CN115715369A

CN115715369A - Use of H-NGAL for detecting peritonitis

Info

Publication number: CN115715369A
Application number: CN202280004351.2A
Authority: CN
Inventors: 纪永生; 黄芳芳; J·E·穆诺茨-席尔瓦; 蒋涛
Original assignee: Fresenius Medical Care Deutschland GmbH; Fresenius Medical Care Holdings Inc
Current assignee: Fresenius Medical Care Deutschland GmbH; Fresenius Medical Care Holdings Inc
Priority date: 2021-05-31
Filing date: 2022-05-30
Publication date: 2023-02-24
Also published as: EP4348253A1; WO2022253173A1; CN115480061A

Abstract

The present invention provides a method for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising: detecting H-NGAL protein in the peritoneal dialysis dialysate of a peritoneal dialysis subject, and indicating that the subject has, or is at risk of having, peritonitis if the H-NGAL protein is greater than or equal to the cut-off value. Also provided are test strips, kits and devices for use in the methods and the use of molecules that specifically bind to H-NGAL protein in the preparation of compositions, test strips or kits for diagnosing peritonitis, preferably rapid diagnosis of peritonitis, by detecting H-NGAL protein in a peritoneal dialysis permeate from a peritoneal dialysis subject.

Description

Use of H-NGAL for detecting peritonitis

Technical Field

The present invention relates to the diagnosis of peritonitis, and in particular to the diagnosis, especially rapid diagnosis, of peritonitis in a subject on peritoneal dialysis by detecting homodimeric Neutrophil gelatinase-associated lipocalin (H-NGAL) in the peritoneal dialysis permeate.

Background

Neutrophil gelatinase-associated lipocalin (NGAL) is a protein involved in innate immunity, which exists in three forms in blood and urine: a 25-kD monomer released primarily by renal tubular cells, a 45-kD homodimer (H-NGAL) synthesized primarily by neutrophils, and a 135-kD heterodimer bound by matrix metalloproteinase 9 (MMP-9).

NGAL is known to be a marker of Acute Kidney Injury (AKI) (Linjun Cai et al, "Assays of levels of HNL/NGAL in tissues underscoring cardiac surgery and The impact of anti-cancer compliance on The third clinical practice", clinical Chimica Acta 403 (2009) 121-125, DOI.

Interleukin-6 (IL-6) is a multifunctional protein produced by a variety of cells. Increase in Peritoneal Dialysis Exudate (PDE) at the onset of peritonitis, during peritonitis, and even after peritonitis.

Peritonitis is a common condition in peritoneal dialysis patients and is one of the leading causes of death and failure of peritoneal dialysis in peritoneal dialysis patients. Early detection and treatment generally leads to a good prognosis of peritoneal dialysis-related peritonitis. Peritonitis is typically detected by leukocyte count laboratory culture (3-7 days) and imaging examinations, and antibiotic therapy is adjusted based on the culture results. These methods of detecting peritonitis require specialized facilities and are long. There is a need in the art for new means to facilitate the detection of peritonitis, and in particular to enable a rapid diagnosis of peritonitis, to detect and treat peritonitis as early as possible, thereby improving peritoneal dialysis treatment.

Disclosure of Invention

The present invention provides a method for diagnosing peritonitis, in particular a rapid diagnosis of peritonitis or the risk of peritonitis, in a peritoneal dialysis subject comprising: detecting H-NGAL in the subject's peritoneal dialysis dialysate, if H-NGAL is greater than or equal to a cutoff value, indicating that the subject has, or is at risk of having, peritonitis; optionally, the method further comprises detecting IL-6 in the peritoneal dialysis permeate, if IL-6 is greater than or equal to the cut-off value, indicating that the subject has, or is at risk of having, peritonitis. By rapidly diagnosing peritonitis or peritonitis risk, peritonitis can be discovered as early as possible, thereby realizing early diagnosis and treatment.

The present invention also provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end comprising, in succession from said first end: a sample pad, a conjugate pad, a Nitrocellulose (NC) membrane, and optionally an absorbent pad, wherein the NC membrane comprises a detection line and a quality control line, wherein: the binding pad comprises a first molecule that specifically binds to H-NGAL protein, optionally labelled; the detection line comprises an immobilized species that detects complexes formed by H-NGAL protein and the first molecule, and the quality control line comprises an immobilized species that binds to the first molecule. Optionally, the binding pad further comprises a third molecule that specifically binds to IL-6 protein, optionally labeled, and the NC membrane further comprises another detection line comprising an immobilized species that detects complexes formed by IL-6 protein and third molecule; and optionally, the control line can comprise an immobilized species that binds the third molecule. The test strip for rapidly diagnosing the peritonitis or the peritonitis risk can be used at home, and rapidly and conveniently diagnoses the peritonitis or the peritonitis risk, so that early diagnosis and treatment of the peritonitis are realized.

The present invention also provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end, comprising in succession, starting from said first end: sample pad, biotinylation pad, gold mark pad, nitrocellulose membrane and absorption pad, wherein nitrocellulose membrane contains detection line and matter control line, wherein: the biotinylated pad comprises biotin-labeled first molecules that specifically bind to H-NGAL protein, the gold-labeled pad comprises colloidal gold-labeled second molecules that specifically bind to H-NGAL protein, the detection line comprises streptavidin, and the quality control line comprises immobilized molecules that specifically bind to the first and/or second molecules, wherein optionally the first and second molecules are not identical.

