⁺ binding properties of the S100A8/A9 proteins additionally have an important influence on their conformation and oligomerization. S100A8 and S100A9 tend to form tetramers in the presence of calcium or zinc ions, while the heterodimer appears to be the predominant form in the absence of calcium (cf. Grabarek Z, Structural basis for diversity of the EF–hand calcium–binding proteins , J Mol Biol (2006 ), 359: 509–25). Mixing purified S100A8 and S100A9 does not automatically form a calprotectin with properties like the heterodimer secreted by granulocytes. Experiments with mutant S100A8 and S100A9 suggest that homo- and hetero-oligomerization of S100A8 and S100A9 is also functionally and diagnostically important.

[004] Calprotectin is expressed in vivo during various stages of myeloid differentiation, in circulating neutrophils and monocytes, while absent in macrophages and normal tissue lymphocytes, and is upregulated in many types of malignant tumors, including malignant tumors of the stomach, esophagus, colon intestine, pancreas, bladder, ovary, thyroid, breast and skin, in non-degenerative disorders, as well as in inflammatory and autoimmune diseases and pathologies. Chronic inflammatory conditions such as psoriasis lead to expression in the epidermis. Calprotectin is found at high concentrations in localized sites of inflammation, as well as in the serum (blood) and faeces of patients with inflammatory diseases, rheumatoid arthritis, cystic fibrosis, inflammatory bowel disease, Crohn's disease, giant cell arteritis, Sjögren's syndrome, systemic lupus erythematosus, and progressive systemic disease. scleroderma. Calprotectin is also involved in the formation and accumulation of amyloids in the aging prostate, known as amyloid body inclusions.

[005] Various roles are attributed to calprotectin. It can induce neutrophil chemotaxis and increase bactericidal activity by stimulating phagocytosis and/or degranulation of neutrophils via a MAPK-dependent mechanism (Simard JC et al, Induction of neutrophil degranulation by S100A9 via a MAPK-dependent mechanism , J Leukoc Biol. (2010) 87( 5):905–14). Antimicrobial, oxidative-utilizing, and apoptosis-inducing activities, as well as pro-inflammatory activities (eg, recruitment of leukocytes) are additionally attributed to calprotectin , which appears to be an inflammatory enhancer in autoimmunity, as well as a stimulator of innate immune cells through their binding to pattern-recognizing receptors. , such as the Toll-like receptor 4 (TLR4) and the glycation end product receptor (AGER). Other activities include stimulation of cytokine and chemokine production, and regulation of leukocyte adhesion and migration. Binding to TLR4 and AGER activates the MAP-kinase and NF-kappa-B signaling pathways, leading to an increase in the pro-inflammatory cascade. The antimicrobial activity against bacteria and fungi appears to result from the binding of Zn ²⁺ and Mn ^{2+ ions} , which are essential for microbial growth. Transnitrosylase activity has also been found for calprotectin , and it has been suggested that the iNOS–S100A8/A9 transnitrosylase complex drives selective inflammatory stimulus-dependent S-nitrosylation of targets containing the [IL]–x–C–x–x–[DE] motif. Calprotectin can further act as an alarmin or a hazard-associated molecular pattern (DAMP) molecule.

[006]Correct and accurate measurementcalprotectin in biological samples and matrices is necessary if the results are to be interpreted for diagnostic purposes and patient care, in particular for monitoring the treatment of acute and chronic inflammatory disorders using biological agents. In particular, the results should be comparable if the pooled values of diagnostic findings and results of clinical trials are to be used. Metrological problem, taking into account the various states of oligomerizationcalprotectin, can be divided into traceability of measured values, measurement inaccuracy and their commutativity. When traceability is not achievable, comparability of results must be realized in other ways, since measurements are the main activity of clinical laboratories. The goal is for patient outcomes to be traceable to the highest possible reference value to improve the quality of diagnostic results.

[007] Conventional methods for measuring calprotectin use polyclonal antibodies, either in free form or bound to a solid phase (cf. WO2012/175616, WO2013/132347, WO2014/037588, US20170108507). However, parallel determination by immunoassay (e.g., ELISA) of purified calprotectin added to sample solutions using UV/visible light, biuret, Bradford reagent, or Coomassie blue does not provide definitive answers to the standard problem, as calprotectin may take the form of dimers (S100A8/A9), tetramers (S100A8/A9) ₂ or even oligomers (T. Vogl et al, Poster: Towards a Reference Material for the Standardization of Calprotectin (S100A8/Ag; MRP8/14) Immunoassay, presented at FOCUS (Association for Clinical Biochemistry and Laboratory Medicine), May 3–5. 2017, Leeds, UK). The use of mutant proteins S100A8 and S100A9 having at least one mutation in the region of high or low affinity calcium binding has been proposed, since these recombinant proteins are no longer capable of oligomerization (WO 2016/116881).

[008] Turbidimetric immunoassays provide the advantages of simple methods and an automated analyzer. In the latex particle-enhanced turbidimetric immunoassay (PETIA), target-specific antibodies are bound to the particles ("sensitized particles") in such a way that the antibody-antigen reaction results in particle agglutination, which can be measured by spectrometry (cf. EP 0 061 857; EP1 205 755 B1; and references in this document). One type of particulate reagent is sufficient when two or more antibodies recognize the target antigen by bridging two or more latex particles (Methods in Enzymology (1981) 74 , 106–139, Academic Press, New York; EP 1 739 430 B1; EP1 573 335 B1). Non-specific reactions between the particulate reagent and the sample can also lead to agglutination and increased turbidity (cf. JP 11 023 573 A, JP 58 144 748 A, EP 1 205 755 B1). Safe and complete inhibition of non-specific reactions is particularly problematic if polyclonal antibodies coupled to a particulate reagent are used, or if the target is isolated from a complex or heterogeneous matrix like faeces (cf. EP 0 038 181 B1). A mutant calprotectin standard ( mut S100A8/A9) is of little use in measuring the presence of endogenous calprotectin in human matrices and samples, eg obtained from appropriate patients, or by means of an agglutination method. Currently, calprotectin is measured in biological samples using latex particle-enhanced turbidimetric immunoassay ( PETIA ) where immobilized polyclonal antibodies bind to multiple epitopes on calprotectin in such a way that any type of protein S100 could lead to agglutination, regardless of whether it is in a state of oligomerization secreted by granulocytes or not. This results in cut-off values that are not suitable for diagnosis, measurement inaccuracies, and undermines commutativity, in particular since polyclonal antisera are prone to the formation of non-specific agglutination.

[009] The observed scatter in method comparisons between ELISA based on monoclonal antibodies and PETIA based on agglutination using polyclonal antibodies, as well as the lack of commutativity of diagnostic cut-off values, are additional reasons for concern. While monoclonal antibodies are easier to standardize and allow quality control to a defined standard throughout the life of the product, there is the combined problem of obtaining an agglutination test, measurement sensitivity, and specificity for calprotectin in the form secreted by granulocytes . The prior art for the determination of calprotectin in biological samples and matrices thus presents a challenge.

SUMMARY OF THE INVENTION

[0010] The solution to these problems is achieved by the in vitro method as described in claim 1. Preferred embodiments of the method are defined in dependent claims 1-14. Another aspect of the invention relates to a kit, for example, for use in a turbidimetric or nephelometric immunoassay, containing two reaction components as defined in claim 15.

[0011] Accordingly, one objective is to provide an in vitro method for measuring the presence of calprotectin in a biological sample from a patient, comprising the steps of: a ) collecting a predetermined amount of said biological sample; b) solubilizing and extracting said biological sample in a predetermined amount of an aqueous organic buffer having i) a pH between 5.0 and 6.0, ii) an osmolality of at least 150 mosmol/kg H ₂ O, iii) 0.01-0, 1% weight percent anionic surfactant, iv) wherein the organic buffer molecules can be coordinated using calcium and zinc ions, and iv) and optionally homogenizing and extracting the matrix of said biological sample followed by removal of any particulate material to obtain sample solution with defined presence of solubilized calprotectin (S100A8/A9) in the form secreted by granulocytes (with intact lysosomal compartments); c) mixing a certain amount of a solution of the specified sample from step (b) with a certain amount of a reagent to obtain a mixture containing nanoparticles having immobilized monoclonal antibodies or fragments thereof that specifically bind any of S100A8 and S100A9 or calprotectin (S100A8/A9), and the reactions associated with antibody-antigen particles for calprotectin (S100A8/A9); d) incubating the mixture from step c) for a period of time; and e) obtaining the optical properties of the mixture and determining a signal indicative of the content of calprotectin (S100A8/A9) based on the optical properties of the mixture; f) associating said content with a calibrated control and assessing the clinical condition of said patient based on the measured presence of calprotectin (S100A8/A9) in said biological sample. If calprotectin is determined as described above, the amount may be metrologically traceable to the measurement of a calprotectin standard isolated from granulocytes with intact lysosomal compartments. The original methods for the determination of calprotectin used polyclonal monospecific antibodies, since the structure, amount and concentration of calcium-binding proteins of the S100 family are variable.

