EP3756013A1 - Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases - Google Patents

Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases

Info

Publication number
EP3756013A1
EP3756013A1 EP19711171.9A EP19711171A EP3756013A1 EP 3756013 A1 EP3756013 A1 EP 3756013A1 EP 19711171 A EP19711171 A EP 19711171A EP 3756013 A1 EP3756013 A1 EP 3756013A1
Authority
EP
European Patent Office
Prior art keywords
antibody
immune complex
naprt
disease
bind
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19711171.9A
Other languages
German (de)
French (fr)
Inventor
Silvia DEAGLIO
Valentina AUDRITO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Italian Institute For Genomic Medicine (iigm)
Universita degli Studi di Torino
Original Assignee
Italian Institute For Genomic Medicine (iigm)
Universita degli Studi di Torino
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Italian Institute For Genomic Medicine (iigm), Universita degli Studi di Torino filed Critical Italian Institute For Genomic Medicine (iigm)
Publication of EP3756013A1 publication Critical patent/EP3756013A1/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Definitions

  • the present invention relates to the field of in vitro diagnosis of tumour disease and acute inflammatory disease.
  • Neoplastic disease primarily solid tumours, as well as inflammatory acute disease, in particular sepsis, are surely among the most difficult diseases to be diagnosed correctly due to the considerable clinical complexity thereof.
  • Tumours are one of the main worldwide causes of morbidity and mortality due to the difficult identification of these diseases at a sufficiently early stage such as to allow effective therapeutic intervention. Therefore, in the cancer field, the majority of diagnostic approaches are still based on radiological and anatomical-pathological methods, which are often invasive for the patient and associated with high costs.
  • circulating biomarkers i.e. those substances whose presence and/or increased concentration in a biological fluid is associated with a specific pathological condition or its particular progress, plays a major role. Since they are present in an easily accessible tissue such as blood, determining the circulating markers allows a sort of "liquid biopsy” to be performed and a result, even in quantitative terms, to be obtained quickly and at low cost, also preventing the patient from being subject to often complex and painful invasive methods.
  • the object of the present invention is to provide a diagnostic method, which allows patients suffering from a tumour disease or an acute inflammatory disease to be identified with a high degree of accuracy, preferably in an initial stage of the disease.
  • NAPRT nicotinic acid phosphoribosyltransferase
  • one object of the present invention is an in vitro method for the diagnosis and/or prognosis of a tumour disease and/or an acute inflammatory disease, comprising the step of determining, by a fluorescence-based suspension immunoassay, in a biological fluid sample from a subject affected by or suspected of being affected by a tumour disease and/or an acute inflammatory disease, the level of the nicotinic acid phosphoribosyltransferase enzyme (NAPRT), wherein the level of the NAPRT enzyme above a predetermined threshold is indicative of a tumour disease and/or an acute inflammatory disease.
  • NAPRT nicotinic acid phosphoribosyltransferase enzyme
  • the NAPRT enzyme is an intracellular enzyme that plays a key role in the NAD + cofactor recycling pathway starting from nicotinic acid, whose expression is mainly located at the hepatic and renal tissue cells.
  • NAPRT EXPRESSION IN HUMAN TISSUES AND TUMORS.
  • Oncotarget 2016; 7(2): 1973-83 also revealed transcriptional expression of the gene encoding the NAPRT enzyme in several human tumour cell lines, including cells from lung cancer and colon cancer.
  • This threshold value is comprised within a concentration range of from 1 to 3 ng/ml. Therefore, the determination, in a biological fluid sample, of a concentration value for the NAPRT enzyme greater than 2 ng/ml is indicative of tumour disease and/or acute inflammatory disease.
  • tumour disease preferably even if not exclusively, of solid tumours, preferably lung, prostate and bladder carcinoma, melanoma, mesothelioma and sarcoma, and blood neoplasms, preferably chronic lymphocytic leukaemia, myeloma and lymphoma.
  • Acute inflammatory diseases include, for example, but not exclusively, sepsis, septic shock and systemic inflammatory response syndrome (SIRS).
  • the method according to the present invention is a suspension immunoassay in which the solid phase, on which the affinity reagents employed for capturing the NAPRT enzyme are immobilized, is dispersed in a solution.
  • this assay fonnat ensures considerable analytical sensitivity as it allows the affinity reactions to be measured within very wide analyte concentration ranges, thereby providing robust results in short times, even in real time.
  • the affinity reagent used in the method of the invention is an antibody, the so-called first antibody or capture antibody, preferably a monoclonal or polyclonal antibody, suitable to selectively bind the above enzyme.
  • a second antibody the so-called detection antibody
  • the detection antibody binds, on the antigen molecule, an epitope different from the epitope that is bound. by the first antibody.
  • the two different antibody populations can be added to the test sample simultaneously or can be reacted in sequential order (the "one step” or "two steps” techniques).
  • said second antibody is a polyclonal antibody.
  • the method according to the invention in addition to a second antibody, uses a third antibody, which is suitable to bind the secondary immune complex formed between the primary immune complex and said second antibody.
  • the third antibody may be, for example, a species-specific antibody, i.e. an antibody able to recognize and bind the antibodies of the host animal species in which the second antibody used in the assay was generated.
  • the use of a third antibody allows an additional level of specificity to be added in the qualitative or quantitative analysis of the NAPRT enzyme present in the biological fluid test sample.
  • the present invention employs fluorescence-based measurements means.
  • the formation of the primary immune complex can be determined by using said second antibody or said third antibody labelled with a fluorescent dye.