The present invention also provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end comprising, in succession from said first end: a sample pad, a conjugate pad, a nitrocellulose membrane, and optionally an absorbent pad, wherein: the binding pad comprises a first molecule that specifically binds to H-NGAL protein, optionally labelled, and optionally further comprises a second molecule that specifically binds to H-NGAL protein, optionally labelled, the nitrocellulose membrane comprises a detection line comprising an immobilised species that detects complexes formed by H-NGAL protein and the labelled first and/or second molecule, and a quality control line comprising an immobilised species that binds to the first and/or second molecule, wherein optionally the first and second molecules are not the same. Optionally, the binding pad further comprises a third molecule that specifically binds to IL-6 protein, optionally labeled, and optionally further comprises a fourth molecule that specifically binds to IL-6 protein, optionally labeled, and the nitrocellulose membrane further comprises another detection line comprising an immobilized substance that detects complexes of IL-6 protein with the third molecule and/or the fourth molecule; and optionally, the control line may comprise an immobilized species that binds the third and/or fourth molecule, wherein optionally the third and fourth molecules are not identical.

The invention also provides a kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient comprising means for detecting H-NGAL protein, and optionally including means for detecting IL-6 protein. Optionally, the kit further comprises a reference color chart, and the result is negative or positive according to the comparison of the detection result with the reference color chart.

The present invention also provides a device for diagnosing, preferably rapidly diagnosing, peritonitis or a peritonitis risk in a peritoneal dialysis subject, comprising: a means for detecting H-NGAL protein and optionally IL-6 protein in the peritoneal dialysis permeate of a peritoneal dialysis subject.

The invention also provides the use of a molecule that specifically binds to H-NGAL protein in the manufacture of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis by detecting H-NGAL protein in the peritoneal dialysis permeate of a peritoneal dialysis subject. The invention also provides an in vitro assay for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis.

The invention also provides the use of a combination of a molecule that specifically binds to H-NGAL protein and a molecule that specifically binds to IL-6 protein in the manufacture of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis by detecting H-NGAL and IL-6 protein in the peritoneal dialysis dialysate of a peritoneal dialysis subject.

The invention also provides the use of a molecule that specifically binds to an IL-6 protein for the preparation of a composition, test strip or kit for the detection of peritoneal dialysis permeate from a peritoneal dialysis subject in combination with a molecule that specifically binds to H-NGAL protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis.

The invention also provides the use of a molecule that specifically binds to H-NGAL protein in the manufacture of a composition, test strip or kit for use in the detection of peritoneal dialysis dialysate of a peritoneal dialysis subject in combination with a molecule that specifically binds to IL-6 protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis.

The invention also provides molecules that specifically bind to H-NGAL protein for use in the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis by detecting H-NGAL protein in the peritoneal dialysis dialysate of a peritoneal dialysis subject.

The invention also provides a combination of a molecule specifically binding to H-NGAL protein and a molecule specifically binding to IL-6 protein for use in diagnosing peritonitis, preferably rapid diagnosis of peritonitis or peritonitis risk, by detecting H-NGAL and IL-6 protein in a peritoneal dialysis dialysate of a peritoneal dialysis subject.

The invention also provides a molecule specifically binding IL-6 protein for use in detecting peritoneal dialysis dialysate of a peritoneal dialysis subject in combination with a molecule specifically binding H-NGAL protein for diagnosis, preferably rapid diagnosis, of peritonitis or peritonitis risk.

The invention also provides the use of a molecule that specifically binds to H-NGAL protein for the detection of peritoneal dialysis dialysate of a peritoneal dialysis subject in combination with a molecule that specifically binds to IL-6 protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis.

The invention also provides the use of a molecule that specifically binds to H-NGAL protein for the preparation of a test strip for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis by detecting H-NGAL protein in the peritoneal dialysis permeate of a peritoneal dialysis patient, wherein the test strip has an H-NGAL cut-off value of 500pg/mL.

The invention also provides the use of a combination of a molecule that specifically binds to H-NGAL protein and a molecule that specifically binds to IL-6 protein for the preparation of a test strip for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of having peritonitis by detecting H-NGAL and IL-6 protein in a peritoneal dialysis permeate of a peritoneal dialysis patient, wherein the test strip has an H-NGAL cut-off of 500pg/mL and an IL-6 cut-off of 200pg/mL.

Brief Description of Drawings

FIG. 1: lateral flow sandwich immunoassay principle

FIG. 2: exemplary reference color chart

FIG. 3: schematic view of assembled test strip

FIG. 4 is a schematic view of: structure schematic diagram of colloidal gold immunochromatographic test strip

FIG. 5 is a schematic view of: schematic diagram of immunochromatographic test strip

FIGS. 6 to 8: schematic diagram of test strip detection result

Detailed Description

Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly known to those skilled in the art. Also, unless otherwise required, singular terms shall include the plural and plural terms shall include the singular. The foregoing techniques and methods are generally performed according to conventional methods well known in the art and described in the references cited in this specification. See, e.g., sambrook et al molecular Cloning, A Laboratory Manual (3 rd ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (2001)) incorporated by reference. All references cited herein, including patents, patent applications, articles, texts, etc., and references cited therein, are hereby incorporated by reference in their entirety.