[0012] The stage of obtaining optical properties includes determining the value of optical density, light transmission, reflection, light scattering, fluorescence or scintillation. Turbidimetric or nephelometric measurements are preferred, and the latex particle enhanced turbidimetric immunoassay (PETIA) is most preferred, where steps b) and c) involve the use of two component reagents.

[0013] Nanoparticles preferably have diameters from 150 to 350 nm to increase sensitivity. More preferred is PETIA, where the antibodies are coupled to two types of particles having homogeneous diameters in the range of i) 150 to 200 nm and ii) 250 to 350 nm to increase the measurement range. Said nanoparticles can be made from carboxylated polystyrene or chloromethyl-activated polystyrene.

[0014] The particle-bound antibody-antigen reaction is preferably carried out in a mixture having a pH between 5.0 and 6.0 and containing an anionic surfactant or sodium dodecyl sulfate and coordinated Ca ²⁺ buffer molecules.

[0015] The biological sample may be a feces or, more specifically, a fecal extract, since the determination of fecal calprotectin has become an integral component of the laboratory study of inflammatory bowel disease. Monitoring of a fecal inflammation parameter and a marker of granulocyte neutrophil activity such as calprotectin facilitates early recognition of a relapse outbreak after remission. Other preferred biological samples are blood, serum or plasma and urine, for example, for diagnosing and distinguishing between prerenal and intrarenal kidney disease, or other inflammatory diseases of the cardiovascular system.

[0016] The buffer composition is preferably composed of at least one salt selected from the group consisting of salts of polycarboxylic acids, tricarboxylic acids, aconitic acids, tricarboxylic acids, dicarboxylic acids, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, alpha-, beta- and gamma-hydroxycarboxylic acids, hydroxydicarboxylic acids, malic acid, citric acid, tartaric acids, malonic acid, gluconic acid, 5-ketogluconic acid, 2-ketogluconic acid, dihydroxymaleic acid, maleic acid, fumaric acid , nitrilotriacetic acid, lactic acid and/or ascorbic acid. The calcium sequestration buffer from step b) preferably contains at least one citrate, acetate or maleate salt, protease-treated serum albumin, and 0.01-0.1 percent by weight anionic surfactants. The addition or presence of a non-specific IgM antiserum in the reaction components may be necessary to initiate and accelerate the agglutination reaction.

[0017] Another object is to provide a test kit for measuring the presence of calprotectin in a biological sample by means of a latex particle enhanced turbidimetric immunoassay as described. The test set may contain

the first reagent component containing

– 20–1000 mmol/l organic buffer with pH in the range from 5.0 to 6.0;

- 50-300 mmol / l sodium, potassium or lithium salts;

- 0.1-1.5% protease-treated serum albumin;

- 0.01-0.1% (w/v) sodium dodecyl sulfate, and

optionally, beta-aldoses, triose, tetroses, pentoses, hexoses, glucan, dextran and/or sugar to achieve an osmolality of at least 200 mosmol/l; and

the second reagent is a component containing

- 0.01-0.5% (w/v) latex particles 150 to 350 nm in diameter bearing immobilized monoclonal antibodies that bind any of S100A8 and S100A9 or calprotectin (S100A8/A9).

[0018] Buffer reagents in said reaction components for sequestering calcium and zinc ions may contain at least one organic acid salt selected from the group consisting of polycarboxylic acids, tricarboxylic acids, aconitic acids, tricarballylic acids, dicarboxylic acids, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, alpha-, beta- and gamma-hydroxycarboxylic acids, hydroxydicarboxylic acids, malic acid, citric acid, tartaric acids, malonic acid, gluconic acid, 5-ketogluconic acid, 2- ketogluconic acid, dihydroxymaleic acid, maleic acid, fumaric acid, nitrilotriacetic acid, lactic acid, ascorbic acid. Such organic acids can be coordinated in water with calcium and zinc ions, in particular when used in combination.

[0019] Without intending to be bound by any theory, the acidic pH of 5.0 and 6.0, and the coordination of buffer reagents by calcium ions, appears to create conditions specific to calprotectin a as it is present in neutrophil granulocytes, where proteins S100A8 and S100A9 have pKIs of 6.3 and 6.5, and strongly bind calcium via bidentate, unidentate, and pseudobridges. The presence of a small amount of an anionic surfactant, such as sodium dodecyl sulfate, may be necessary to keep proteins in solution while not inhibiting the reaction between particle-bound antibodies and the target. The composition of the buffer, in the context of the invention, is, in terms of pH and osmolality, similar to the cellular environment in which calprotectin is present in granulocytes. The granulocyte has an internal pH between 5.0 and 6.0, and the concentration of calcium ions is 100-1000 times lower than in the environment.

[0020] A preferred aspect of the description relates to a kit with two reaction components capable of being determined by turbidimetry. This may be a latex particle-enhanced turbidimetric immunoassay (PETIA) comprising two component reagents to support steps b) and c). The most preferred embodiment relates to the determination of fecal calprotectin together with a device for transferring a certain amount of feces into a buffer for dilution and extraction of calprotectin from the fecal matrix, for example, as described in DE10 2012 109 457 B4, DE10 2008 057 866 B4 US5246669 A, JP-H10 –300 642 A, DE10 2007 07 760 B3.

[0021] Additional objects, features, and advantages are apparent from a consideration of the drawings and the following description of representative examples, which are provided by way of illustration and not limitation, as potential modifications may also be considered by one skilled in the art. The scope of the description is defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In the drawings: -

1 is a graph showing a calibration curve for a turbidimetric immunoassay using a mouse monoclonal antibody against calprotectin (S100 A8/A9) covalently bound to latex particles having a diameter of 180 nm;

Figure 2 shows a calibration curve for a turbidimetric assay using a mouse monoclonal antibody against calprotectin and the prozone limit of the immunoassay;

Figure 3 is a graph showing the correlation of a turbidimetric immunoassay using one monoclonal antibody and a conventional ELISA using two monoclonal antibodies as binding and detecting antibodies to determine standard calprotectin from granulocytes with intact acidic compartments;

Figure 4 is a graph showing the correlation of a turbidimetric assay using two monoclonal antibodies and a conventional ELISA assay for standard calprotectin isolated from granulocytes with intact lysosomal membranes.

Figure 5 is a graph showing the measurement correlation of a turbidimetric assay for the determination of calprotectin according to the invention using two monoclonal antibodies and one monoclonal antibody.

Figure 6 is a graph showing the measurement correlation of the described assay using two types of particles of different sizes and a conventional ELISA assay for the determination of calprotectin.

DETAILED DESCRIPTION OF THE INVENTION

[0023] In the past, it has been difficult to determine a specific diagnostic threshold for fecal calprotectin, and marked interassay differences exist between different commercial assays due to different extraction methods and buffers (Sipponen T, Kolho KL. Faecal calprotectin in children with clinically quiescent inflammatory bowel disease Scand J Gastroenterol 2010, 45 :872–877 Gisbert JP, Bermejo F, Perez–Calle JL, et al Fecal calprotectin and lactoferrin for the prediction of inflammatory bowel disease relapse Inflamm Bowel Dis 2009 15 :1190 -1198; Walkiewicz D, Werlin SL, Fish D, et al. Fecal calprotectin is useful in predicting disease relapse in pediatric inflammatory bowel disease . Inflamm Bowel Dis. 2008, 14 : 669–673; D'Inca R, Dal Pont E, Di Leo V, et al Can calprotectin predict relapse risk in inflammatory bowel disease Am J Gastroenterol 2008 103 :2007–2014 Tibble JA, Sigthorsson G, Bridger S, et al Surrogate markers of intestinal inflammation are predicti ve of relapse in patients with inflammatory bowel disease . gastroenterology. 2000;119:15–22). The described method provides the advantage that the extraction and measurement buffers provide an environment similar to that found in the destruction of granulocytes, but keeping the lysosomal membranes and acidic compartments intact. In other words, the environment is physiologically similar to the environment into which granulocytes release their physiological calprotectin.

