HK1174972B - Use of a mixture of synthetic and cellular substrates in the manufacture of a diagnostic kit - Google Patents

Description

Use of a mixture of synthetic and cellular substrates for the preparation of a diagnostic kit

The present invention relates to methods and systems for disease diagnosis that simultaneously detect antibodies that bind to cellular and/or tissue substrates and antibodies that bind to synthetic substrates, such as microparticles or beads coated with specific antigens, thereby providing a "one-step" method for simultaneously detecting and identifying disease-associated antibodies with low specificity (cells and/or tissues) and high specificity (antigens).

Background

Detection of antibodies by Indirect Immunofluorescence (IIF) using different cell and tissue substrates as multiple antigen sources has been achieved in routine diagnostics. Cell-based immunofluorescence assays such as the detection of antinuclear antibodies using HEp-2-cells have been widely used as an industrial standard in clinical diagnostics and research (Tan EM, adv. immunology 1982;33: 167-. Methods for diagnosing autoimmune diseases using indirect immunofluorescence-based methods are known in the art. WO97/06440 discloses a method for diagnosing rheumatoid arthritis by the presence of antibodies against microtubule tissue centers or microtubules extending therefrom in a sample of a body fluid of a patient. The cell line used as substrate is preferably an IT-1 macrophage line, which is used as substrate for binding of autoantibodies present in a patient sample.

The increasing demand for automated reading and interpretation of fluorescence patterns for improved standardization and cost effectiveness has been met by currently available sophisticated pattern recognition software and fully automated reading instruments (Hou YN, et al, radial Res.2009;171(3):360-7, Hiemann R, et al, Ann N Y Acad Sci 2007;1109:358-71, Bocker W, et al, radial Res.2006;165(1):113-24.Hiemann R, et al, Autoimmun Rev.2009;9: 17-22). Indirect immunofluorescence has additionally been used in quantitative and semi-quantitative methods to determine antibody titers, for example in the diagnosis of diseases characterized by the presence and/or amount of autoantibodies (WO 2009/062479a 2).

However, advances in assay development and recombinant technology pave the way to detect the specificity of autoantibodies for individual antigenic targets, thereby improving the diagnostic capabilities of antibody tests. The increasing diversity of antibodies found in different diseases, such as infectious and rheumatic diseases, requires innovative technologies to overcome the drawbacks of single antibody detection and to reduce the cost and time to report the results. Therefore, a multiplex platform has recently been disclosed to meet the need to determine the specificity of multiple antibodies simultaneously in one sample. Multiplex assays based on fluorescent bead-based flow cytometry and microarray systems have proven to be powerful tools, supporting higher throughput analysis and more extensive detection of patient samples (Avaniss-Aghajani E, et al, Clin Vaccine Immunol.2007;14:505-9, Lai G, et al, J Immunol Methods 2005;296: 135-47). Computer-assisted pattern recognition has also been used to analyze antigen arrays whereby antibodies that bind a panel of disease-associated antigens can be detected and correlated with a particular disease diagnosis (Binder SR et al, Clinical and Diagnostic Laboratory Immunology, Dec.2005, 1353-1357). Furthermore, for specific antibodies for the assessment of rheumatic diseases, multiplex detection using polychromatic fluorescence using microbeads encoded by immobilized fluorescence can be used. This multicolor fluorescence analysis using pattern detection algorithms provides a common platform technology for screening for ANA (GroBmann K, et al., Cytometry A.2011Feb;79(2): 118-25).

For example, disease-specific autoantibodies (AAB) are a serological phenomenon of systemic rheumatic and autoimmune liver disease. In particular, detection of ANA by IIF is one of the first techniques for serological diagnosis of systemic rheumatism in routine laboratories. Screening for ANA by IIF assays remains the standard method in current multi-stage diagnostic methods, although enzyme linked immunosorbent assay (ELISA) and multiplex (multiplex) techniques for detecting disease-specific AAB are disclosed. Recombinant or purified antigens can be provided on beads and subsequently analyzed for antibodies that specifically bind to the selected antigen using an enzyme immunoassay. However, this method presents significant drawbacks due to the apparently different sensitivity and specificity between different antigens (Hayashi N, et al,2001, Clinical Chemistry, 47: 91649-one 1659). A variety of bead-based fluorescence assays have been disclosed which show appropriate correlation with cell-based autoantibody diagnostic methods (Smith J, et al,2005, Ann.N.Y.Acad.Sci.1050:286-294), but the lower sensitivity to identify IIF positive control samples indicates that the IIF method is still an advantageous method (Nifli AP, et al, Journal of Immunological methods 311(2006), 189-197).

Several substrates have been proposed for performing ANA IIF assays, however, screening for non-organ specific AAB on HEp-2 cells is the most mature method. Typically, evaluation of ANA is followed by detection of specific AABs against e.g. Extractable Nuclear Antigens (ENA) and cytoplasmic antigens by immunoassay using purified native or recombinant antigens. This two-stage approach presents the following benefits: (i) highly sensitive screening of the most common, clinically relevant non-organ specific AABs; (ii) optimally combining other assay techniques to distinguish downstream AAB reactivities (e.g., SS-A/Ro and SS-B/lA) based on the detected IIF pattern and suspected diagnosis, (iii) assessing clinically relevant AABs without further testing (e.g., anti-centromere AABs), and (iv) assessing AABs that can only be detected by IIF in the case of unknown autoantigen targets or unavailable commercial assays.