  • the use of the second labelled antibody can only occur in the absence of the third antibody, since the presence of a fluorochrome conjugated to the second antibody and the consequent steric hindrance resulting therefrom would impair the subsequent binding of the third antibody.
  • fluorescent labels most commonly used in immunoassays, exemplary, but not limiting, fluorescent labels are phycoerythrin, fluorescein isothiocyanate and tetramethylrhodamine, which give signal stability and strength, usually measurable in real time, to the labelled moiety.
  • the quantitative determination of the NAPRT enzyme in biological fluid samples can occur by using a standard curve set up by employing known quantities of the NAPRT protein, preferably a recombinant NAPRT protein, and then interpolating on the above curve fluorescence signals measured in the examined samples.
  • a standard curve set up by employing known quantities of the NAPRT protein, preferably a recombinant NAPRT protein, and then interpolating on the above curve fluorescence signals measured in the examined samples.
  • any method for quantitative analytical assessment in immunoassays is within the scope of the present invention, and the selection of which method is within the skills of those of ordinary skill in the art.
  • the suspended solid phase immobilizing the primary antibody used in the method of the invention is a magnetic bead, on whose surface the formation of the primary immune complex and the immunometric assay occur.
  • the use of magnetic beads as a solid support in an immunoassay facilitates their separation from the solution in which they are suspended by means of a magnet.
  • this embodiment of the method according to the invention allows operations on small sample volumes as well as a significantly larger analyte dynamic range to be measured compared to the prior art solid- support immunoassays, among which ELISA immunoassays are mentioned, for example.
  • the method according to the invention is a multiple format suspension immunoassay, wherein the determination of the NAPRT enzyme takes place in parallel with the measurement of other analytes occurring in the same biological sample at concentrations that can also be very different from each other.
  • the magnetic beads acting as a solid phase for immobilizing the primary capture antibodies are conjugated to two fluorochromes having different emission spectra, occurring in varying ratios.
  • the particular combination of fluorochromes therefore represents a sort of labelling, which, once detected by means of a detection laser, allows beads conjugated to different antibodies to be distinguished from one another after they have been mixed together within the same sample.
  • the formation of specific immune complexes on the conjugated beads is detected by using an antibody labelled with a fluorescent dye that has spectral properties different from those possessed by the two fluorochromes used for the labelling/identification of the beads.
  • the diagnostic method of the invention may employ any type of biological fluid sample suitable for assessing the protein content, preferably blood samples and derivatives thereof, such as serum and plasma or cell culture supernatants.
  • kit including means suitable to perform the method according to the ihvention, as defined in claim 11, is also included within the scope of the present invention.
  • EXAMPLE 1 NAPRT suspension immunoassay The present inventors have set up a suspension immunoassay suitable for assessing the NAPRT enzyme levels in clinical biological fluid samples, according to the scheme described in Figure 1, In short, a set of magnetic beads (Bio-Rad) was chemically conjugated to a monoclonal antibody (ProteinTech) capable of specifically binding the C- terminal region of the NAPRT protein.
  • a set of magnetic beads Bio-Rad
  • a monoclonal antibody ProteinTech
  • the suspension of conjugated beads (100 beads/pl Bio-Rad reaction buffer) was distributed in a 96-well plate (50 pl/well) and subjected to two subsequent washing steps.
  • the analysis was performed on plasma and serum samples obtained from patients with cancer (312 patients), inflammatory disease (93 patients), as well as healthy subjects (121 individuals).
  • the above samples were diluted 1 :4 by using the Bio-Rad buffer.
  • a standard curve was set up by using known decreasing concentrations of the recombinant NAPRT protein, from 200 ng/ml to 0.035 ng/ml, in Bio-Rad dilution buffer mimicking the human serum.
  • clinical samples and standard samples were distributed in duplicate in the 96-well plate containing the conjugated beads (25 pl/well), according to a predefined scheme. The plate was then incubated for 3 hours at room temperature under stirring and subsequently subjected to three washing rounds in the buffer.
  • each well of the plate was then added with the solution containing the polyclonal detection antibody (antibody concentration 5 pg/ml), and the plate was incubated for 90 minutes at room temperature under stirring and subsequently subjected to three washing rounds in the buffer.
  • the detection antibody used in the assay specifically recognizes the central portion of the NAPRT protein.
  • each well of the plate was added with the solution containing the polyclonal anti-detection antibody species (rabbit) antibody conjugated to the Phycoerythrin (PE) fluorochrome (antibody concentration 3 pg/ml), and the plate was incubated for 30 minutes at room temperature under stirring.
  • Bio-Rad buffer a 125 pl-volume of Bio-Rad buffer was dispensed into each of the 96 wells of the plate. The plate was incubated for five minutes at room temperature under stirring, and subsequently subjected to cytofluorimeter high sensitivity reading by analysing the red fluorescence (phycoerythrin).
  • EXAMPLE 2 interpretation of the results
  • the present inventors have set up a standard curve by using known decreasing concentrations of the recombinant NAPRT protein, from 200 ng/ml to 0.035 ng/ml, in Bio-Rad dilution buffer mimicking the composition of human serum.
  • the fluorescence values measured in the clinical samples according to the above-described method have been interpolated on the values obtained for the standard curve and translated into a corresponding protein concentration value (ng/ml), taking into account the dilution factor to which the samples had been subjected prior to the immunoassay.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