[0024] The buffer used in step b) may contain at least one citrate, acetate, or maleate salt, protease-treated serum albumin, and 0.01-0.1 percent by weight of one or more anionic surfactants. To improve and accelerate agglutination, one or more of the reaction components may additionally contain an agglutination facilitator, such as non-specific IgM, rheumatoid factor (RF). Although RF can exist in the form of IgG, IgM, IgA, and IgE isotypes, the IgM class of RF is the preferred clinical measurement of calprotectin.

[0025] The second reagent component may contain one or more anti-calprotectin monoclonal antibodies immobilized on the nanoparticles. At least two different monoclonal antibodies are preferred for general safety reasons. If only one monoclonal antibody is used, there is always the risk of an atypical immune response and false diagnosis, which can be reduced by using a second specific anti-calprotectin monoclonal antibody. In addition, the agglutination reaction is more reliable with two specific antibodies because they have different epitopes and binding determinants.

[0026] Nanoparticles can have diameters from 150 to 350 nm to improve sensitivity. Nanoparticles with a homogeneous diameter are preferred. To increase the measurement range, it may be preferable to use anti-calprotectin monoclonal antibodies immobilized on two types of particles having diameters in the ranges i) from 150 to 200 nm and ii) from 250 to 350 nm. Said particles of one or more sizes may be particles of carboxylated polystyrene or chloromethyl-activated polystyrene.

[0027] Another aspect relates to a kit of two component reagents for measuring calprotectin in a physiological fluid or sample by latex particle enhanced turbidimetric immunoassay. The first component reagent can be an extraction buffer, as described above, and/or containing 20-1000 mmol/l salt from a buffer reagent, as described, with a pH in the range from 5.0 to 6.0; 50–300 mmol/l of sodium, potassium or lithium ions; 0.1-1.5% protease-treated serum albumin; 0.01-0.1% (w/v) anionic detergent, preferably sodium dodecyl sulfate, and optionally beta-aldoses, triose, tetroses, pentoses, hexoses, glucan, dextran and/or sugar to achieve an osmolality of at least measure 200 mosmol/l. The second component reagent may contain 0.01-0.5% (w/v) latex particles 150 to 350 nm in diameter bearing immobilized anti-human calprotectin monoclonal antibodies that bind any of S100A8 and S100A9 or calprotectin (S100A8/ A9) at pH between 5.0 and 6.0.

[0028] A method for measuring the presence of calprotectin in a biological sample from a patient, comprising the steps of: (a) collecting a predetermined amount of said biological sample, which may be feces, serum, or synovial fluid; b) solubilizing and extracting said biological sample in a predetermined amount of an organic buffer having i) a pH between 5.0 and 6.0, ii) an osmolality of at least 150 mosmol/kg H ₂ O, iii) where the organic acid can coordinate or sequester zinc and calcium ions, and iv) 0.01-0.1% weight percent anionic surfactant and optionally homogenizing and extracting said biological sample matrix followed by removal of any particulate material to obtain a sample solution containing calprotectin (S100A8/A9); c) mixing a certain amount of a solution of said sample from step (b) with a certain amount of a particulate reagent to obtain a mixture having i) a pH between 5.0 and 6.0, ii) an osmolality of at least 150 mosmol/kg H ₂ O, iii) organic salts and acids sequestering calcium and zinc ions, and iv) containing nanoparticles having diameters of at least 150-350 nm and immobilized on them monoclonal antibodies or their fragments that specifically bind any of the proteins S100A8 and S100A9 or calprotectin (S100A8 /A9); d) incubating the mixture from step c) for a first time interval; and e) obtaining the optical properties of said mixture and determining a signal indicative of the content of calprotectin (S100As/A9) based on the optical properties of said mixture; f) associating said content with a calibrated control from calprotectin in the same buffer solution and assessing the clinical condition of said patient based on the measured presence of calprotectin (S100A8/A9) in said biological sample.

[0029] Another aspect relates to a set of reagents for immunological latex turbidimetry for automated analysis, as well as the use of these reagents. Said embodiment may be a two-reagent system consisting of a first component reagent containing a buffer to stabilize the dimeric form of calprotectin in said sample solution from step b) and a second component reagent containing particles carrying immobilized one or more monoclonal antibodies against S100A8 and S100A9 or calprotectin (S100A8/A9). The method described may include transferring or diluting the biological sample extract in the first component reagent, optionally after removing any solid material present. The first component reagent may contain protease-treated bovine or human serum albumin, preferably in an amount of 0.1-1.5%, in an organic buffer system having a pH in the range of 5.0 to 6.0 and containing organic buffer reagents, sequestering calcium and zinc ions.

[0030] As mentioned, the nanoparticles used can be carboxylated polystyrene particles with diameters ranging from 150 to 350 nm, preferably from 150 to 200 nm. In the most preferred embodiment, said carboxylated polystyrene nanoparticles have diameters ranging from 160 to 180 nm. In the most preferred embodiment, the nanoparticles are substantially uniform in size, preferably with diameters ranging from 160 to 180 nm. This particle size allows improved presentation of the antibody molecules and minimizes detection difficulties under antigen-excess conditions. Since such nanoparticles have a large interaction surface with the antigen, fewer antibody-coated particles are needed, and the antibody-antigen response becomes comparable to a surface-mediated immune response, which is generally faster. Such large particles are also preferred for better metrologically traceable association of measured values with previous measurements of calprotectin using immunosorbent assay (eg ELISA) and for their commutativity. Traceability, reliability and commutativity cannot be achieved when the antibody-antigen reaction is 3D in liquid and is not surface dependent. Therefore, the authors of the present invention believe that the use of larger particles than usual improves the metrological traceability of the results. Comparability of results must be realized and represents the main activity of clinical laboratories.

[0031] Nanoparticle sizes are preferred that are suitable for the specific two-dimensional reaction between said monoclonal antibodies and the substantially heterodimeric calprotectin, and are optimized such that fewer antibodies are needed. A particle-enhanced immune response is stimulated, while steric effects seem to reduce the contribution of the calprotectin structure. The increase in particle size is not associated with a high degree of spontaneous haze, if it is in this range of particle sizes.

[0032] In a preferred embodiment, these nanoparticles can be of two different sizes, having diameters i) from 250 to 350 nm and ii) from 160 to 250 nm. The combination of immunosensitized particles of different sizes can further increase the accuracy as well as the measurement range.

[0033] Calprotectin is expressed by granulocytes, a subgroup of white blood cells, and is stored in cytoplasmic granules, probably some form of endoplasmic reticulum compartment and trans-Golgi network, before being released or secreted, but this is still under investigation. However, granulocytes get their name from the presence of granules in their cytoplasm. Granulocytes are also called polymorphonuclear leukocytes in accordance with the different forms of their nuclei. The term polymorphonuclear leukocyte usually refers to "neutrophilic granulocytes", which make up approximately 95% of granulocytes. Other granulocytes are subdivided into eosinophilic and basophilic granulocytes, and mast cells having smaller numbers. Granulocytes are formed by granulopoiesis in the bone marrow and remain in the bloodstream for approximately 6 hours, where they carry out their biological functions. The levels of calcium ions in the cell cytosol are kept relatively constant, being 100,000 times lower inside the cell than outside the cell. It is well known that an increase in the amount of calcium ions inside the cell can lead to important cellular changes, so the levels of calcium ions and intracellular calcium are called second messengers. It is vital for the function of granulocytes and their calcium-binding proteins, such as calprotectin, to keep intracellular calcium ion levels under tight control. Strict control of the calcium environment available for binding by calprotectin thus seems to be critical for its function and structure, and for the reproducible determination of biologically effective calprotectin in physiological fluids (serum, synovial fluid, etc.) as well as in biological matrices. such as tissue and feces.