The present invention provides a method, product and device for diagnosing, in particular rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis patient, so that the peritonitis or the risk of peritonitis can be diagnosed rapidly and conveniently for the corresponding treatment or prevention. By using the method, the product and the device, the peritonitis of the patient can be quickly diagnosed after the peritonitis occurs, and the auxiliary diagnosis of the peritonitis related to the peritoneal dialysis is provided. The method, product and device for rapid diagnosis of peritonitis or peritonitis risk can be applied at home, and diagnosis can be rapidly and conveniently carried out, so that peritonitis can be detected and treated early.

In one aspect, the present invention provides a method for diagnosing, preferably rapidly diagnosing, peritonitis or a peritonitis risk in a peritoneal dialysis subject, comprising: detecting H-NGAL protein in the peritoneal dialysis dialysate of the subject, and if H-NGAL protein is greater than or equal to the cut-off value, indicating that the subject has peritonitis or is at risk of having peritonitis. In a further embodiment, the method further comprises detecting IL-6 protein in the peritoneal dialysis dialysate, if IL-6 protein is greater than or equal to the cut-off value, indicating that the subject has peritonitis or is at risk of having peritonitis.

As used herein, "subject" or "patient" are used interchangeably and include both human and non-human mammals, such as mice, rats, sheep, cattle, dogs, cats, rabbits, etc., particularly humans.

As used herein, "cutoff value", or "cutoff value" are used interchangeably and are sometimes also referred to as "positive judgment values".

Peritoneal dialysis is a process in which peritoneal dialysis solution is injected into the abdominal cavity of a subject for dialysis, and the dialysis solution, i.e., peritoneal dialysis dialysate, is taken out from the abdominal cavity after dialysis. Peritoneal dialysis includes both continuous ambulatory peritoneal dialysis (CAPD for short) and automated peritoneal dialysis (APD for short). Continuous ambulatory peritoneal dialysis is achieved by manually replacing the dialysate in the abdomen every few hours. Automatic peritoneal dialysis is the peritoneal dialysis that needs with the help of external small-size machine, and through setting up in advance, the machine can be automatically toward the peritoneal cavity perfuse the dislysate of great volume, again with its external process of drainage gradually, need not manual change dislysate, can treat night. For example, in various countries there are "standard practice for peritoneal dialysis" or similar industry documents in which detailed instructions are typically given to the prescription and adjustment of peritoneal dialysis and the operation of peritoneal dialysis.

Typically, the subject performs dialysis every several hours using a prescribed volume of dialysate, e.g., every 2, 3, 4, 5, 6, 7, or 8 hours using about 1, 2, 3, 4, 5, 6, 7, or 8 liters of dialysate, depending on the physician's judgment and the prescribed prescription. Herein, the default is CAPD if not specifically indicated or otherwise determinable from context, and the dialysate volume used at a single time is about 2 liters.

According to the International GUIDELINES for the Peritoneal Dialysis (ISPD GUIDELINES/RECOMMENDATION, peritoneal analysis International, vol.36, pp.481-508), normally, for APD patients who do not exchange fluid during the day, no fluid can be drawn off when abdominal pain occurs. In this case, 1L of peritoneal dialysis solution should be infused and left in the abdomen for at least 1-2 hours, then drained and checked for turbidity and relevant tests and cultures. In some cases of suspected infection, or those with clear effluent but systemic or abdominal symptoms, a second peritoneal fluid exchange should be made and left in the abdomen for at least two hours before being submitted for inspection. Reference is made herein to the recommended method for transudate sample acquisition of APD patient subjects.

As used herein, "peritoneal dialysis dialysate" (PDE) refers to the fluid that is drained from the peritoneal cavity without further treatment after peritoneal dialysis using a peritoneal dialysis solution for a patient undergoing stable CAPD or APD dialysis according to the standard procedures for peritoneal dialysis. In one embodiment, the peritoneal dialysis dialysate is obtained by subjecting a peritoneal dialysis solution (e.g., 2 liters of dialysate) to a peritoneal dialysis solution for at least 2 hours. In one embodiment, PDE efflux is used directly to detect the H-NGAL and/or IL-6.

As used herein, "peritonitis" is an inflammation of the peritoneum due to, for example, bacterial/fungal infection, chemical irritation or injury, manifested primarily as abdominal pain, abdominal muscle tone, and nausea, vomiting, fever, which can lead to a drop in blood pressure and a systemic toxic response when severe, e.g., failure to treat in a timely manner can lead to toxic shock.

Push according to the International guide of peritoneal dialysis societyRecommendations (ISPD GUIDELINES/RECOMMENDIMENTS, peritoneal analysis International, vol.36, pp.481-508) were confirmed if the subjects met 2 out of the following 3 strips: (1) Clinical features consistent with peritonitis, i.e., abdominal pain and/or dialysis dialysate turbidity; (2) Dialysis dialysate leukocyte count>100/. Mu.L or>0.1×10 ⁹ L (dwell time of at least 2 hours) (dwell time) and (3) dialysis permeate culture positive.

As used herein, "rapid diagnosis" refers to the ability to diagnose peritonitis or peritonitis risk within, for example, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 8 minutes, 6 minutes, 4 minutes, 2 minutes, or 1 minute or even less. By "early diagnosis" is meant that the peritonitis or peritonitis risk can be diagnosed early after the subject has developed peritonitis, e.g., within 0-4 days, 0-3 days, 0-2 days, 0-1 day, 0-24 hours, 0-18 hours, 0-12 hours, 0-11 hours, 0-10 hours, 0-9 hours, 0-8 hours, 0-7 hours, 0-6 hours, 0-5 hours, 0-4 hours, 0-3 hours, 0-2 hours, 0-1 hour, or even less.