A method for in vitro diagnosis and/or prognosis of a tumour disease or an acute inflammatory disease and the related kit are disclosed. The method of the invention is based on the quantitative determination of the nicotinic acid phosphoribosyltransferase enzyme (NAPRT) in a biological fluid sample from a subject suspected of being affected by a tumour disease or an acute inflammatory disease, wherein the level of said enzyme above a predetermined threshold is indicative of a tumour disease and/or an acute inflammatory disease. The biological fluid sample is blood, plasma or serum or cell culture supernatant.

Description

Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases
The present invention relates to the field of in vitro diagnosis of tumour disease and acute inflammatory disease.
Neoplastic disease, primarily solid tumours, as well as inflammatory acute disease, in particular sepsis, are surely among the most difficult diseases to be diagnosed correctly due to the considerable clinical complexity thereof.
Tumours are one of the main worldwide causes of morbidity and mortality due to the difficult identification of these diseases at a sufficiently early stage such as to allow effective therapeutic intervention. Therefore, in the cancer field, the majority of diagnostic approaches are still based on radiological and anatomical-pathological methods, which are often invasive for the patient and associated with high costs.
Early diagnosis of acute inflammatory disease has also received increased attention in recent years. In particular, there has been a significant increase in the incidence and mortality rates of sepsis, understood as the pathological picture characterized by systemic inflammatory response syndrome and infection simultaneously. The increase in the frequency of aggressive surgical interventions, the spread of multiresistant microorganisms as well as the ever-increasing presence of immunocompromised patients are among the factors that have certainly affected the rapid increase in the incidence of sepsis. The clinical-epidemiological significance of acute inflammatory disease concerns all its various manifestations (sepsis, severe sepsis and septic shock), generally associated with high patient management costs.
In the field of diagnostics in general and of tumour disease and acute inflammatory disease in particular, the use of circulating biomarkers, i.e. those substances whose presence and/or increased concentration in a biological fluid is associated with a specific pathological condition or its particular progress, plays a major role. Since they are present in an easily accessible tissue such as blood, determining the circulating markers allows a sort of "liquid biopsy” to be performed and a result, even in quantitative terms, to be obtained quickly and at low cost, also preventing the patient from being subject to often complex and painful invasive methods.
Despite the above diagnostic value, the majority of biomarkers occurring in biological fluids and currently used in the field of diagnosis of tumour disease and acute inflammatory disease find poor application substantially due to poor sensitivity and specificity of the above mentioned markers. In fact, said application is often limited to particular patient subgroups in which the risk of the onset of the tumour or acute inflammatory disease is particularly high.
In this context, therefore, new biomarkers for the primary diagnosis and the prognostic evaluation of a tumour or acute inflammatory disease, which do not have the drawbacks mentioned above, are dramatically needed.
Therefore, the object of the present invention is to provide a diagnostic method, which allows patients suffering from a tumour disease or an acute inflammatory disease to be identified with a high degree of accuracy, preferably in an initial stage of the disease.
This and other objects are achieved by means of the in vitro method for diagnosis and/or prognosis of a tumour disease or an acute inflammatory disease as defined in appended claim 1 and of the related kit as defined in appended claim 11. Preferred embodiments of the method and the kit of the invention are defined in the dependent claims.
The appended independent and dependent claims form an integral part of the present specification.
As will be explained in more detail in the experimental section that follows, the present inventors have surprisingly found that serum and plasma samples collected from patients suffering from a tumour disease or from sepsis exhibit a significant increase in the levels of the nicotinic acid phosphoribosyltransferase (NAPRT) enzyme compared to control samples from healthy subjects. The NAPRT enzyme, therefore, is an ideal circulating biomarker associated with tumour disease or acute inflammatory disease, in particular sepsis, thereby allowing the set up of diagnostic methods aimed at identifying patients in an early stage of the tumour or acute inflammatory disease, for which an early surgical and/or therapeutic intervention significantly correlates with the subsequent survival rates.
Therefore, one object of the present invention is an in vitro method for the diagnosis and/or prognosis of a tumour disease and/or an acute inflammatory disease, comprising the step of determining, by a fluorescence-based suspension immunoassay, in a biological fluid sample from a subject affected by or suspected of being affected by a tumour disease and/or an acute inflammatory disease, the level of the nicotinic acid phosphoribosyltransferase enzyme (NAPRT), wherein the level of the NAPRT enzyme above a predetermined threshold is indicative of a tumour disease and/or an acute inflammatory disease.