[0034] On the other hand, the use of phosphates, such as sodium tripolyphosphate (STP), in detergent formulations can lead to precipitation if calcium is present. At low pH, S100A8 and S100A9 are strong calcium binders and have isoelectric points at pH 6.3 and pH 6.5. Therefore, these proteins are already bound to the calcium ion, and any additional calcium present in biological samples (serum, faeces, synovial fluid, etc.) may interfere with their quantification. Without intending to be bound by theory, it seems beneficial to inhibit additional calcium binding and oligomerization by changing the pH below the isoelectric points, more specifically below pH 6.0, and such a mild sequestering agent is necessary to avoid calcium precipitation. This can be achieved using citrate, maleate or malic buffers. Alternatively, polymers and copolymers of acrylate and maleate can be added to inhibit precipitation and calcium ion-induced agglutination. The formation of complexes with calcium depends on the pH of the solution. In the case of citric acid, the concentration of uncomplexed calcium remains low and constant above pH 6.5, when complete deprotonation of the three carboxylic groups is effective. In the case of malic and lactic acids, and at a pH higher than the latter's deprotonation constant, the free calcium concentration is more important and fluctuating. A number of polycarboxylic acids containing acetal functional groups were investigated and their behavior in relation to calcium sequestration was compared. Calcium sequestration by oxidized carbohydrates is generally less than by the corresponding polycarboxylate esters.

[0035] When calprotectin is determined in faeces, the amount of calcium to bind to calprotectin depends on calcium levels in the gastrointestinal (GI) tract and is highly variable between samples. Most calcium salts, in fact natural sources of calcium in supplements and food, have a pH-dependent solubility. The solubility of the four predominant calcium salts (calcium oxalate hydrate, calcium citrate tetrahydrate, calcium phosphate, calcium glycerophosphate) increases in each case with increasing pH. Therefore, low pH is preferred for extraction and measurement because any free calcium can lead to the formation of atypical tetramers or oligomers of the low molecular weight calcium binding proteins S100A8 and S100A9.

[0036] The second reagent component may be a suspension of latex particles bearing the immobilized antibody. One embodiment of an immunological latex turbidimetry reagent may be a first component reagent containing 0.1 to 1.5% protease-treated human serum albumin, 20-1000 mmol/L citrate buffer, pH 5.0, 50-300 mmol /l sodium chloride and 0.1% (w/v) sodium dodecyl sulfate. The second component reagent may contain 20-1000 mmol/l citrate buffer, pH 5.0; 50–300 mmol/L sodium chloride and 0.01–0.5% (w/v) latex particles 150 to 350 nm in diameter bearing immobilized monoclonal antibodies that bind any of S100A8 and S100A9 or the calprotectin heterodimer (S100A8 /A9).

[0037] Such component reagents provide high sensitivity and specificity for calprotectin (S100A8/A9) and improved diagnostic reliability in a latex particle-enhanced turbidimetric immunoassay using monoclonal antibodies against calprotectin (MRP 8/14, S100A8/A9), for example , commercially available from Immundiagnostik AG, Bensheim, DE (Art. K6927, K6936). More specifically, diagnostic results using such a homogeneous immunoassay are comparable, even more accurate, than established ELISA methods and point-of-care immunochromatographic tests. A diagnostic cut-off value of 50 micrograms/g faeces can be set for the presence of inflammation. The latex-enhanced turbidimetric immunoassay uses, compared to the prior art, standardized monoclonal antibodies against the calprotectin heterodimer (S100A8/A9) immobilized on large nanoparticles having diameters ranging from 150 to 350 nm.

[0038]Because the calprotectin occurs predominantly in physiological fluids and faeces in the form of a heterodimer, agglutination should not destroy these complexes. It seems that large nanoparticles in the range of 150 to 350 nm make the agglutination kinetically dependent on the surface ("two-dimensional"), so that the turbidity becomes homogeneous and has a metrologically traceable relationship with the concentration of calprotectin (S100A8/A9), as determined by standard ELISA. Thus, diagnostic results and assessments are commutative, which is necessary for the work of the clinical laboratory. Therefore, the set diagnostic cut-off values can thus be used.

[0039] Briefly, the diagnostic results are comparable and commutative with the results of the developed ELISA using monoclonal antibodies, while the turbidimetric immunoassay is not only a homogeneous analysis. The amount of calprotectin (S100A8/A9) present in a sample can be determined by determining the agglutination or opacity/haze of the sample compared to the standard. The analysis can thus be carried out in any commercial automatic analyzer such as Hitachi or Cobas®. The use of recombinant antibodies and fragments is preferred, given the required amounts of antibodies.

[0040] Stabilization of calprotectin during and after extraction from faeces can be achieved with Ca ²⁺ in extraction and reaction buffers. However, biological samples (serum, blood, synovial fluid or faeces) contain endogenous calcium. The use of a buffer that gently chelates calcium ions is preferred. Fecal calprotectin (S100A8/A9) is only protected against proteases when it has bound calcium ions, while an excess of calcium ions leads to the formation of tetramers and oligomers. It appears that an acidic calcium sequestering buffer can stabilize and maintain the calprotectin heterodimer (S100A/A9). The acidic stabilizing buffer may have a pH between 5.0 and 6.0, and up to 0.5%, preferably 0.1%, ionic and anionic surfactant. The ionic surfactant may be SDS and the non-ionic surfactant may be Tween®20. pH between 5.0 and 6.0 and ionic strength above 150 mOsm/l are also observed in granulocyte granules, so that the conditions for solubilization and measurement are similar to those in secretory granules.

[0041] In the following; some terms, as used in the description, are further explained and defined:

[0042] The term "body sample" or "body fluid " describes material from a human or animal, including blood, serum, plasma, feces, urine, saliva, excreta, body and tissue fluids. Body samples are also extracts of material and specimens for diagnostic testing or evaluation and identification of a medical condition. The sample may be a liquid or a solid. In the case of a solid or semi-solid matrix (faecal sample), the sample must be subjected to extraction. Any commercial faecal sample extraction kit can be used, such as the Sample Obtaining System (SSPS) pre-filled with IDK Extract® (Immundiagnostik AG, Bensheim, DE), or it can be done manually using the first reaction component as described herein. . The amount needed to perform a turbidimetric test is in the range of 5–20 mg of fecal extract. The sample may be urine for examination of diseases of the intestinal tract, liver, gallbladder, pancreas or kidney. The sample may be blood, plasma or serum for cardiovascular disease testing.

[0043] Calibrators are (off-the-shelf) solutions with known analyte (calprotectin) concentrations and equivalence required for accurate laboratory performance. To interpret the signal strength and thus determine the presence or concentration of the analyte (calprotectin) in the sample, the result for the sample from the organism is compared with the results obtained for the serial dilution of the calibrator.

[0044] The limit of detection is defined as the lowest amount of analyte that can be reliably detected in an assay in which the total error is in line with accuracy requirements. According to the method described, a detection limit of ≤ 10 micrograms calprotectin/gram feces can be obtained, which is reasonably below the cut-off threshold (50 micrograms calprotectin/gram feces) for diagnosing inflammation in feces.

[0045] Turbidimetry is the measurement of the loss of transmitted light intensity due to the effect of scattering by particles suspended in a solution. Light of a known wavelength (nm) is passed through a cuvette containing a solution of particles and collected in a photovoltaic cell. The measured value (mU) gives the amount of absorbed light. Two types of turbidimetric assays are currently in use, direct and latex particle-enhanced immunoturbidimetry (PETIA). In direct immunoturbidimetry, antibodies form an immune complex by interacting directly with their respective antigens. The particle-enhanced immunoturbidimetric method is based on coating nanoparticles with antibodies, in the present application, with two mouse monoclonal antibodies against calprotectin. Particle-enhanced immunoturbidimetric assays are useful when the analyte is present at low concentrations because particles enhance the signal and provide increased sensitivity.