Another example is the detection of anti-neutrophil cytoplasmic antibodies (ANCA) for the differential diagnosis of systemic vasculitis (Bosch X, et al, Lancet 2006;368: 404-18). Due to the development of ANCA in these systemic autoimmune diseases, the term ANCA-associated vasculitis (AASV) has been established for these clinical realities. ANCA was revealed by IIF, which is still the recommended method to detect the reactivity of these antibodies. ANCA typically exhibits two different staining patterns of fixed granulocytes in IIF: punctate cytoplasmic (cANCA) pattern and perinuclear (pANCA) pattern. cANCA, commonly found in Wegener's Granulomatosis (WG) patients, is predominantly directed to protease 3(PR3) among other targets, whereas pANCA occurs predominantly in Microscopic Polyangiitis (MPA) and is predominantly directed to Myeloperoxidase (MPO) among other targets (Van der Woude FJ, et al, Lancet 1985;1:425-9; Csernok E, et al, Nat Clin practice rheumatol.2006apr;2(4): 174-5).

It has been proposed that estimating antibody concentrations is helpful for diagnosing and addressing these clinical realities. Antibody values are often correlated with the severity of the disease (Cohen Tervaert JW, et al, Arch Intern Med 1989;149: 2461-5). However, other target antigens of ANCA are described in IIF, which can be found in non-AASV patients (Savige J, et al, best. practice. Res. Clin. Rheumatotol. 19:263-76, Savige J, et al, Am J Clin Path 2003;120: 312-8). Thus, according to the most recently established consensus guidelines, a different technique for detecting ANCA, such as ELISA, is recommended in addition to IIF.

However, techniques for determining certain antibodies by the two or more stages described above involve assays of individually labeled multiple components. For cost-effective serological diagnosis, it is clearly necessary to combine the detection of antibodies against cellular and tissue antigenic targets on the one hand and purified and characteristic proteins thereof on the other hand in a single method, using one label.

The current identification of proteins by microarray technology alone does not provide a satisfactory solution. As the number of antigenic targets detected on a single microarray increases, the associated manufacturing equipment, miniaturized and specialized materials and processing requirements add complexity and cost to the production of such microarrays. Other techniques, including enzyme linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), chromogenic assays, High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and Thin Layer Chromatography (TLC), present the disadvantage of a limited number of analytes in antigenic form that can be simultaneously assessed. These techniques are also time consuming and require expensive equipment. In contrast, the use of HEp-2 cell substrates for ANA detection provided more than 1200 antigenic targets for antibody identification.

Summary of The Invention

In view of the technical difficulties in the prior art, the present invention provides a disease diagnosis method that reduces multi-step diagnosis to a one-step diagnosis method. This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the appended claims. It is therefore an object of the present invention to provide a disease diagnostic method and system which detects antibodies bound to cells and/or tissues and simultaneously detects antibodies bound to synthetic substrates, such as microparticles coated with specifically purified natural and/or recombinant antigens, thereby providing a "one-step" method for the simultaneous detection of antibodies associated with a disease with both low (cell or tissue specificity) and high (antigen specificity) specificity.

The invention also relates to a device and a kit for carrying out the method of the invention. The method is also suitable for screening methods whereby the reactivity of a plurality of patient sera with any given number of specific antigens can be detected.

It is therefore an object of the present invention to provide a method for the diagnosis of a disease comprising the simultaneous detection of antibodies binding to one or more cellular substrates and antibodies binding to one or more synthetic substrates, characterized in that:

a) providing a mixture of a cellular substrate and a synthetic substrate,

b) incubating the substrate mixture with a sample containing the antibody to be detected,

c) detecting and/or identifying cellular and synthetic substrates and antibodies binding to said substrates using fluorescence microscopy, and

d) the immunofluorescence image data is evaluated, preferably using an automated pattern recognition interpretation system.

By cellular substrate is meant an antigen-binding biological substrate, such as a mammalian cell, so that different tissue types or combinations of cells can be used. Tissue substrates, including a plurality of cells of similar type obtained from organ tissue (organic tissue), may also be used as "cell substrates" in the methods of the invention.

In a preferred embodiment of the above method, the cell or tissue substrate may be HEp-2 cells, human granulocytes, and/or organ tissue, preferably pancreatic tissue.

In a preferred embodiment, the synthetic substrate is a particle or bead coated with purified natural and/or recombinant antigens. The antigen may be a protein, peptide, nucleic acid, such as DNA, a multimolecular cell substrate such as a centromere, a protein complex or a protein-membrane structure, or any other cellular component to which an antibody can bind. The synthetic substrate refers to a carrier that is a mixture of specific or specific purified and/or recombinant antigens. Microparticle and bead supports of various substances and materials are known in the art and are suitable for the methods of the invention, for example microparticles, particles or beads composed of natural or artificial polymers, agarose, cellulose, glass or metal oxides.

In a preferred embodiment of the invention, the diagnostic method is characterized in that the antigen coating the synthetic substrate is bound by an antibody associated with the presence of a disease. The antigen coating the synthetic substrate is selected based on its association with a known disease. The identification of antibodies that bind to specific antigens thus allows a "high specificity" diagnosis of disease in addition to a "low specificity" detection of antibodies that bind to cellular and/or tissue substrates.