H-NGAL protein refers to the neutrophil gelatinase associated lipocalin 45-kD homodimeric form. NGAL is a 25kDa glycosylated single chain monomer (GenBank ID: CAA67574.1 of human NGAL) and is a member of the lipocalin family that binds to transport small lipophilic molecules. NGAL is released from activated neutrophils and tissues such as renal tubular epithelial cells, cardiomyocytes, uterus, prostate, salivary glands, etc., and can be detected in serum and urine. NGAL can form dimers, including 45kDa disulfide-linked homodimers and 135kDa heterodimers with metalloproteinase 9 (MMP 9, gelatinase B). In one embodiment, the NGAL is human NGAL and the homodimeric H-NGAL is human H-NGAL.

IL-6 belongs to gp130 cytokine family. IL-6 is preferably a protein produced from a single gene encoding a 212 amino acid product, more preferably a 184 amino acid IL-6 peptide that is cleaved at the N-terminus of the 212 amino acid peptide (Song M, kellum JA. Interleukin-6.Crit Care Med 2005 (Suppl 12): 463-465, NCBI sequence accession No. NP-000591 of the 212 amino acid long IL-6 precursor. In one embodiment, the IL-6 is human IL-6. Herein, IL-6 preferably covers IL-6 variants, preferably human IL-6 variants. In one embodiment, the IL-6 variants are naturally occurring variants in a subject, e.g., a human, such as allelic variants, species variants, and the like. One skilled in the art would know how to identify and validate IL-6 and its variants.

In one embodiment, the H-NGAL protein has a cutoff of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000pg/mL, preferably 500pg/mL.

In one embodiment, the IL-6 protein has a cutoff of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000pg/mL, preferably 200pg/mL.

Herein, detection of H-NGAL and/or IL-6 protein may use any suitable method known in the art for detecting H-NGAL and/or IL-6 protein, such as chemiluminescence, biochemical methods, immunological techniques (including, for example, enzyme-linked immunosorbent assay (ELISA), alphaLISA, immunoblotting, dot blotting, co-immunoprecipitation, colloidal gold immunochromatography, etc.), mass spectrometry techniques, and the like. In one embodiment, H-NGAL and/or IL-6 protein using specific antibodies (e.g., monoclonal antibodies) or antigen binding fragments thereof, ligands, aptamers detection. In one embodiment, the H-NGAL and/or IL-6 protein is detected using a specific antibody, such as a lateral flow sandwich immunoassay (e.g., strip).

Lateral flow assay ("LFA") (also known as lateral flow immunoassay ("LFIA")) is a simple, easy-to-use assay for confirming the presence of a target analyte, such as a human or animal pathogen or biomarker. Lateral flow assays typically have a quality control line along with one or more target or detection lines to confirm that the assay is working properly. They may be qualitative, visual, or in combination with reader technology (e.g. in the case of a reader device)

) When used in combination, is quantitative. Although "competitive assays" are applicable herein (e.g., by using enrichment methods such as centrifugation to increase detection sensitivity), sandwich (or "non-competitive") assays are generally preferred for use herein. The principle of lateral flow sandwich immunoassay can be seen in FIG. 1. In non-competitive immunoassays, a positive test is typically indicated by the presence of a colored line at the detection line location. Non-competitive immunoassays are an ideal choice for large molecular weight analytes with multiple antigenic sites. In general, non-competitive immunoassays have a lower detection limit (higher assay sensitivity) than competitive immunoassays. Typically, noncompetitive immunoassays can detect on the order of picograms/milliliter, while competitive assays are nanograms/milliliter.

Lateral flow devices typically utilize immunoassay techniques with nitrocellulose membranes, colored latex nanoparticles (or labels such as magnetic beads or colored styrene spheres), and antibodies for rapid analysis. When a sample from a PDE is added to a lateral flow device, the sample flows along the assay device, through the conjugate pad into the nitrocellulose membrane, and then into the absorbent pad to absorb excess sample. The sample pad serves as the first stage of the absorption process and in some cases contains a filter to ensure accurate and controlled flow of the sample. A conjugate pad storing a conjugate tag and an antibody receives the sample. If the target is present, the immobilized binding antibody and label bind to the target and continue to migrate along the device. As the sample moves along the device, the binding reagents located on the nitrocellulose membrane bind to the target at the detection line. The form of the colored lines and the line density vary depending on the number of targets present. Some targets may require quantification to determine target concentration, for example, by using a reader. More sophisticated systems can be used to obtain quantitative data, for example immunofluorescence techniques and readers can be used to measure the number of markers in the PDE, providing more information to the patient and clinician by comparing the values obtained with a predetermined cutoff value.

US20050250141A1 (11/10/2005) discloses a multiplex lateral flow assay, related methods and devices, the contents of which are incorporated herein by reference. US 6924153 B1, US 128871B2 and US 7785899 B2, the contents of which are incorporated herein by reference, disclose various lateral flow test kits capable of detecting multiple analytes.

For example, the gold immunochromatography fixes a specific antibody (or antigen) in a strip shape to a membrane, adsorbs a gold-labeled reagent (antibody) to a conjugate pad, moves forward by capillary action after a sample to be tested is applied to the sample pad at one end of a test strip, dissolves the gold-labeled reagent on the conjugate pad and reacts with each other, and when moving to a region where the antibody (or antigen) is fixed, the conjugate of the sample and the gold-labeled reagent is specifically bound thereto and trapped, and is collected on a detection zone, and a color development result can be observed by naked eyes. The schematic structure of the colloidal gold immunochromatographic test strip is shown in FIG. 4.