[0046]As a rule, they use nanoparticles, whose average diameter is usually measured in nanometers (nm). The size (diameters) of nanoparticles can be reliably determined by dynamic light scattering (DLS), photon correlation spectroscopy (PCS), or quasi-elastic light scattering (QELS). Diameters, within the scope of the invention, are measured using a Malvern Zetasizer Nano (Malvern Panalaytical Ltd., Malvern, GB) or Horiba SZ-100, where the Gaussian deviation from this mean is less than 10 nm. Since the surface of the nanoparticle is conjugated with biomolecules (HAS) and, of course, immunoglobulins (Ig) and monoclonal antibodies, the diameters must be accurately determined and controlled. The large surface area to volume ratio of the nanoparticle-Ig bioconjugate provides advantages for the interaction of immunoglobulins with target biomolecules. The preparation of antibody-bearing nanoparticles is well known in the art. The antibodies may be absorbed by a suitable coating on the particles, or may be chemically bonded to them via a bridging agent. The antibodies can also be bound directly to the polymer on the latex particle itself. These nanoparticles can be stored before use in a buffer containing tribasic sodium citrate dihydrate, bovine serum albumin, Tween 20, sucrose, sodium azide (storage buffer). When using the storage buffer of the invention, anti-calprotectin monoclonal antibody latex immunoparticles are highly stable in suspension, i. without spontaneous aggregation, for periods up to 12 months at 2–8°C prior to immunoassay, while maintaining antibody stability

[0047] Calprotectin is released by granulocytes and, prior to release, is stored within cellular granules, more specifically, probably in non-lysosome trans-Golgi compartments. Calcium ion levels in the cytosol and within these acidic compartments (pH 5.0–6.0) are kept relatively constant, where the calcium ion concentration is 100,000 times lower than outside the cell. It is vital for the function of granulocytes, as well as other cells, that the levels of intracellular calcium ions be kept under strict control. Strict control of calcium ions available for calprotectin binding and pH is likely to be critical, not only for its function, but also for the metrologically traceable determination of calprotectin in body samples. Therefore, it is necessary to use anti-calprotectin monoclonal antibodies that bind at low pH. Thus, there is a need to stabilize the soluble form of calprotectin during extraction from the matrix sample and in the assay solution where the immunoreaction and subsequent agglutination occurs. This can be achieved by adding up to 0.5%, preferably 0.1% ionic and non-ionic surfactant. The preferred anionic surfactant is SDS, while the prior art seems to have worked predominantly with nonionic surfactants such as Tween®20. A pH between 5.0 and 6.0 is used for extraction and this pH is also observed in acidic compartments and granulocyte granules.

[0048] To reduce non-specific immunoreactions, the sample can be dissolved in a buffer solution containing antiserum against IgM. The presence of anti-IgM antisera can contribute to correcting the effect of IgM on the immunoassay so that uniform results can be obtained regardless of sample origin. Anti-IgM antiserum may contain endogenous calprotectin, so that each batch of anti-IgM antiserum requires, as appropriate, correction for serum IgM-associated calprotectin content.

[0049] The present invention further relates to a test kit for the quantitative determination of calprotectin. The kit may contain homogeneous nanoparticles coated with at least one monoclonal antibody against calprotectin or fragments of this antibody, binding at a pH between 5.0 and 6.0, where these nanoparticles have diameters in the range from 160 to 180 nm. To increase the sensitivity and range of measurements, the kit may contain a second type of homogeneous nanoparticles having other homogeneous diameters. The present invention also relates to a method for the quantitative detection of calprotectin in faeces, comprising steps a) extracting a certain amount of feces using the first reaction component to solubilize calprotectin and obtain a liquid sample, where the first reaction component has a pH of 5.0-6.0 and an osmolality of at least measure 150 mOsm/l; b) bringing said liquid sample into contact with nanoparticles having immobilized at least one anti-calprotectin monoclonal antibody (S100A8/A9) binding at pH 5.0-6.0; and c) assessing the amount of calprotectin in the sample by turbidimetry, where the diameters of these nanoparticles lie in the range from 160 to 350 nm. In a preferred embodiment, said nanoparticles may be carboxylated polystyrene particles, and for the sake of sensitivity and detection range, two types of homogeneous sized nanoparticles may be present, the homogeneous diameters of which lie in the range i) from 150 to 250 nm and ii) from 250 to 350 nm. The latex particles can be particles of organic high molecular weight materials, for example, latex particles of polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate or styrene-styrene sulfate copolymer.

[0050] Such a test and method can be adapted for automated analyzers in clinical laboratories. The method is characterized by reduced non-specific agglutination due to the use of monoclonal antibodies that are not specific to the lot and bind to only one single epitope. Conventional turbidimetric assays use avian polyclonal antibodies (chicken IgY against the MRP8/MRP14 heterodimer), which lack spontaneous aggregation in the turbidimetric assay. In addition, the prior art uses a reaction buffer at physiological pH (3-(N-morpholino)propane sulfone (MOPS) buffer pH 7.2) that does not interfere with the aggregation of S100A9 and S100A9 proteins into homo- and heteromultimers (Nilsen T et al, A new turbidimetric immunoassay for serum calprotectin for fully automized clinical analysers , J. Inflammation, 2015, 12:45). The same can be observed when calprotectin is diluted in buffer at physiological pH 8 R1: pH 7.2; R2, pH 8.1) as used in the Bühlmann fCal® turbo using the same avian polyclonal antibodies. However, the turbidimetric method and reaction determine the amount of target molecules (complex) in solution, and not the amount of protein (amino acids), as determined for the calibration method using biuret. Therefore, conventional turbidimetric methods for the determination of calprotectin using high pH and polyclonal antibodies can give metrologically non-traceable results. Commutability of results between conventional methods can only be achieved by means of correction factors (backdating), which is not available in the daily practice of the clinical laboratory. In addition, lot variability occurs between different lots of antibodies, and purification using different lots of calprotectin antigen in different lots is critical.

[0051] In one embodiment of the invention, the nanoparticles may be latex particles. In a preferred embodiment, the latex nanoparticles are carboxylated polystyrene particles with a particle size of 150 to 250 nm (250 to 350 nm), a surface charge density of 40 to 60 μC/cm ² ; surface charge density: 150–250 µEq/g, solids content 10.0 (%); stabilized with 0.05% sodium azide.

[0052] The turbidimetric method of the invention does not require a specially designed analyzer as it has a flexible applicability to conventional photometric analyzers. In accordance with the present invention, the additional cost of purchasing a specially designed device or consumables is no longer necessary. The method of the present invention facilitates fully automated processing, without time-consuming sample splitting, thus providing higher sample throughput and increased clinical laboratory efficiency. Based on the present disclosure, accurate quantification, metrological traceability, commutativity of results, and differential diagnosis based on a cut-off value (eg, 50 micrograms calprotectin/gram faeces) are feasible. This is necessary for therapeutic monitoring of patients. Automation and standardization are applicable and preferred options because manual testing involves a high risk of contamination for the user and the analyzed sample.

[0053] One object of the present invention is to provide a particle-enhanced turbidimetric immunoassay based on mammalian anti-human calprotectin monoclonal antibodies that overcomes the disadvantage of reduced assay sensitivity that avian antibodies typically experience. Another purpose is assay for the determination of calprotectin, which may be applicable to any standard chemical analyzer, regardless of manufacturer. Another aspect relates to a buffer for storing latex particles with immobilized antibodies, eliminating the natural tendency of latex particles to spontaneously aggregate and precipitate upon storage. The reaction buffer described can also be used as a storage buffer.

[0054] White blood cell count and/or measurement of C-reactive protein (CRP) is currently used to diagnose a bacterial infection requiring antibiotic treatment. It is estimated that approximately 40% of such cases are misclassified as bacterial infections and other causes (XuS et al, Lipocalins as biochemical markers of disease , Biochim Biophys Acto 2000, 1482:298–307). The neutrophil activation marker HNL/NGAL may distinguish between acute bacterial and viral infections, but is not a useful marker due to its very low concentration in body samples. The calprotectin assay of the present invention reflects granulocyte activation and is shown to be elevated in inflammatory conditions. However, the human calprotectin molecule is described as a 24 kDa heterodimer containing the S100A8 (MRP8) and S100A9 (MRP14) subunits, but there is no consensus on the actual structure of calprotectin in vivo , while measurement by turbidimetry requires a specific standard and molecules. Calprotectin can, according to the authors of the studies, be found in the form of a heterodimer, heterotrimer, or even heterotetramer when calcium is present. Calprotectin is a calcium-binding protein found in neutrophilic granulocytes that becomes available in the intestinal lumen through leukocyte release, active secretion, cell injury and cell death. During intestinal inflammation, neutrophil granulocytes migrate to the intestinal mucosa and increase fecal calprotectin a levels.