The substrate of the invention is detected and/or identified by one or more distinguishable characteristics or parameters. In one embodiment, the method of the invention is characterized in that the optical, fluorescent and/or physical characteristics of the substrate are used to detect and/or identify said substrate. For example, various substrates can be identified by their size, shape, fluorescent properties, or other parameters during microscopic analysis. Different parameters can also be combined for different substrates, thereby representing one code for substrate, antigen and/or antibody identification.

In one embodiment of the invention, the method is characterized by fluorescence characteristics of the fluorophore concentration, such as the rhodamine concentration, and/or physical size characteristics for identifying synthetic substrates, wherein the bead or microparticle is preferably between 1-100 μm in size, such as 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100 μm, or any similar value. Multiple beads or microparticles of different sizes may be combined, where each size of bead is coated with a different antigen, whereby an array-like approach of multiple antigens can be used simultaneously with the cell substrate.

In a preferred embodiment of the invention, multicolor fluorescence microscopy is used for identifying said substrate and/or bound autoantibody, wherein the substrate and/or bound antibody exhibits different fluorescence colors, preferably blue, green and red.

In one embodiment of the invention, the cellular substrate is stained with a blue emitting (blue emission) fluorescent dye, such as DAPI, the synthetic substrate is labeled with a green emitting (green emission) fluorescent dye, such as rhodamine or FITC, and/or the specifically bound antibody is detected by an anti-human immunoglobulin specific antibody, such as Cy5 and/or Allophycocyanin (APC), labeled with a red emitting (red emission) fluorescent dye.

The methods of the invention are particularly directed to diseases or disorders in which antibody detection provides an effective diagnosis. In a preferred embodiment, the method is characterized in that the disease is an autoimmune or infectious disease, preferably a systemic rheumatic disease, an autoimmune liver disease, type 1 diabetes, lyme disease or a herpes simplex virus infection.

Although autoimmune diseases are difficult to diagnose, the identification of the specific antigen or antigens to which the autoantibody binds, in addition to the pattern of binding of the autoantibody on the cellular substrate, allows more accurate diagnosis in a shorter time than previously known methods. The present invention allows diagnostic methods to combine arrays with cell-based methods, which has never been considered possible at present.

In one embodiment of the invention, the antibody detected is an antinuclear antibody (ANA) or an anti-neutrophil cytoplasmic antibody (ANCA).

In a further embodiment of the invention, the method is characterized in that the HEp-2 cells are used for analyzing the staining of antinuclear antibodies (ANA).

In a further embodiment of the invention, the method is characterized by the ANA cell staining pattern shown in table 3, table 4 and/or table 5, the corresponding antigen and the associated disease for use in disease diagnosis.

In a further embodiment of the invention, the method is characterized in that human granulocytes are used for the analysis of the staining of anti-neutrophil cytoplasmic antibodies (ANCA).

In a further embodiment of the invention, the method is characterized in that the ANCA cell staining pattern shown in table 1 and/or table 2, the corresponding antigen and the associated disease are used for disease diagnosis.

In a further embodiment of the invention, the method is characterized in that the anti-neutrophil cytoplasmic antibody (ANCA) exhibits perinuclear staining (p-ANCA) or punctate cytoplasmic staining (cANCA) of human granulocytes.

In a further embodiment of the invention the method is characterized in that the antibody targets the protease 3(PR3) or Myeloperoxidase (MPO) protein or antigen thereof.

In a further embodiment of the invention the method is characterized in that the antibody exhibits a spotted cytoplasmic staining (cANCA) of human granulocytes in combination with binding of protease 3(PR3), wherein the protein or antigen thereof is provided on a synthetic substrate, thereby providing a diagnosis of Wegener's Granulomatosis (WG).

In a further embodiment of the invention the method is characterized in that said antibody exhibits perinuclear staining of human granulocytes (p-ANCA) and binds Myeloperoxidase (MPO), wherein said protein or antigen thereof is provided on a synthetic substrate, thereby providing a diagnosis of ANCA-associated vasculitis (AASV).

In a further embodiment of the invention the method is characterized in that the sample comprises blood, serum, cerebrospinal fluid, synovial fluid or saliva obtained from a subject, wherein the subject is understood as a patient suspected of having a disease or disorder diagnosed using the method of the invention.

The invention also covers a method in which the interpretation system is controlled by specially designed software, as has been disclosed software particularly for use in the method of the invention, which comprises modules for device and autofocus control, image acquisition, image analysis and/or pattern recognition algorithms.

In one embodiment, the method of the invention is characterized in that the interpretation system is used to evaluate immunofluorescence images according to the following hierarchy (hierarchy):

i) a signal of a positive staining is given,

ii) localization of the staining, preferably by classifying the staining pattern into the classes of cell substrates or synthetic substrates, and

iii) determining a cellular pattern, preferably a perinuclear and/or cytoplasmic pattern for granulocytes; nuclear, cytoplasmic, and/or chromatin pattern of mitotic cells for HEp-2 cells.

This hierarchy facilitates rapid and accurate analysis of the acquired microscope images, so that diagnosis can be made based on multiple parameters within the immunofluorescence data.

In one embodiment, the method of the invention is characterized in that the classification of the staining patterns into cell or synthetic substrate classes and the determination of the cell patterns is achieved by a combination of structural and textural features of the immunofluorescence image by defining a set of rules for each pattern. This classification can be done by specially designed software, as has been developed specifically for use in the method of the present invention.