As used herein, "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). An "antigen-binding fragment" refers to a polypeptide fragment of an antibody, e.g., a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated by reference herein in its entirety non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (e.g., from Ablynx as a technology), domain antibodies (e.g., from Domantis as a technology), and polypeptides comprising at least a portion of an antibody sufficient to have antigen-specific binding capability.

As used herein, "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. The term "specific binding" or "specific recognition" as used herein in reference to the interaction of an antibody with its binding partner (e.g., an antigen) means that the interaction is dependent on the presence of a particular amino acid sequence or structure (e.g., an antigenic determinant or epitope) on the binding partner. In other words, an antibody preferentially binds or recognizes a binding partner even if the binding partner is present in a mixture of other molecules or organisms. Binding may be mediated by covalent or non-covalent interactions or a combination of both. In other words, the term "specifically binds" or "specifically recognizes" means that the antibody is specifically immunoreactive with an antigenic determinant or epitope, but not with other antigenic determinants or epitopes. Antibodies that specifically or immunospecifically bind to an antigen may bind with low affinity to other peptides or polypeptides as determined by, for example, radioimmunoassay ("RIA"), enzyme-linked immunosorbent assay ("ELISA"), BIACORE, or other assays known in the art. An antibody or fragment thereof that specifically binds an antigen can cross-react with a related antigen bearing the same epitope. Preferably, the antibody or fragment thereof that specifically binds to the antigen does not cross-react with other antigens.

The strength or affinity of a specific binding interaction can be expressed in terms of the dissociation equilibrium constant (KD) of the interaction. The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody has less than about 10 ^-8 M, e.g. less than about 10 ^-8 M、10 ^-9 M、10 ^-10 M or 10 ^-11 M or less KD binds to the antigen. In some embodiments, when KD ≦ 10 × 10 ^-8 M, the antibodies or antigen binding fragments thereof of the present invention are believed to specifically bind to H-NGAL or IL-6 protein. The technicians in this field can obtain H-NGAL protein, NGAL protein and IL-6 protein specific antibody.

Detection of H-NGAL protein may be carried out using any suitable method or means. In one embodiment, can directly detect H-NGAL protein, for example by mass spectrometry or use of H-NGAL protein specific antibody or antigen binding fragment thereof. In another embodiment, the H-NGAL protein may be detected indirectly, for example by first detecting total NGAL (e.g., including monomeric and homodimeric forms, or including monomeric, homodimeric and heterodimeric forms) protein and then detecting the monomeric or monomeric and heterodimeric forms, thereby determining the H-NGAL protein.

For example, an IgG1 monoclonal antibody that specifically binds to the homodimeric form of NGAL rather than the heterodimeric form is provided as "N457" and is commercially available from HyTest, inc. of Turku, finland (Cat. #4NG 7-N457). Other antibodies provided by HyTest (including N308 and N432) have similar binding specificity to N457. Antibodies that bind monomeric, homodimeric and heterodimeric forms of NGAL include N316, N417, N422 and N461 of HyTest limited. An enzyme immunoassay ("sandwich immunoassay") used only for the detection of NGAL homodimer form contained N316 as NGAL capture antibody and N457 as detection antibody, see "neutrophile gelatinase-associated lipocalin (NGAL)" technnotes, p.2 (jan.2020 by HyTest ltd.), the contents of which are incorporated herein by reference. Thus, in certain embodiments, the detection antibody for H-NGAL in the assay may be selected from N457, N308, N432, and any combination thereof, and the capture antibody may be selected from N316, N417, N422, N461, and any combination thereof.

In one embodiment, the present invention provides a method of diagnosing peritonitis or the risk of peritonitis in a peritoneal dialysis patient comprising:

(a) Detecting H-NGAL protein in the peritoneal dialysis dialysate; or

(b) Detecting total NGAL, including monomeric, H-NGAL and heterodimeric forms, in the peritoneal dialysis permeate and detecting the monomeric and possibly heterodimeric forms to determine the amount of H-NGAL.

In one embodiment, the present invention provides a method of diagnosing peritonitis or a risk of peritonitis in a peritoneal dialysis patient comprising:

-obtaining a peritoneal dialysis dialysate sample of the patient:

-contacting a first molecule, such as an antibody, that specifically binds to H-NGAL protein, optionally immobilized on a solid support, such as an elisa plate, with a sample to be tested; and

-detecting the formed complex.

-obtaining a peritoneal dialysis dialysate sample of the patient:

-contacting a first molecule, such as an antibody, that specifically binds to H-NGAL protein, optionally immobilized on a solid support, such as an elisa plate, with a sample to be tested;

-adding a second molecule, such as an antibody, that specifically binds to the H-NGAL protein, optionally labelled, such as with an enzyme (e.g. horseradish peroxidase, alkaline phosphatase, glucose oxidase, β -D-galactosidase), wherein the second molecule binds to a different epitope on the H-NGAL protein than the first molecule;

optionally, adding a substance that specifically binds to the second molecule, e.g. an antibody against the second molecule, optionally labeled, e.g. labeled with an enzyme (e.g. horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase), and

-detecting the formed complex.