[0055] Fecal calprotectin a levels correlate with histological and endoscopic assessment of disease activity in Crohn's disease and ulcerative colitis. Fecal isolation of indium-111 labeled neutrophilic granulocytes has been proposed as the gold standard for disease activity in inflammatory bowel disease (IBD). Because patients with active inflammatory bowel disease (IBD) may have up to a 10-fold increase in fecal calprotectin levels, a commutative determination of increased fecal calprotectin concentrations is necessary. Fecal calprotectin is also used to differentiate between IBD and irritable bowel syndrome. Due to its resistance to enzymatic degradation, but only if calcium is bound, calprotectin can be extracted and measured in faeces. Calprotectin can also indicate other inflammatory conditions of the gastrointestinal tract, such as colorectal cancer, gastroenteritis, and food intolerance, but its levels vary with age and on different days in the same individuals. Serum calprotectin is a useful inflammatory marker for diagnosing sepsis and acute appendicitis. However, there is no international standard for calprotectin. Thus, domestically developed calibration standards are used to avoid inaccurate determinations due to inter-batch variability. This also results in different levels of standards between manufacturers, depending on the method chosen.

[0056] However, the determination of calprotectin as described provides metrologically traceable results. One related aspect, therefore, is to validate the throughput of turbidimetric assays with calprotectin calibrators from 0 to 2000 µg/ml sample, which is necessary to overcome the high variability in calprotectin content among different biological samples. A large range eliminates overruns or antigen excess problems, and can be achieved using the method described.

[0057] Figure 1 shows a calibration curve for mouse monoclonal antibody coated latex particles. Optical density values (Y-axis) correspond to calprotectin values in micrograms calprotectin/g faeces (X-axis). Results maintain consistent behavior over a range of 0 to 2000 µg/g. Immunoassay performance loses linearity when calprotectin concentrations in the sample exceed 2000 µg/g faeces, as shown in Figure 2 .

[0058] The immunoparticles of the invention are coated with mouse monoclonal antibodies raised against calprotectin MRP8/MRP14. While avian antibodies are less reactive with rheumatoid factor or human anti-mouse IgG antibodies or the human complement system (see EP 1 573 335 B1)—well-known causes of measurement error—polyclonal avian antibodies are less specific than mammalian monoclonal antibodies. Another object of the present invention, therefore, is to provide a turbidimetric assay for calprotectin based on highly specific antibodies without the disadvantage of non-specific reactivity. This is achieved by the analysis system of the present invention.

[0059] Sensitive methods for measuring calprotectin are available. However, available ELISA assays are associated with long test processing times and are more labor intensive than turbidimetric assays. The present invention relates to an assay system where samples can be processed as they enter the laboratory so that any medical condition associated with elevated calprotectin levels can be diagnosed in a timely manner. The suitability of the turbidimetric method was tested on 36 faecal samples and correlated with a standard ELISA assay as a reference. Figure 3 shows the correlation between a turbidimetric assay using one particle size coated with a monoclonal antibody and an ELISA assay for calprotectin. For turbidimetric analysis, a Hitachi 912 application was used, in accordance with the manufacturer's manual. Statistical analysis revealed commutative results, thus confirming the suitability of the described method for the determination of fecal calprotectin (regression P/BY=0.914 * X - 21.403; med. (95)=212.169; n=36; r=0.9018, t= 0.7693). It should be noted that the correlation coefficient r ranges from -1 (0) to 1. A value of 1 indicates that the relationship between X and Y is exactly described by a linear equation. A value of 0 refers to a non-linear correlation between variables.

[0060] In the following experiment, in which latex particles of the same size were coated with two different anti-calprotectin monoclonal antibodies, the turbidimetric method was tested using 36 fecal samples, and the detection results were correlated with a standard ELISA assay as a reference. Figure 4 shows the correlation of measurements of turbidimetric analysis using one and two monoclonal antibodies, and the corresponding ELISA for calprotectin. Statistical analysis of these analyzes did not reveal significant differences between the use of one (Fig. 3) or two antibodies (Fig. 4). The slight increase in sensitivity of the two-antibody assay is likely due to higher signal intensity due to increased antibody loading (regression P/BY=1.063 * X - 22.849; med(95)=258.068; n=36; r=0 .9023, t=0.7781).

[0061] In addition, the same size latex particles were coated with a) two monoclonal antibodies and b) one single monoclonal antibody (mAB No. 1062, 1067, 1068, 1089 from Immundiagnostik AG, Bensheim, DE). 39 faecal samples were extracted and incubated with latex particles under each condition. The turbidimetric assay of the invention was performed under both conditions using the Hitachi 912 application . Figure 5 shows the correlation analysis of the turbidimetric assay (regression P/BY=1.151*X+0.0; med.(95)=39.409; n=39; r=0.9989; t=0.9605). Additional experiments resulted in similar statistical values as shown in Table 1.

TABLE 1

Monoclonal antibody number Y X honey. (95) n r t 1067 Vs 1062+1089 1.151 0.0 39.409 39 0.9989 0.9605 1067 Vs 1067+1068 1.014 0.0 14.472 61 0.9997 0.9748 1068 Vs 1067+1068 1.008 +9.051 22.799 61 0.9994 0.9617

[0062] The performance of a turbidimetric assay using immunoparticles of two different sizes (heterogeneous) coated with a single monoclonal antibody was tested in 36 fecal samples and correlated with a standard ELISA assay as a reference. For turbidimetric analysis, a Hitachi 912 application was used, in accordance with the manufacturer's manual. The figure 6 shows the correlation analysis of the turbidimetric analysis. Statistical analysis of the test revealed commutative results, thus confirming the way to accurately determine fecal calprotectin (regression P/BY=1.102 * X – 24.963; med. (95)=289.067, n=36; r=0.9012; t=0 .7847).

[0063] The turbidimetric method was tested further and calprotectin measurements were performed using latex particles of two different sizes (heterogeneous), and compared with the method using particles of the same size (homogeneous). Latex particles of 250 nm and 175 nm were coated with one monoclonal antibody against calprotectin. Samples with calprotectin concentrations ranging from 0 to 2000 µg/g were used as calibrators for turbidimetric analysis using the Hitachi 912 application, according to the manufacturer's instructions. As shown in Table 2 (mU (mA): optical density in milliunits), the performance of the analysis using latex particles of two different sizes leads to increased detection sensitivity, compared with particles of the same size. Accordingly, a combination of at least two particle sizes may result in an improvement in the detection range of calprotectin compared to using particles of the same size.

[0064] Latex nanoparticles tend to self-aggregate. The storage buffer described may contain tribasic sodium citrate dihydrate, pH 5.0, bovine serum albumin, 1% SDS, sucrose, sodium azide. The storage buffer of the present invention contributes to increasing the storage stability of latex immunoparticles over extended periods of time. The description of the stability of the immunoparticles means that the spontaneous aggregation (haze) of the latex particles is reduced such that the second reaction component can be stored for up to 12 months at 2-8°C prior to use. The storage buffer keeps the particles stable at 37°C for several days. Not only is aggregation reduced, but also the stability of the antibody can be extended, resulting in measurement values largely independent of storage conditions.

TABLE 2

Calprotectin (mcg/g) 110 nm +175 nm (mU) 175 nm (med) 0 11.3 9.2 100 52.1 47.5 200 111.4 103.9 400 218.1 212.6 1000 456.1 443.7 2000 681.9 672.2 4000 768.7 757.9 8000 748.2 724.3 12000 715.3 695.1 16000 629.7 689.8

[0065] Table 3 shows absorbances (mU) measured on storage days 1 and 30 at 2-8°C using immunoparticles coated with two monoclonal antibodies in the turbidimetric assay of the invention. Immunoparticles i) stored at 2-8°C for 30 days and ii) stored at 37°C for 3 days and an additional 30 days at 2-8°C. Given that monoclonal antibodies are temperature sensitive, this suggests that the storage buffer of the present invention contributes to the accurate determination of calprotectin at various levels, namely immunoparticle aggregation and particle-bound antibody stability.