Another aspect of the invention relates to a system for simultaneously detecting antibodies binding to one or more cellular substrates and to one or more synthetic substrates according to the method of any one of the claims, comprising:

a) fluorescence microscope with camera, motorized scanning stage and multi-channel Light Emitting Diode (LED), and

b) a computer device with software containing modules for device and automated focus control, automated image acquisition, automated image analysis, and automated pattern recognition algorithms, wherein three color channels are analyzed, preferably blue, green, and red.

The system of the invention has been developed for use in particular in the disease diagnosis method of the invention. Such a system may comprise a fluorescence microscope in combination with a data processing computer such as a PC.

One aspect of the invention is a kit for simultaneously detecting and/or identifying antibodies binding to one or more cellular substrates and one or more synthetic substrates according to the method of claims 1-21, comprising: a) a slide with immobilized cellular substrate mixed with antigen-coated synthetic substrate, wherein the cellular substrate is preferably HEp-2 cells and/or human granulocytes and the synthetic substrates differ from each other according to their optical, fluorescent and/or physical characteristics, and b) a conjugate with immunoglobulin-specific antibodies conjugated with a fluorescent label, preferably FITC, Cy5 and/or APC, and optionally washing buffer, coverslip, cover medium, uncoated synthetic substrate (with or without fluorescent label), and/or additional fluorescent label of the synthetic substrate.

The kits of the invention are very simple and economical diagnostic methods compared to the standards known in the art. The kit slide may contain multiple synthetic substrates with multiple epitopes, especially in combination with array-based and cell substrate methods, thus providing a more complete and efficient diagnostic tool than any diagnostic tool known in the art, in an economically feasible compact format.

Detailed Description

The method of the invention allows the diagnosis of diseases which usually require multiple steps for diagnosis. The combination of multiple diagnostic steps to assess increased specific antibody binding this method can provide faster, more efficient and more accurate diagnosis in a diagnostic assay. This method is particularly relevant to diseases or disorders where antibody detection provides an effective diagnosis, such as infectious diseases and autoimmune diseases. The method makes it possible to screen not only for the presence and pattern of antibodies that bind to cells or tissues, but also to provide more specific information about the individual antigens to which the antibodies are directed. This facilitates the diagnosis of a particular disease in a one step process.

The term diagnosis refers to the identification and/or determination of the nature and cause of a disease by assessing the characteristics of a patient. The present invention provides a diagnostic method which, when carried out, provides an indication that a particular disease or diseases may be present in a patient. For example, multiple autoimmune diseases may be present in a subject, whereby the methods of the invention identify one disease without exception to the presence of other diseases, e.g., multiple diseases may be detected or diagnosed simultaneously or separately using the methods of the invention.

The method of the invention is particularly suitable for diagnosing systemic rheumatic inflammatory diseases, such as autoimmune systemic vasculitis. The disease is associated with antibodies to antigens within granulocytes (ANCA), such as Microscopic Polyangiitis (MPA), Myeloperoxidase (MPO) and Wegener's Granulomatosis (WG), and protease 3(PR3), and can be diagnosed using the methods of the present invention. Systemic lupus erythematosus can be diagnosed, for example, when associated with antibodies to double-stranded DNA. Polymyositis can also be diagnosed using the methods of the invention, for example, when associated with an antigen within HEp-2 cells, such as an antibody to tRNA synthetase.

Autoimmune liver disease can also be diagnosed using the methods of the invention, for example when associated with antibodies to antigens within HEp-2 cells. Disorders associated with antibodies to the fibrillin (fibrillary) actin-asialoglycoprotein receptor or other antigens within hepatocytes may also be diagnosed.

Table 1 lists the types of relevant anti-neutrophil cytoplasmic antibodies and the relevant cell patterns in granulocytes. Table 1 provides examples of antibodies, staining patterns, and their associated clinical relevance. This table does not limit the scope of the invention.

TABLE 1

Table 2 lists relevant antigens that can be used in the synthetic substrates of the invention. This table does not limit the scope of the invention. It can be seen from table 1 that for each cellular pattern, a number of diseases can be represented. To provide a more specific diagnosis, more detailed information about the specific antigen to which the autoantibody binds is required. As can be seen from table 2, each specific antigen exhibits a close association with a disease, and thus combining the cellular pattern for positive signals for this antigen in the methods of the invention may provide improved diagnostic methods compared to those known in the art.

The present invention thus shows a new combination of analytical techniques, providing an improved diagnostic method. The present invention relates to a combination invention whereby the novelty of the individual ingredients is not important, but rather the specific combination as claimed achieves surprising results and synergy. The present invention exhibits a synergistic effect between the components, such that simultaneous analysis of patient antibodies with low (cell and/or tissue) and high (antigen) specificity provides a faster method and a more accurate diagnostic method than those known in the prior art.