In one embodiment, the detection may be a chromogenic reaction known in the art, for example, using the substrates o-phenylenediamine (OPD), tetramethylbenzidine (TMB), nitrophenyl phosphate (pNPP), diaminobenzidine (DAB), and the like.

In another embodiment, the present invention provides a method of diagnosing peritonitis or a risk of peritonitis in a peritoneal dialysis patient comprising:

-obtaining a peritoneal dialysis dialysate sample of a peritoneal dialysis patient:

-contacting a first antibody molecule specifically binding to H-NGAL protein with a sample to be tested, wherein said first antibody molecule is labeled with biotin;

-adding a second antibody molecule that specifically binds to H-NGAL protein, wherein the second antibody molecule is colloidal gold labeled and the second antibody molecule is a different antibody that binds to a different epitope on H-NGAL protein than the first antibody molecule, optionally the first and second antibody molecules are independently selected from N316 and N457;

adding streptavidin, wherein the streptavidin is immobilized on a solid support, such as nitrocellulose, and

-detecting the formed complex.

In one embodiment, the first and second molecules that specifically bind to H-NGAL protein are antibodies independently selected from N316 and N457 (marine peptide biotechnology (shanghai) ltd). In one embodiment, the first molecule is antibody N457 and the second molecule is antibody N316.

In a further embodiment, the method of diagnosing peritonitis or at risk of peritonitis further comprises detecting IL-6 protein in the peritoneal dialysis dialysate sample. Means for detecting IL-6 are known in the art, e.g., anti-IL-6 antibodies, including polyclonal and monoclonal antibodies and fragments thereof, e.g., M-BE8 (EP 0430193; KLEIN, B., et al 1991, murine anti-endogenous antibody therapy for a patient with plasma cell leukemia, blood 78, 1198-1204) or M-23C7, or antibodies that bind to an epitope recognized thereby.

The protein concentration in the peritoneal dialysis permeate can be determined according to any suitable method known in the art, for example, methods including, but not limited to, chemiluminescence, biochemistry, affinity chromatography, immunoblotting, and mass spectrometry. Methods for determining the concentration of a particular protein in a sample are well known to those skilled in the art.

The test strip may also be used to detect whether an analyte (e.g., H-NGAL and/or IL-6 protein) in a sample is greater than or equal to a cut-off value, for example, using a test strip (strip) having a desired cut-off value. It is known to those skilled in the art how to prepare test strips as described herein, for example test strips having a particular cut-off value.

In certain embodiments, a test strip having a given cutoff value (e.g., 500pg/mL for H-NGAL protein; 200pg/mL for IL-6 protein) is used to detect H-NGAL and/or IL-6 protein in the peritoneal dialysis dialysate and based on the detection, a determination is made as to whether the H-NGAL and/or IL-6 protein in the peritoneal dialysis dialysate is greater than or equal to the cutoff value.

In one aspect, the invention provides a method of treating peritonitis in a peritoneal dialysis subject, comprising: detecting H-NGAL protein in the peritoneal dialysis permeate of the subject; determining the level of H-NGAL; comparing the H-NGAL level to a cutoff value; administering an antibiotic treatment if the H-NGAL level exceeds a cutoff value.

In one embodiment, the invention provides a method of treating peritonitis in a peritoneal dialysis subject comprising: detecting H-NGAL protein and IL-6 protein in the peritoneal dialysis dialysate of the subject; determining the level of H-NGAL and the level of IL-6; comparing the H-NGAL level and IL-6 level to cut-off values; administering an antibiotic treatment if both the H-NGAL level and the IL-6 level exceed cut-off values.

The cut-off values for H-NGAL and IL-6 are as described herein. In one embodiment, the H-NGAL protein has a cutoff of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or pg10000/mL, preferably 500pg/mL. In one embodiment, the IL-6 protein has a cutoff of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000pg/mL, preferably 200pg/mL.

The antibiotic treatment may be a single drug antibiotic or a combination antibiotic. In one embodiment, the single drug antibiotic comprises ertapenem (INVANZ) ^TM ) Cefoxitin and Doripenem (DORIBAX) ^TM ) Imipenem/cilastatin (PRIMAXIN) ^TM ) Meropenem (MERREM) ^TM ) Moxifloxacin (AVELOX) ^TM ) Piperacillin/tazobactam (ZOSYN) ^TM ) Ticarcillin/clavulanate (TIMENTIN) ^TM ) And Tigecycline (TYGACIL) ^TM ). In one embodiment of the process of the present invention,the antibiotic for combined therapy comprises cefepime (MAXIPIME) ^TM ) Cefotaxime (CLAFORAN) ^TM ) Ceftazidime (FORTAZ) ^TM ) Or Ceftriaxone (ROCEPHIN) ^TM ) Combined with metronidazole (FLAGYL) ^TM ) Or cefazolin, cefotaxime, ceftriaxone, ciprofloxacin (CIPRO) ^TM ) Levofloxacin (LEVAQUIN) ^TM ) Combined with metronidazole, or combined with cefepime, ceftazidime, ciprofloxacin, levofloxacin and metronidazole, or combined with gentamicin or tobramycin and clindamycin (CLOOCIN) ^TM ) Or metronidazole (with or without ampicillin).