TABLE 3

Storage Buffer, 2-8°C Storage Buffer 37°C Calprotectin (µg/g) Day 1 Day 30 Day 1 Day 30 0 3.4 2.1 3.4 -1.1 100 58.5 53.6 58.5 59.7 200 108.4 114.3 108.4 119.1 400 220.3 229.8 220.3 240.4 1000 487.7 500 487.7 531.6 2000 707.9 698.2 707.9 732.9 4000 818.4 790.1 818.4 814.7

[0066] If the analyte exceeds a certain concentration in the sample, saturation of the antibody occurs, followed by a decrease in aggregation. Reduced aggregation results in lower signal intensity. This effect is described as "antigen excess" and can lead to misinterpretation of falsely low values and misdiagnosis. As described, an excess of the target antigen does not necessarily interfere with the turbidimetric determination as the nanoparticles are optimally dispersed and separated from each other, also due to their charged surfaces, leaving the entire surface of the particle free for antigen binding. Increases in antibody binding capacity are achieved by the described particle storage buffer and test reaction.

[0067] In a preferred embodiment, the method of the invention is carried out using antibody-coated nanoparticles with sizes ranging from 150 to 350 nm. The prior art gives information that nanoparticles for turbidimetric immunoassays cannot be larger than 140 nm in diameter, since an increase in particle size may correlate with non-specific aggregation, in particular of latex particles. This information should, however, apply to particles bearing less specific or cross-reactive polyclonal antibodies. The present invention, however, relates to nanoparticles having diameters from 150 to 350 nm and to the fact that large particles contribute to an accurate determination. Enhancement of the reaction between the monoclonal antibody and the target antigen (calprotectin) has also been achieved due to the increased area of interaction and because the reaction is partly surface-dependent. It is important that the increase in size is not associated with a high degree of spontaneous turbidity.

[0068] In this area, however, there are no reliable ways to increase the sensitivity of the measurement and accelerate the dispersion of suspended latex particles without disturbing the immunological response. The method of the invention solves this problem by using a buffer composition ( test buffer or first reaction component ) containing sodium citrate, pH 5.0-6.0, 150 mM NaCl, bovine serum albumin, SDS/Tween® 20, sucrose, sodium azide . The method of the present invention can be used in combination with commercially available faecal extraction buffers (eg IDK Extract® ^{Immundiagnostik} AG, Bensheim, DE) when mixed with the first reaction component R1. For comparison, various concentrations of calprotectin calibrators from 0 to 2000 μg/g were separately diluted in test buffer as described and in IDK ^Extract® buffer. Immunoturbidimetric analyzes were performed using two different monoclonal antibodies associated with latex particles. As shown in Table 4, a measurable increase in detection sensitivity can be achieved using the test buffer (mA: absorbances in milliunits), indicating "more available target molecules" and less multimer formation.

[0069] For a further comparison, 20 faecal samples were individually extracted using the test buffer of the invention and a commercially available extraction buffer from Bühlmann ( Bühlmann fCal Turbo® ) having a measured physiological pH of 7.2 (Bühlmann Laboratories AG, Basel, Switzerland ). Turbidimetric analysis was performed as described above using the Hitachi 912 application, following the manufacturer's instructions. The optical density values under any conditions correlated with each other. Statistical analysis showed a correlation between both conditions (regression P/BY= 1.004 * X - 1.552; med(95)= 30.37; n= 20; r=0.9932; t=0.9524). The test buffer of the invention thus provided at least as good an extraction of fecal calprotectin as commercially available buffers, but provided the benefit of reduced non-specific immunoparticle aggregation.

TABLE 4

Calprotectin (mcg/g) Test buffer (mU) IDK extract(honey) 0 11.5 0.4 100 48.1 44.5 200 103.3 93.9 400 206.6 194.6 1000 456.1 435.6 2000 656 630.8 4000 759.4 730.5 8000 751.8 724.8 12000 712.8 675.6 16000 673 638

[0070] Feces contain about 75% water, and the remaining solid fraction contains 84-93% organic solids. These organic solids are composed of: 25–54% bacterial biomass, 2–25% protein or nitrogenous matter, 25% carbohydrates or undigested plant material, and 2–15% lipids. These proportions vary considerably with diet and body weight. The remaining solids consist of calcium and iron phosphates, intestinal secretions, epithelial cells, and mucus. Components of body samples, in particular faeces, may represent a source of interference affecting the sensitivity of immunoassays and PETIA. Such interference can lead, for example, to negative optical density values. By the method of the present invention, interference due to the above-described components of the faecal samples is reduced.

[0071] Immunoglobulin M (IgM) is the main antibody produced by B cells. IgM is the largest antibody in the human circulatory system and the first antibody produced in response to initial exposure to an antigen. The presence of antiserum IgM may contribute to interference with the sample matrix. However, IgM antiserum may contain endogenous calprotectin. Analysis of each lot of IgM antiserum is recommended. Table 5 shows absorbance values of mU obtained by turbidimetric measurements of calprotectin calibrators and faecal samples diluted in reaction components with or without addition of IgM antiserum. Negative optical density values were discarded, and an overall increase in sensitivity and detection reliability was achieved.

TABLE 5

Calprotectin (mcg/g) without IgM (honey) IgM (honey) Calibrators 0 4.2 8.5 40 31.7 27.6 100 74.2 61 200 160.5 134.4 400 392 351.3 2000 1094.1 1082.1 4000 1211.1 1183 8000 1140.3 1128.9 Fecal samples fourteen -10.4 2.3 22 -6.5 11.7 31 5.6 17.5 40 82.6 67.7 57 29.5 43.3

EXAMPLES

EXAMPLE 1 Preparation of immunoparticles against calprotectin

[0072]used latex particles from well known manufacturers such as MERCK, Bangs Laboratories. The latex was carboxylated polystyrene or chloromethyl latex. The latex particles had the following parameters: surface charge density 62 µC/cm2; surface charge density 163 μEq/g pol.; solids content 9.0%, stabilized using 0.05% sodium azide. Immunoparticles were obtained by covalent attachment of purified mouse monoclonal antibodies (mAB 1062, 1067, 1069, 1089 and binding in the first reaction component) targeted against purified human calprotectin from granulocytes with intact lysosomal membranes. The carboxylated polystyrene particles preferably had a uniform size (175 nm). Alternatively, nanoparticles of two different sizes (160-175 nm and 250-275 nm) were coated with one monoclonal antibody (see figure 6). Also, latex particles of the same size 175 nm were coated with two different monoclonal antibodies.

[0073] Particles were stored until use in storage buffer containing sodium citrate, pH 5.0-6.0, 50 mM maleate buffer, pH 5.0, 150 mM NaCl, bovine serum albumin, 150 mM sucrose, sodium azide, at 2–8°C.

EXAMPLE 2 Extraction of faecal samples

[0074] Fecal samples were subjected to extraction as follows. Every 15 mg of faeces were diluted 1:100 in 1.5 ml of buffer. Empty sample tubes were filled with 1.5 ml test buffer, 50 mM maloeat pH 5.0, 150 mM NaCl, sodium azide, 0.1% sodium dodecyl sulfate, bovine serum albumin, in the presence or absence of IgM antiserum. For comparison, samples were also extracted using ready-to-use IDK Extract® extraction buffer (Cat. No. K 6967) and Bühlmann fCal Turbotm extraction buffer at room temperature. Fecal samples were collected and stored up to 48 h at 2–8°C. For extended periods (up to 12 months), storage at -20°C is recommended. At the beginning of the turbidimetric analysis, the frozen samples were slowly thawed, preferably at 2–8°C. In some cases, heterogeneous samples were homogenized mechanically. The IDK® Stool Sample Application System (SAS) faecal processing system (SAS) (Cat. No. K 6998SAS) was used to collect samples. The tip of the SAS probe, having notches to hold a fixed amount of raw material, was inserted into the faecal sample. The probe was placed back into the tube with extraction buffer. When the probe was placed back into the tube, the excess material was flushed away. Then 15 mg of the faecal sample remaining in the dipstick was diluted in extraction buffer. The tubes were tightly closed and shaken well until no more faecal samples remained in the notches. 10 minutes was needed to allow the formation of a precipitate. After confirming that the precipitate did not disperse again, the extracted sample was diluted 1:25 in test buffer. For example, 40 µl of the extracted fecal sample was added to 960 µl of test buffer.

EXAMPLE 3 Turbidimetric immunoassay for calprotectin

[0075] Samples extracted using any extraction buffer as described above were used for turbidimetric determination using the Roche Hitachi 912 application, in accordance with the manufacturer's recommendations. 10 µl of the extracted sample was added to 200 µl of test buffer and 50 µl of storage buffer containing sodium citrate pH 5.0, bovine serum albumin, Tween 20, sucrose, sodium azide. The automated analyzer gently mixed the immunoparticles with the extracted samples during incubation at 37 degrees Celsius for 5 minutes. The second component of the reaction with particles bearing immobilized monoclonal antibodies was added with gentle stirring. The agglutination time was 5 minutes at 37 degrees Celsius, at which time the optical density was measured. The measurements were carried out in duplicate. Absorbance values were reads taken at 570 nm.