TABLE 2

Table 3 lists the relevant antinuclear antibodies and the relevant clinical relevance. This table does not limit the scope of the invention. The table shows the antinuclear antibody incidence of systemic autoimmune disease. The incidence of antinuclear antibodies and their association with a particular disease are known in the art (Arbuckle MR et al.New Engl J Med 2003;349:1226-33, Bradwell AR and Hughes RG.atlas of HEp-2patterns.3rd ed.2007, Dalakas MC et al.Lancet 2003,362:971-82, Fox Rl.Lancet 2005;366:321-31, Rahman A et al.New Engl J Med 2008;358:929-39, Tan EM et al.Arthritis Rheum 1997;40:1601-11, Zisweler H-R et al.Swiss Med Wkly 2007;137: 586-90).

Abbreviations: SLE: systemic lupus erythematosus; and (4) MCTD: mixed connective tissue disease (Sharp-syndrome); DM/PM: leatherMyositis/polymyositis; RA: rheumatoid arthritis; SSc: systemic sclerosis/scleroderma; LSSC: localized systemic scleroderma (CREST-syndrome); LE: lupus erythematosus caused by drugs; and (3) AIC: autoimmune cholangitis; PBC: primary biliary cirrhosis.

The diseases provided in tables 1-4 are only exemplary of the pathologies, conditions and/or diseases diagnosed by the methods of the present invention.

TABLE 3

Table 3 notes:

^aspecificity 95%

^bU1-snRNP-complex: IgG against Sm and/or U1-snRNP p68/A and/or tertiary epitopes

^cThe pathogenesis in neonatal lupus with av-Block is significant in up to 5% of cases.

SS-A/Ro52IgG recognizes the cardiac 5-HT 45-hydroxytryptamine receptor and inhibits calcium flux L (ICA) activated by 5-hydroxytryptamine

^dSS-A/Ro p52IgG together with Jo-1IgG in inflammatory myopathies

^erRNP: ribosomal RNPs with proteins P0, P1 and P2

^fIn DM/PM with SSc

Table 4 shows the nuclear pattern (IIF) of antinuclear antibodies when bound to HEp-2 cells. As can be seen from table 4, similar or identical cell/nuclear patterns were observed for different diseases. Table 5 shows a further cellular pattern (cytoplasm) of antibodies binding to HEp-2 cells. While cellular assays may provide evidence or support for the presence or absence of autoimmune disease, indirect immunofluorescence assays using only cellular substrates do not provide the best specific diagnosis. Specific antigens that can be applied to the synthetic substrates of the invention are also listed in tables 4 and 5. The combination of cellular patterns (interphase, mitosis and cytoplasm) with specific antigens (Antigen) leads to an improvement and a more definitive diagnosis of the disorder or disease.

TABLE 4

Detection of antinuclear antibodies (ANA) (ANA IIF HEp 2) on Hep-2 cells

TABLE 5

Type 1 diabetes can also be diagnosed using the methods of the invention, for example, when associated with pancreatic islet cells of the pancreas, glutamate decarboxylase, or an antibody to tyrosine phosphatase IA-2.

Another class of diseases that can be diagnosed by the methods of the invention relates to infectious diseases. Such diseases include Borrelia disease (Borreliaiosis) or Lyme disease, wherein the disease is associated with Borrelia (Borrelia) or antibodies to Borrelia proteins such as p21, p41, p100, OSPC or OSPA.

Herpes simplex virus infection is another infectious disease that can be diagnosed by using the methods of the invention, for example when it is associated with antibodies to herpes simplex virus type 1 (herpes labialis virus) and type 2 (herpes genitalis virus). Herpes virus infected cells can be used as cellular substrates, together with synthetic substrates containing specific herpes virus antigens such as gG1 and gC1 for type 1 virus and gG2 for type 2 virus.

The use of antigen-coated small supports such as microparticles encoded by their optical or physical characteristics or by the attachment of a reporter molecule in a test environment and the use of cellular and/or tissue substrates together surprisingly provides a suitable basis for the simultaneous assessment of multiple antibody activities outlined in the multi-step methods described above.

The best reported method for this detection technique is multicolor fluorescence, which can be readily applied to particle coding and simultaneously used as a marker to assess multiple specific antibody-antigen interactions. In addition to the fluorescent signal generated by detection of target-bound antibodies on particles, cell substrates and/or tissue substrates, a fully automated microscope with pattern recognition software reads the fluorescent signal, which encodes the particles coated with the respective specific antigen.

The pattern recognition software can determine both cellular and/or tissue substrates and synthetic substrates. The defined set of rules for each pattern (tissue and substrate pattern) allows the software to identify specific substrates for each fluorescent signal, which occurs through the unique optical, fluorescent, and/or physical characteristics of the different substrates. These features serve as coding for software to recognize the substrate to which the antibody binds (and thus the specific antigen to which it binds). Automated determination of specific cellular patterns (e.g., pericyclic and cytoplasmic for granulocytes; nuclear, cytoplasmic, punctate (cytoplasmic) chromatin for mitotic cells of HEp-2 cells) provides additional information that allows diagnosis of MPA and WG diseases as described above.

The use of complex pattern recognition does not require highly trained personnel and knowledge of the settings of several different systems and the fluorescence pattern specific to a particular antibody. This requirement usually requires expensive costs in personnel training. In terms of quality standards and validation instructions, repeated testing is often required, requiring supervision by experts or other highly trained personnel. Consistent reproducibility and high quality are particularly required for cell-based IIF.

Interpretation of immunofluorescence patterns is influenced by the knowledge and individual qualifications of the investigator. Thus, variability is common both inside and between laboratories, which is a major diagnostic problem, especially in non-professional laboratories.