In one aspect, the present invention provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or having a risk of peritonitis in a peritoneal dialysis patient, comprising a solid support having a first and a second end comprising, in succession from said first end: a sample pad, a binding pad comprising a first molecule (optionally labelled) that specifically binds H-NGAL protein, a nitrocellulose membrane (comprising a detection line comprising an immobilised substance that detects complexes formed by H-NGAL protein and the first molecule), a quality control line comprising an immobilised substance that binds the first molecule, and optionally an absorbent pad (e.g. as shown in figure 5). In further embodiments, in the test strip, the binding pad further comprises a third molecule (optionally labeled) that specifically binds to an IL-6 protein, the nitrocellulose membrane further comprises another detection line comprising a substance that detects a complex formed by an IL-6 protein and a third molecule, and the quality control line can comprise a substance that binds to the first molecule and/or third molecule.

In one embodiment, the solid support comprises, in succession from the first end: a sample pad, a conjugate pad, a nitrocellulose membrane (containing a detection line and a quality control line), and an absorbent pad.

In one aspect, the present invention provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or having a risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end comprising, in succession from said first end: sample pad, biotinylation pad, gold mark pad, nitrocellulose membrane and absorption pad, wherein nitrocellulose membrane contains detection line and matter control line, wherein: the biotinylation pad comprises a biotin-labeled first molecule specifically binding to H-NGAL protein, the gold-labeled pad comprises a colloidal gold-labeled second molecule specifically binding to H-NGAL protein, the detection line comprises immobilized streptavidin, and the quality control line comprises an immobilized molecule specifically binding to the first molecule and/or the second molecule, wherein optionally the first molecule and the second molecule are different.

As used herein, the sample pad is used to apply a sample to be tested thereto and may be prepared according to any suitable method known in the art.

As used herein, the conjugate pad comprises capture molecules, optionally labeled, that can bind to the molecules to be detected in the sample.

As used herein, a biotinylated pad comprises biotinylated molecules that can specifically bind to the molecules to be detected in the sample. As used herein, a gold-labeled pad comprises a colloidal gold-labeled molecule, such as an antibody, that can specifically bind to a molecule to be detected or a complex formed by the molecule to be detected and a capture molecule. See regions B and D in fig. 3.

As used herein, the nitrocellulose membrane comprises one or more detection lines comprising immobilized detection molecules that can bind to complexes formed by the test molecule and the capture molecule. The two detection lines are spaced apart by a distance, e.g. a distance of 0.5-5mm, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0mm, so as not to interfere with the detection results. It is known in the art how to set up the individual test lines of the test strip.

As used herein, a quality control line is used to confirm whether a test is valid. The distance between the quality control line and the detection line is a certain distance, for example, 0.5-5mm, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0mm, which can implement detection and determine whether the detection is effective. It is known in the art how to arrange the quality control and detection lines.

As used herein, an absorbent pad is used to allow liquid to flow over other pads and/or absorb excess sample by absorbing the liquid.

As used herein, continuous means that adjacent regions are in contact with each other, preferably with an overlap, so that sample can diffuse from one region to an adjacent region. The person skilled in the art can determine how much the overlap is, for example, 0.5-4.0mm, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0mm, according to the knowledge of the person skilled in the art.

For example, the test strip may have at least one of the following characteristics: (a) The sample pad and conjugate pad have overlapping portions, wherein preferably the sample pad is above the conjugate pad; (b) The conjugate pad and the nitrocellulose membrane have an overlap, wherein preferably the conjugate pad is on the nitrocellulose membrane, and (c) the nitrocellulose membrane and the absorbent pad have an overlap, wherein the absorbent pad is on the nitrocellulose membrane. Further, when including biotinylated and gold-labeled pads, the test strip may also have at least one of the following characteristics: (d) The biotinylation pad and the gold-labeled pad have overlapping portions, wherein preferably the biotinylation pad is on top of the gold-labeled pad; and (e) the gold-labeled pad and the nitrocellulose membrane have an overlapping portion, wherein preferably the gold-labeled pad is on top of the nitrocellulose membrane.

As used herein, the molecule that specifically binds to H-NGAL or IL-6 protein is any suitable molecule that can specifically bind to H-NGAL or IL-6 protein, including, for example, an antibody (preferably a monoclonal antibody) or an antigen-binding fragment thereof, a ligand, an aptamer, and the like. Antibodies that specifically bind to H-NGAL or IL-6 protein are known in the art.

As used herein, the label is any molecule or means that can be used to label the H-NGAL and/or IL-6 protein, including, for example, but not limited to, biotin, fluorescence, radioactivity, luminescence, chemistry, enzymes, particulate labels such as latex particles, colloidal gold, and the like. In one embodiment, the label is selected from the group consisting of a colloidal gold label and a biotin label.

As used herein, the substance that detects the complex is a suitable substance that can indicate the presence or absence of the complex, for example, including but not limited to an antibody or antigen binding fragment thereof, a ligand, an aptamer, preferably selected from a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody or an antigen binding fragment thereof, more preferably a monoclonal antibody or an antigen binding fragment thereof. The skilled person can select suitable substances for detecting the complexes according to the knowledge of the skilled person. For example, where the label is an enzymatic label, the substance may be a substrate for the enzyme, which is catalyzed by the enzyme to produce a signal that can be observed (e.g., visually or by a detection device), e.g., color development, luminescence, fluorescence, etc.; in the case where the label is a label such as colloidal gold, an isotope, luminescence, or the like, the substance may be a substance (e.g., an antibody) that binds (e.g., specifically binds) the complex or a molecule in the complex, or a substance (e.g., an antibody) that binds to a label carried by the molecule, so that the presence or absence of the complex can be detected by detecting a signal from the label. In one embodiment, the label is biotin and the substance of the detection complex is streptavidin.