[0076] Commercially available calprotectin was diluted in citrate buffer pH=5.4 in the range of 0 to 2000 μg/g. (6 calibration points) for calibration of turbidimetric analysis. A linear range of 30 to 2000 µg/g was obtained.

[0077] Fecal samples with known amounts of calprotectin were used as controls and test samples. Samples containing calprotectin were diluted 1:100 in reaction buffer to give measurement ranges of 0.1 to 20 μg calprotectin per 1 g faeces. Taking into account the dilution factor, the actual measurement range was up to 2000 µg/g.

[0078] The range of reference values for 1 g of feces is equivalent to 1 ml. The median value in healthy adults is approximately 25 µg calprotectin/g faeces. Samples with calprotectin concentrations below 50 µg/g were considered negative. Samples with calprotectin concentrations between 50 µg/g faeces and 100 µg/g faeces were considered borderline positive. Samples with calprotectin concentrations above 100 µg/g faeces were considered positive.

EXAMPLE 4 Calprotectin ELISA Assay

[0079] ELISA IDK® calprotectin (MRP8/14) selected for comparison with the turbidimetric method of determination according to the invention. The assay uses a two-site sandwich method with two selected monoclonal antibodies that bind to human calprotectin. The calibrator, controls, and diluted patient samples are added to the wells of a microplate coated with anti-human calprotectin monoclonal antibody. During the first stage of incubation, the immobilized antibody molecules bind calprotectin in the samples. The peroxidase-labeled conjugate is then added to each well and a complex is formed. Tetramethylbenzidine (TMB) is used as a peroxidase substrate. Finally, an acidic stop solution is added to stop the reaction. The color changes from blue to yellow in the presence of calprotectin.

[0080] The color intensity was directly proportional to the concentration of calprotectin in the sample. The samples were quantified in relation to their optical density. A general calibration curve using a calprotectin calibrator was built in each test. Performed two repetitions. Optical density was measured immediately using an ELISA reader at 450 nm versus 620 nm (or 690 nm) as a control. Alternatively, absorbance was measured at 405 nm against 620 nm as a control when the extinction of the highest standard exceeded the range of the photometer. It should be noted that the intensity of the color change is temperature sensitive. The methods followed the ELISA protocols IDK® calprotectin (MRP8/14) from Immundiagnostik AG, Bensheim, Germany).

[0081] Comparative results were evaluated using the Analyse-it software for Excel (Analyse-It Software, Ltd, Leeds UK). Passing–Bablok regression fitting was used to analyze the commutativity of turbidimetric assays (and ELISA).

[0082] In general, the present invention relates to an immunoturbidimetric assay for calprotectin, in particular for the determination of calprotectin in fecal matrix or in serum and other fluids and body samples, based on mammalian monoclonal antibodies specific for calprotectin in the form in which it is present. in acid granules of neutrophilic granulocytes. Such monoclonal antibodies are formed against calprotectin subunits at low pH and also bind these subunits at this pH. Specific calibration of the calprotectin analyte is necessary because this analyte can (self)aggregate and form complex multimers in the presence of calcium, which interferes with turbidimetric measurement. Metrological traceability and commutativity of results is essential in the work of a clinical laboratory. The present inventors have found that the disclosed mouse monoclonal antibodies bind calprotectin predominantly in the form of a heterodimer (S100A8/A9) and that the formation of multimers can be inhibited by an organic calcium ion coordinating buffer. The presence of a surfactant is recommended, and the addition of an anionic surfactant is most preferred because the calprotectin subunits have pIs of 6.1 and 6.3 and the immunoturbidimetric reaction is carried out at pH below 6.0, preferably between pH 5.0 and 6.0. The reaction components must have an osmolality above 150 mOsm/kg for further agglutination. Metrologically traceable relationship and commutativity of results with other conventional immunoassays using monoclonal and polyclonal antibodies (but using conventional buffers at physiological pH) has been proven.

Claims (24)

1. A method for in vitro measurement of the presence of calprotectin in a biological sample from a patient using a latex particle-enhanced turbidimetric immunoassay, comprising the steps of: a) collecting a predetermined amount of said biological sample; b) extracting said biological sample in a predetermined amount of an aqueous organic buffer having i) a pH between 5.0 and 6.0, ii) an osmolality of at least 150 mosmol/kg H ₂ O, iii) 0.01-0.1% percent by weight of an anionic surfactant, iv) where the organic buffer is coordinating and sequestering calcium and zinc ions, and homogenizing the matrix of said biological sample, followed by removal of any particulate material to obtain a sample solution with the presence of solubilized heterodimeric calprotectin; c) mixing a certain amount of a solution of said sample from step (b) with a certain amount of a reagent containing nanoparticles having immobilized two or more monoclonal antibodies or their fragments that specifically bind calprotectin to obtain a particle-bound antibody-antigen reaction for calprotectin , which is present as heterodimer S100A8 and S100A9; d) incubating the mixture from step c) for a period of time; and e) obtaining the optical properties of the mixture and determining a signal indicative of the content of calprotectin based on the optical properties of the mixture; f) associating said content with a calibrated control and assessing the clinical condition of said patient based on the measured presence of calprotectin in said biological sample. 2. The method of claim 1, wherein step e) of obtaining the optical properties comprises determining a value for optical density, translucency, reflectance, light scattering, fluorescence, or scintillation. 3. The method according to claim 1, including the use of nanoparticles having diameters from 150 to 350 nm to increase sensitivity. 4. The method of claim 3, wherein the antibodies are coupled to two types of particles having diameters in the range i) 150 to 200 nm and ii) 250 to 350 nm to increase the measurement range. 5. The method according to any one of claims 1 to 4, wherein said particles are particles of carboxylated polystyrene or chloromethyl-activated polystyrene. 6. The method according to any one of claims 1 to 5, wherein the biological sample is feces or fecal extract. 7. The method according to any one of claims 1 to 6, wherein the buffer composition is composed of at least one salt selected from the group consisting of salts of polycarboxylic acids, tricarboxylic acids, aconitic acids, tricarboxylic acids, dicarboxylic acids, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, alpha-, beta- and gamma-hydroxycarboxylic acids, hydroxydicarboxylic acids, malic acid, citric acid, tartaric acids, malonic acid, gluconic acid, 5-ketogluconic acid, 2-ketogluconic acid , dihydroxymaleic acid, maleic acid, fumaric acid, nitrilotriacetic acid, lactic acid and/or ascorbic acid. 8. The method according to any one of claims 1 to 7, wherein the particle-bound antibody-antigen reaction is carried out in a mixture having a pH between 5.0 and 6.0 and containing an anionic surfactant or sodium dodecyl sulfate and coordinated by Ca ²⁺ buffer molecules. 9. The method of claim 8, wherein the calcium sequestration buffer from step b) contains at least one citrate, acetate, or maleate salt, protease-treated serum albumin, and 0.01-0.1 percent by weight of anionic surfactants . 10. The method according to any one of claims 1 to 9, comprising the addition or presence of a non-specific IgM antiserum. 11. The method according to any one of claims 1-6 and 8-10, wherein said biological sample is blood, serum or plasma. 12. Test kit for implementing the method according to claim 1 for measuring the presence of heterodimeric calprotectin in a biological sample by means of turbidimetric immunoassay with latex particle enhancement, containing the first aqueous reagent component containing – 20-1000 mmol/l organic buffer with a pH in the range of 5.0 to 6.0, which coordinates and sequesters calcium and zinc ions; - 50-300 mmol / l sodium, potassium or lithium salts; - 0.1-1.5% protease-treated serum albumin; – 0.01–0.1% w/v sodium dodecyl sulfate, and beta-aldoses, triose, tetroses, pentoses, hexoses, glucan, dextran and/or sugar to achieve an osmolality of at least 200 mosmol/l; and the second reagent is a component containing 0.01–0.5% wt./vol. latex particles with a diameter of 150 to 350 nm, carrying two or more immobilized monoclonal antibodies or their fragments that bind heterodimeric calprotectin.

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