The NAPRT enzyme is an intracellular enzyme that plays a key role in the NAD+ cofactor recycling pathway starting from nicotinic acid, whose expression is mainly located at the hepatic and renal tissue cells. The research described in Duarte-Pereira S et al, EXTENSIVE REGULATION OF NICOTINATE PHOSPHORIBOSYLTRANSFERASE
(NAPRT) EXPRESSION IN HUMAN TISSUES AND TUMORS. Oncotarget 2016; 7(2): 1973-83 also revealed transcriptional expression of the gene encoding the NAPRT enzyme in several human tumour cell lines, including cells from lung cancer and colon cancer.
As shown in the graph in Figure 2, the determination of an increase in the levels of the circulating NAPRT enzyme by immunological test on a patient cohort representative of the tumour disease (312 patients) and the acute inflammatory disease (93 patients), compared to the levels measured in the healthy controls (121 subjects), allowed the present inventors to determine a concentration threshold value for the circulating NAPRT enzyme, thereby enabling discrimination between healthy subjects and affected subjects. This threshold value is comprised within a concentration range of from 1 to 3 ng/ml. Therefore, the determination, in a biological fluid sample, of a concentration value for the NAPRT enzyme greater than 2 ng/ml is indicative of tumour disease and/or acute inflammatory disease.
With reference to the tumour disease, mention can be made, for example, even if not exclusively, of solid tumours, preferably lung, prostate and bladder carcinoma, melanoma, mesothelioma and sarcoma, and blood neoplasms, preferably chronic lymphocytic leukaemia, myeloma and lymphoma. Acute inflammatory diseases include, for example, but not exclusively, sepsis, septic shock and systemic inflammatory response syndrome (SIRS).
The method according to the present invention is a suspension immunoassay in which the solid phase, on which the affinity reagents employed for capturing the NAPRT enzyme are immobilized, is dispersed in a solution. In addition to the high degree of specificity due to the immunological nature of the reaction, this assay fonnat ensures considerable analytical sensitivity as it allows the affinity reactions to be measured within very wide analyte concentration ranges, thereby providing robust results in short times, even in real time.
According to a preferred embodiment, the affinity reagent used in the method of the invention is an antibody, the so-called first antibody or capture antibody, preferably a monoclonal or polyclonal antibody, suitable to selectively bind the above enzyme.
In order to increase the specificity of the method according to the invention, in a preferred embodiment, a second antibody, the so-called detection antibody, is used, which is suitable to detect the immune complex formed between the test antigen and the first antibody. Typically, the detection antibody binds, on the antigen molecule, an epitope different from the epitope that is bound. by the first antibody. The two different antibody populations can be added to the test sample simultaneously or can be reacted in sequential order (the "one step" or "two steps” techniques). Preferably, said second antibody is a polyclonal antibody.
According to another preferred embodiment shown in Figure 1, the method according to the invention, in addition to a second antibody, uses a third antibody, which is suitable to bind the secondary immune complex formed between the primary immune complex and said second antibody. The third antibody may be, for example, a species-specific antibody, i.e. an antibody able to recognize and bind the antibodies of the host animal species in which the second antibody used in the assay was generated. The use of a third antibody allows an additional level of specificity to be added in the qualitative or quantitative analysis of the NAPRT enzyme present in the biological fluid test sample.
As regards the detection of the NAPRT enzyme, more specifically of the immune complexformed between this protein and the primary antibody, the present invention employs fluorescence-based measurements means. For example, the formation of the primary immune complex can be determined by using said second antibody or said third antibody labelled with a fluorescent dye. Of course, the use of the second labelled antibody can only occur in the absence of the third antibody, since the presence of a fluorochrome conjugated to the second antibody and the consequent steric hindrance resulting therefrom would impair the subsequent binding of the third antibody. Among the fluorescent labels most commonly used in immunoassays, exemplary, but not limiting, fluorescent labels are phycoerythrin, fluorescein isothiocyanate and tetramethylrhodamine, which give signal stability and strength, usually measurable in real time, to the labelled moiety.
In the method according to the present invention, the quantitative determination of the NAPRT enzyme in biological fluid samples can occur by using a standard curve set up by employing known quantities of the NAPRT protein, preferably a recombinant NAPRT protein, and then interpolating on the above curve fluorescence signals measured in the examined samples. Of course, the use of any method for quantitative analytical assessment in immunoassays is within the scope of the present invention, and the selection of which method is within the skills of those of ordinary skill in the art.
According to a preferred embodiment, the suspended solid phase immobilizing the primary antibody used in the method of the invention is a magnetic bead, on whose surface the formation of the primary immune complex and the immunometric assay occur. Typically, the use of magnetic beads as a solid support in an immunoassay facilitates their separation from the solution in which they are suspended by means of a magnet.