Automated reading of immunofluorescence patterns to detect antibodies to cellular and purified antigenic targets through automated interpretation systems and intelligent pattern recognition provides a reliable and cost-effective basis for serological diagnostics, particularly for laboratories that detect large numbers of samples. Applying the conditions of modern electronic data analysis and processing can significantly reduce the heavy workload in such laboratories.

Description of the drawings

The invention is further described by the accompanying drawings. These do not limit the scope of the invention.

FIG. 1: simultaneous immunofluorescence analysis of antibodies to ANCA and MPO.

FIG. 2: simultaneous immunofluorescence analysis of antibodies to ANCA and PR 3.

FIG. 1: the results of the simultaneous immunofluorescence analysis of antibodies to ANCA and MPO are shown in fig. 1. One serum sample tested positive for panca (MPO) and another serum sample tested positive for cANCA (PR3) were tested on slides with human granulocytes and MPO-coated microparticles. The location of granulocytes on the slide was detected by DAPI staining, while antigen-coated microparticles were assessed by rhodamine or FITC fluorescence. pANCA positive sera confirmed positive signals for both the immobilized granulocytes and MPO coated microparticles. Antibodies binding to MPO located within granulocytes and immobilized on microparticles (shown by arrows) were shown by specific staining with Cy5 conjugate. In contrast, cANCA-positive sera gave positive reactions only with granulocytes.

FIG. 2: the results of the simultaneous immunofluorescence analysis of the antibodies to ANCA and PR3 are shown in fig. 2. One serum sample tested positive for panca (mpo) and another serum sample tested positive for cANCA (PR3) were tested on slides with human granulocytes and PR3 coated microparticles. The location of granulocytes was detected by DAPI staining, while antigen-coated microparticles were assessed by rhodamine or FITC fluorescence. cANCA positive serum confirmed positive signals for both fixed granulocytes and PR3 coated microparticles. Antibodies binding to PR3 located within granulocytes and immobilized on microparticles (shown with arrows) are shown by specific staining with Cy5 conjugate. In contrast, pANCA-positive sera gave a positive reaction only with granulocytes.

Examples

The following methods were used in the practice of the invention, as demonstrated in the examples. These examples are merely illustrative to further describe the present invention and do not limit the scope of the present invention.

Patient's health

Serum samples from 10 cANCA-positive WG patients in IIF (PR3 positive) and 10 pANCA-positive other AASV patients (MPO positive) were collected and stored at-20 ℃. The diagnosis of WG is based on Chapel Hill Consensus Definitions for WG. 10 patients who met the diagnostic criteria for Systemic Lupus Erythematosus (SLE) served as disease controls in this study. Sera from 10 donors were used as healthy controls. All samples were taken at the time of consent and enrollment.

ANCA detection by conventional IIF

ANCA was detected by working patient samples on ethanol and formalin fixed human granulocytes according to the manufacturer's instructions (GA genetic Assays GmbH, Dahlewitz, Germany). Briefly, the fixed granulocytes were incubated in a humidified chamber for 30 minutes at Room Temperature (RT) with 25. mu.l of serially diluted serum (starting from 1:20 dilution). After washing, the immune complexes were detected by incubating the sample with fluorescein conjugated sheep anti-human IgG for 30 minutes at room temperature. The samples were then washed, embedded, and manually analyzed by fluorescence microscopy.

Detection of MPO and PR3 antibodies by ELISA

Protease 3 and MPO autoantibodies were detected in patient sera using different generations of ELISA according to the manufacturer's instructions, using purified human PR3 and MPO as solid phase antigens, respectively (GA Generic Assays GmbH, Dahlewitz, Germany; Aesku. diagnostics GmbH, Wendelsheim, Germany).

Antibody for simultaneously detecting ANCA and MPO and PR3

Human granulocytes were separated by density gradient. The granulocyte-rich band was collected. After erythrocyte lysis, granulocytes were washed with PBS. The isolated granulocytes were mixed with rhodamine-labeled MPO or PR-3 coated beads. The mixture was fixed on the surface of a 6-well diagnostic slide. The slides were fixed with ethanol. For IIF, the combined granulocyte/bead slides were incubated with 25. mu.l of 1:20 diluted serum in a humidified chamber for 30 minutes at Room Temperature (RT). After washing, bound immune complexes were detected by incubating the samples with Cy 5-conjugated goat anti-human IgG antibody (Dianova, Hamburg, Germany). The samples were then washed, embedded and analyzed by an automated pattern recognition system for fluorescent signals (see below).

Automated pattern recognition of fluorescent signals

The fluorescence pattern of serum samples simultaneously multiplexed detection of antibodies binding to cellular antigens and antibodies binding to antigens coated on microparticles was evaluated automatically by applying a motorized inverted microscope (Olympus 1X81, Olympus corp., Japan) with a motorized scanning stage (IM120, Marzhauser, Germany),400nm, 490nm, 525nm and 635nm Light Emitting Diodes (LEDs) (precis exact, CoolLED, UK) and a grayscale camera (PS4, Kappa, Germany). The interpretation system is controlled by specially designed software, and comprises a device and an automatic focusing control, image analysis and pattern recognition algorithm module. The new automated focusing is based on Haralick image identification of the target by grey scale conversion, using 4', 6-diamidino-2-phenylindole (DAPI) as a fluorescent dye for target identification and focusing. To eliminate artifacts, additional qualitative image analysis is performed by dividing the image into equally sized sub-objects.

Object segmentation is performed using a histogram-based thresholding algorithm followed by a watershed transform. Object segmentation is characterized by regionality, topology, and texture/surface descriptors. A goal description criterion exceeding 1.400 is implemented.

Immunofluorescence image data were evaluated according to the following hierarchy: i) positive staining signal, ii) staining location (cells or microparticles), and iii) determining cell staining pattern: perinuclear, cytosolic.

Cells were identified by DAPI staining and microparticles by rhodamine fluorescence. FITC fluorescence is also used to identify synthetic substrates. Cy 5-specific immunofluorescence was analyzed in the third fluorescence channel specifically binding to the antibody. Features are classified by a combination of structure and texture via defining rules for each object.

The Reactivity Index (RI) is calculated by combining absolute image intensity, contrast (contrast), and grayscale evaluation image data of the entire image. Since RI is affected by exposure time, which depends on the highest image signal after excluding artifacts, even a pattern with weak absolute signals can be detected. The threshold for distinguishing positive signals was determined based on the RI values of 200 normal blood donors.

The invention is further described by the following examples. These examples do not limit the scope of the present invention.

Example 1: antibody for simultaneously detecting ANCA and MPO

For antibodies that detect ANCA and MPO simultaneously, suspended human granulocytes and MPO-coated beads were fixed in the wells of a 6-well diagnostic slide. The nuclei of granulocytes were detected by staining with DAPI, while the coated beads were localized by rhodamine labeling. For IIF, the combined granulocyte/bead slides were incubated with 1:20 diluted serum. After washing, the immune complexes formed were detected by incubating the samples with Cy 5-conjugated goat anti-human IgG.

Slides with granulocytes and MPO coated beads were analyzed (fig. 1). In the upper part of the figure, staining images of pANCA sera taken against three different channels DAPI (left), FITC/rhodamine (middle) and Cy5 (right) were confirmed. Below the figure, slides of the combination stained with cANCA serum are shown. In a particular Cy5 channel, images stained with pANCA serum alone also showed positive staining of MPO-coated microparticles.

Thus, perinuclear staining of granulocytes, typically due to MPO antibodies, was confirmed by staining of MPO-coated beads.

Example 2: antibodies for simultaneous detection of ANCA and PR3

For antibodies that simultaneously detect ANCA and PR3, suspended human granulocytes and PR3 coated beads were fixed in the wells of a 6-well diagnostic slide. The nuclei of granulocytes were detected by staining with DAPI, while the coated beads were localized by rhodamine labeling. For IIF, the combined granulocyte/bead slides were incubated with 1:20 diluted serum. After washing, the immune complexes formed were detected by incubating the samples with Cy 5-conjugated goat anti-human IgG.

Slides with granulocytes and PR3 coated beads were analyzed (fig. 2). In the upper part of the figure, staining images of pANCA sera taken against three different channels DAPI (left), FITC/rhodamine (middle) and Cy5 (right) were confirmed. Below the figure, slides of the combination stained with cANCA serum are shown. FIG. 2 shows a dyed image using PR 3-coated microparticles instead of MPO-coated microparticles of example 1. In contrast to example 1, cANCA serum was shown below the figure to confirm a positive reaction with PR3 coated beads. Thus, cytoplasmic staining of granulocytes, typically due to PR3 antibody, was confirmed by staining of PR3 coated beads.

Example 3: assessment of disease specificity and control sera

To test the specificity of detection of ANCA, anti-PR 3 and-MPO antibodies by IIF on slides with granulocytes and antigen-coated microparticles, 10 anti-PR 3 positive WG patient sera, 10 anti-MPO positive AASV patient sera, 10 SLE patient sera and 10 donor sera were evaluated. The diagnosis of WG is based on Chapel Hill Consensus Definitions for WG. 10 patients who met the SLE diagnostic criteria were enrolled in this study as disease controls. Sera from 10 donors were used as healthy controls.

ANCA reactivity was determined from immunofluorescence patterns of positive granulocyte staining. Reactivity of MPO or PR3 coated microparticles was simultaneously assessed by detecting positive microparticle staining.

Almost all WG sera were cANCA positive in IIF, indicating positive reactivity with PR3 coated beads (90%) and cANCA pattern with fixed granulocytes (100%) of the combination slides. In contrast, all AASV sera positive for pANCA in IIF demonstrated positive reactivity with MPO-coated beads and a pANCA pattern with immobilized granulocytes. All control patients were negative for either fixed granulocytes or antigen coated microparticles. The results are shown in Table 6. Further studies using the methods of the invention to diagnose diseases associated with ANA revealed similar effective results.

TABLE 6

Simultaneous detection of ANCA on granulocytes and MPO and PR3 antibodies on antigen-coated beads

Claims (31)

1. Use of a mixture of a cellular substrate and a synthetic substrate selected from microparticles or beads coated with purified natural and/or recombinant antigens for the preparation of a kit for the diagnosis of a disease comprising the simultaneous detection of antibodies binding to one or more cellular substrates and to one or more synthetic substrates, characterized in that:

a) providing a mixture of a cellular substrate and a synthetic substrate,

b) incubating the substrate mixture with a sample containing the antibody to be detected,

c) detecting and/or identifying cellular substrates from synthetic substrates and antibodies bound to said substrates using fluorescence microscopy, and

d) the immunofluorescence image data is evaluated.