Thanks to the outstanding binding ability of magnetic beads in combination with the considerable accuracy of quantitative determinations, this embodiment of the method according to the invention allows operations on small sample volumes as well as a significantly larger analyte dynamic range to be measured compared to the prior art solid- support immunoassays, among which ELISA immunoassays are mentioned, for example.
According to a further embodiment, the method according to the invention is a multiple format suspension immunoassay, wherein the determination of the NAPRT enzyme takes place in parallel with the measurement of other analytes occurring in the same biological sample at concentrations that can also be very different from each other.
In this embodiment, the magnetic beads acting as a solid phase for immobilizing the primary capture antibodies are conjugated to two fluorochromes having different emission spectra, occurring in varying ratios. The particular combination of fluorochromes therefore represents a sort of labelling, which, once detected by means of a detection laser, allows beads conjugated to different antibodies to be distinguished from one another after they have been mixed together within the same sample. The formation of specific immune complexes on the conjugated beads is detected by using an antibody labelled with a fluorescent dye that has spectral properties different from those possessed by the two fluorochromes used for the labelling/identification of the beads.
The diagnostic method of the invention may employ any type of biological fluid sample suitable for assessing the protein content, preferably blood samples and derivatives thereof, such as serum and plasma or cell culture supernatants.
As previously stated, a kit including means suitable to perform the method according to the ihvention, as defined in claim 11, is also included within the scope of the present invention.
The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.
EXAMPLE 1 : NAPRT suspension immunoassay The present inventors have set up a suspension immunoassay suitable for assessing the NAPRT enzyme levels in clinical biological fluid samples, according to the scheme described in Figure 1, In short, a set of magnetic beads (Bio-Rad) was chemically conjugated to a monoclonal antibody (ProteinTech) capable of specifically binding the C- terminal region of the NAPRT protein.
Then, the suspension of conjugated beads (100 beads/pl Bio-Rad reaction buffer) was distributed in a 96-well plate (50 pl/well) and subjected to two subsequent washing steps.
The analysis was performed on plasma and serum samples obtained from patients with cancer (312 patients), inflammatory disease (93 patients), as well as healthy subjects (121 individuals). The above samples were diluted 1 :4 by using the Bio-Rad buffer. In parallel, a standard curve was set up by using known decreasing concentrations of the recombinant NAPRT protein, from 200 ng/ml to 0.035 ng/ml, in Bio-Rad dilution buffer mimicking the human serum. After dilution, clinical samples and standard samples were distributed in duplicate in the 96-well plate containing the conjugated beads (25 pl/well), according to a predefined scheme. The plate was then incubated for 3 hours at room temperature under stirring and subsequently subjected to three washing rounds in the buffer. Each well of the plate was then added with the solution containing the polyclonal detection antibody (antibody concentration 5 pg/ml), and the plate was incubated for 90 minutes at room temperature under stirring and subsequently subjected to three washing rounds in the buffer. The detection antibody used in the assay specifically recognizes the central portion of the NAPRT protein. Lastly, each well of the plate was added with the solution containing the polyclonal anti-detection antibody species (rabbit) antibody conjugated to the Phycoerythrin (PE) fluorochrome (antibody concentration 3 pg/ml), and the plate was incubated for 30 minutes at room temperature under stirring. After three successive washing rounds in the buffer, a 125 pl-volume of Bio-Rad buffer was dispensed into each of the 96 wells of the plate. The plate was incubated for five minutes at room temperature under stirring, and subsequently subjected to cytofluorimeter high sensitivity reading by analysing the red fluorescence (phycoerythrin).
EXAMPLE 2: interpretation of the results In order to quantitatively assess the NAPRT enzyme occurring in clinical test samples, the present inventors have set up a standard curve by using known decreasing concentrations of the recombinant NAPRT protein, from 200 ng/ml to 0.035 ng/ml, in Bio-Rad dilution buffer mimicking the composition of human serum. The fluorescence values measured in the clinical samples according to the above-described method have been interpolated on the values obtained for the standard curve and translated into a corresponding protein concentration value (ng/ml), taking into account the dilution factor to which the samples had been subjected prior to the immunoassay.
The results of the suspension immunoassay, shown in the graphs in Figure 2, in addition to confirming the presence of the NAPRT enzyme in the blood of a validation cohort consisting of 96 healthy donors (Figure 2A), show a significant increase in the levels of this enzyme in blood samples from patients suffering from tumour disease or sepsis, compared to healthy controls (Figure B), thus supporting the important diagnostic value of the NAPRT enzyme.