2. Use according to claim 1, characterized in that the cellular substrate is a HEp-2 cell, a human granulocyte and/or an organ tissue.

3. Use according to claim 2, characterized in that the organ tissue is pancreatic tissue.

4. Use according to claim 1 or 2, characterized in that the synthetic substrate is a particle or bead coated with purified natural and/or recombinant antigens.

5. Use according to any one of the preceding claims, characterized in that the antigen coating of the synthetic substrate is bound to an antibody associated with the presence of a disease.

6. Use according to any one of the preceding claims, characterized in that the optical, fluorescent and/or physical properties of the substrate are used for identifying the substrate.

7. Use according to the preceding claim, characterized in that the physical properties of fluorescence properties and/or size of the fluorophore concentration are used to identify the synthetic substrate.

8. Use according to the preceding claim, characterized in that the particle size is between 1 and 100 μm.

9. Use according to claim 7, characterized in that the fluorophore concentration is a rhodamine concentration.

10. Use according to any one of the preceding claims, characterized in that multicolor fluorescence microscopy is used for identifying the substrate and/or the bound autoantibodies, wherein the substrate and/or the bound antibody exhibit different fluorescence colors.

11. Use according to the preceding claim, characterized in that the different fluorescent colours are blue, green and red.

12. Use according to any one of the preceding claims, characterized in that the cellular substrate is labeled with a fluorescent dye emitting blue light, the synthetic substrate is labeled with a fluorescent dye emitting green light, and/or the specifically bound antibody is detected by an anti-human immunoglobulin specific antibody labeled with a fluorescent dye emitting red light.

13. Use according to claim 12, characterized in that the blue-emitting fluorescent dye is DAPI.

14. Use according to claim 12, characterized in that the green-emitting fluorescent dye is rhodamine or FITC.

15. Use according to claim 12, characterized in that the red-emitting fluorescent dye is Cy5 and/or Allophycocyanin (APC).

16. Use according to any one of the preceding claims, characterized in that the disease is an autoimmune or infectious disorder, autoimmune liver disease, type 1 diabetes, lyme disease or herpes simplex virus.

17. Use according to the preceding claim, characterized in that the autoimmune disorder is a systemic rheumatic disease.

18. Use according to any one of the preceding claims, characterized in that the antibody to be detected is an antinuclear antibody (ANA) or an anti-neutrophil cytoplasmic antibody (ANCA).

19. Use according to any of the preceding claims, characterized in that HEp-2 cells are used for analyzing staining of antinuclear antibodies (ANA).

20. Use according to claim 18, characterized in that human granulocytes are used for the analysis of the staining of anti-neutrophil cytoplasmic antibodies (ANCA).

21. Use according to claim 18, characterized in that the anti-neutrophil cytoplasmic antibody (ANCA) exhibits perinuclear staining (pANCA) or punctate cytoplasmic staining (cANCA) of human granulocytes.

22. Use according to the preceding claim, characterized in that the antibody is targeted to the protease 3(PR3) or Myeloperoxidase (MPO) protein, or an antigen thereof.

23. Use according to the preceding claim, characterized in that the antibody exhibits a spotted cytoplasmic staining (cANCA) of human granulocytes and binds to protease 3(PR3), wherein the protease 3 protein or antigen thereof is provided on a synthetic substrate, thereby providing a diagnosis of Wegener's Granulomatosis (WG).

24. Use according to any one of claims 20-22, characterized in that the antibody exhibits perinuclear staining (pANCA) of human granulocytes and binds Myeloperoxidase (MPO), wherein the myeloperoxidase or an antigen thereof is provided on a synthetic substrate, thereby providing a diagnosis of ANCA-associated vasculitis (AASV).

25. Use according to any one of the preceding claims, characterized in that the sample comprises blood, serum, cerebrospinal fluid, synovial fluid or saliva obtained from a subject.

26. Use according to any of the preceding claims, characterized in that the immunofluorescence image data evaluation is performed using an automated pattern recognition interpretation system, wherein the interpretation system is controlled by specially designed software, comprising modules for device and autofocus control, image acquisition, image analysis and/or pattern recognition algorithms.

27. Use according to any of the preceding claims, characterized in that the classification of staining patterns into cell or synthetic substrate classes and the determination of cell patterns is achieved by a combination of structural and textural features of the immunofluorescence image by defining a set of rules for each pattern.

28. A kit for the simultaneous detection of antibodies binding to one or more cellular substrates and one or more synthetic substrates according to claims 1-27, comprising:

a) a slide having immobilized cellular substrate mixed with antigen-coated synthetic substrate, wherein the synthetic substrates differ from each other by their optical, fluorescent and/or physical properties,

b) conjugates having an immunoglobulin-specific antibody conjugated to a fluorescent label.

29. Kit according to claim 28, characterized in that the cellular substrate is a HEp-2 cell and/or a human granulocyte.

30. Kit according to claim 28, characterized in that the fluorescent label is FITC, Cy5 and/or APC.

31. Kit according to any one of the preceding claims, characterized in that the kit further comprises a wash buffer, a cover slip, a cover medium, an uncoated synthetic substrate with or without a fluorescent label, and/or an additional fluorescent label for the synthetic substrate.