Claims

1. An m vitro method for the diagnosis and/or prognosis of a tumour disease and/or an acute inflammatory disease, comprising the step of determining, by a fluorescence-based suspension immunoassay, in a biological fluid sample from a subject affected by or suspected of being affected by a tumour disease and/or an acute inflammatory disease, the level of the nicotinic acid phosphoribosyltransferase enzyme (NAPRT), wherein the level of the NAPRT enzyme above a predetermined threshold is indicative of a tumour disease and/or an acute inflammatory disease.
2. The method according to claim 1 , wherein the immunoassay is carried out with at least one first antibody suitable to bind the NAPRT enzyme, thereby forming a first immune complex.
3. The method according to claim 2, wherein the antibody is a polyclonal or a monoclonal antibody.
4. The method according to claim 2 or 3, further comprising the step of detecting tile formation of said first immune complex by employing a second antibody suitable to bind said first immune complex, thereby forming a second immune complex.
5. The method according to claim 4, further comprising the step of detecting the formation of said second immune complex by employing a third antibody suitable to bind said second immune complex, thereby forming a third immune complex.
6. The method according to claim 4 or 5, wherein said second antibody or said third antibody is fluorescently labelled, with the proviso that said second antibody is fluorescently labelled when the third antibody is not employed.
7. The method according to any one of claims 2 to 6, wherein said at least one first antibody suitable to bind the NAPRT enzyme is immobilized on a magnetic bead.
8. The method according to any one of claims 1 to 7, wherein the biological fluid sample is blood, plasma, serum or cell culture supernatant.
9. The method according to any one of claims 1 to 8, wherein the tumour disease is selected from the group consisting of lung carcinoma, prostate carcinoma, bladder carcinoma, melanoma, mesothelioma, sarcoma, chronic lymphocytic leukemia, myeloma and lymphoma.
10. The method according to any one of claims 1 to 8, wherein the acute inflammatory disease is selected from the group consisting of sepsis, septic shock and systemic inflammatory response syndrome (SIRS).
1 1. A kit for in vitro diagnosis of a tumour disease and/or an acute inflammatory disease, comprising at least one first polyclonal or monoclonal antibody suitable to bind the NAPRT en2yme, thereby forming a first immune complex, as well as means suitable for detecting the formation of said first immune complex, wherein said at least one first antibody is immobilized on a magnetic bead and wherein said suitable means comprise a fluorescent label.
12. The kit according to claim 11 , wherein the means for detecting the first immune complex comprise a second antibody suitable to bind the first immune complex, thereby forming a second immune complex, and, optionally, a third antibody suitable to bind said second immune complex, thereby forming a third immune complex, said second antibody or said third antibody being fluorescently labelled.
EP19711171.9A 2018-02-20 2019-02-19 Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases Pending EP3756013A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102018000002866A IT201800002866A1 (en) 2018-02-20 2018-02-20 Immunological procedure and kit for the in vitro diagnosis of tumor and / or inflammatory pathologies.
PCT/IB2019/051314 WO2019162821A1 (en) 2018-02-20 2019-02-19 Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases

Publications (1)

Publication Number Publication Date
EP3756013A1 true EP3756013A1 (en) 2020-12-30

Family

ID=62143519

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19711171.9A Pending EP3756013A1 (en) 2018-02-20 2019-02-19 Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases

Country Status (3)

Country Link
EP (1) EP3756013A1 (en)
IT (1) IT201800002866A1 (en)
WO (1) WO2019162821A1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8912184B1 (en) * 2010-03-01 2014-12-16 Alzheimer's Institute Of America, Inc. Therapeutic and diagnostic methods
KR101941054B1 (en) * 2016-07-20 2019-01-23 연세대학교 산학협력단 Composition for predicting prognosis of cancer and kit comprising the same

Also Published As

Publication number Publication date
IT201800002866A1 (en) 2019-08-20
WO2019162821A1 (en) 2019-08-29

Similar Documents

Publication Publication Date Title
JP6749337B2 (en) Prostate antigen standards and their use
US20090298097A1 (en) Methods for the diagnosis of lung cancer
US20120295814A1 (en) CA-125 Immune Complexes as Biomarkers of Ovarian Cancer
US20140141985A1 (en) Biomarkers of therapeutic responsiveness
US20180180617A1 (en) Intergrin alpha v beta 6 for diagnosis/prognosis of colorectal carcinoma
EP4045912A1 (en) Apparatuses and methods for detection of pancreatic cancer
CN106680515B (en) It is combined for the polymolecular marker of pulmonary cancer diagnosis
US8053198B2 (en) Diagnostic methods
Sánchez-Carbayo et al. Urinary tissue polypeptide-specific antigen for the diagnosis of bladder cancer
CN109154612B (en) Method for quantitative assessment of phosphorylated and non-phosphorylated forms of RKIP in biological samples
DK2551673T3 (en) Methods for detecting cancer infiltration in the central nervous system
US20220299514A1 (en) Biomarkers of therapeutic responsiveness
WO2016071321A2 (en) Lung cancer sub-typing method
EP3756013A1 (en) Immunological method and kit for in vitro diagnosis of tumour and/or inflammatory diseases
US11791043B2 (en) Methods of prognosing early stage breast lesions
JP5818817B2 (en) Cancer prognosis assay
US7611845B2 (en) Serum levels of HER2/neu as an indicator of clinical state and prognosis of prostate cancer
US20230400466A1 (en) Methods and systems for risk stratification and management of bladder cancer
KR101861055B1 (en) Method for Diagnosis of Pulmonary Disease by Detecting ROMO1
KR20230018174A (en) An integrated magneto­electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma
KR20240068432A (en) A kit for diagnosing cancer comprising protein biomarker in blood
El-Sharkawy et al. Circulating Tumor Cells in Breast Cancer: A Step Toward Precision Medicine for Real-Time Monitoring of Metastasis
US20100120080A1 (en) Cancer diagnosis using ki-67
WO2023193109A1 (en) Biomarkers for the determination of sample adequacy and lung cancer metastases
EP2812701A1 (en) Biomarker set for identifying a severe form of cancer

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20200918

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20240216