In one embodiment, the present invention provides a test strip for diagnosing peritonitis or a risk of having peritonitis in a peritoneal dialysis patient comprising a solid support having a first and second end comprising, in succession from said first end: a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad, wherein the binding pad comprises a first molecule (optionally labelled) that specifically binds to H-NGAL protein, and a second molecule (optionally labelled) that specifically binds to H-NGAL protein, the nitrocellulose membrane comprises a detection line comprising an immobilised substance that detects complexes formed by H-NGAL protein and the first and/or second molecules, and a quality control line comprising an immobilised substance that binds to the first and/or second molecules, wherein optionally the first and second molecules are not the same. In further embodiments, in the test strip, the binding pad further comprises a third molecule (optionally labeled) that specifically binds to IL-6 protein, and a fourth molecule (optionally labeled) that specifically binds to IL-6 protein, the nitrocellulose membrane further comprises another detection line comprising an immobilized substance that detects complexes formed by IL-6 protein with the third molecule and/or the fourth molecule, and the quality control line can optionally comprise an immobilized substance that binds to the third molecule and/or the fourth molecule, wherein optionally the third molecule and the fourth molecule are not identical.

In one embodiment, the present invention provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or having a risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end comprising, in succession from said first end: a sample pad, a binding pad, a nitrocellulose membrane, and optionally an absorbent pad, wherein the nitrocellulose membrane comprises a detection line and a quality control line, wherein:

the conjugate pad comprises:

(a) A first molecule that specifically binds to H-NGAL protein, optionally labelled, and optionally further comprising a second molecule that specifically binds to H-NGAL protein, optionally labelled, wherein optionally the first and second molecules are not the same;

(b) A third molecule that specifically binds to an IL-6 protein, optionally labeled, and optionally further comprising a fourth molecule that specifically binds to an IL-6 protein, optionally labeled, wherein optionally the third and fourth molecules are not the same;

the detection line comprises a detection line comprising an immobilized substance detecting a complex formed by the H-NGAL protein and the first molecule and/or the second molecule and a detection line comprising an immobilized substance detecting a complex formed by the IL-6 protein and the third molecule and/or the fourth molecule, and

the control line comprises an immobilized species that specifically binds to the first, second, third, and/or fourth molecule.

In one embodiment, the present invention provides a test strip for diagnosing, in particular rapidly diagnosing, peritonitis or having a risk of peritonitis in a peritoneal dialysis patient, comprising a solid support having a first and a second end comprising, in succession from said first end: sample pad, biotinylation pad, gold mark pad, nitrocellulose membrane and absorption pad, wherein biotinylation pad contains biotin-labeled specific binding H-NGAL protein's first antibody molecule, the gold mark pad contains colloidal gold-labeled specific binding H-NGAL protein's second antibody molecule, nitrocellulose membrane contains detection line and quality control line, the detection line contains immobilized streptavidin, the quality control line contains the immobilized anti-molecular (for example anti-IgG antibody molecule) that combines first antibody molecule and/or second antibody molecule, wherein first antibody molecule and second antibody molecule are different. In one embodiment, the biotinylated first antibody molecule is antibody N457 (HyTest) and the colloidal gold-labeled second antibody molecule is antibody N316 (HyTest). In a further embodiment, in the test strip, the biotinylated pad further comprises a third antibody molecule (optionally labeled with a label other than biotin) that specifically binds IL-6 protein, the gold-labeled pad further comprises a fourth antibody molecule (optionally labeled, e.g., colloidal gold-labeled) that specifically binds IL-6 protein, the nitrocellulose membrane further comprises another detection line comprising an immobilized substance that detects complexes of IL-6 protein with third and/or fourth molecules, and the quality control line may comprise an immobilized substance (e.g., anti-IgG antibody molecule) that binds the third and/or fourth antibody molecule, wherein optionally the third and fourth antibody molecules are not identical.

Methods for preparing the various parts of the test strip (e.g. solid support, sample pad, binding pad, biotinylation pad, gold-labeled pad, nitrocellulose membrane and absorbent pad) and suitable materials are known in the art, for which reference may be made to, for example, gong Pin et al, "colloidal gold immunochromatographic strip technology and its application development in food safety assays", food industry technology, 2019, 40 (13): 358-364, article number: 1002-0306 (2019) 13-0358-07; yan Ganoderma lucidum, research progress of immunochromatographic test strip technology in the field of food safety, food industry science and technology, DOI:10.13386/j. Issn1002-0306.2020110079.

In one embodiment, the test strip has a cutoff value described herein, e.g., the cutoff value for H-NGAL protein can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000pg/mL, and/or the cutoff value for IL-6 protein can be 50, 60, 70, 80, 90, 100, 200, 300, 400, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000/mL. In one embodiment, the H-NGAL protein has a cut-off value of 100, 200, 300, 400, 500pg/mL, preferably 500pg/mL. In one embodiment, the IL-6 protein has a cut-off value of 50, 60, 70, 80, 90, 100, 150, 200pg/mL, preferably 200pg/mL.

One skilled in the art would know how to prepare test strips with the desired cut-off based on knowledge of the art. For example, a commercially available or prepared standard solution of the analyte with a known concentration can be diluted to obtain a solution of the analyte with a certain concentration; or determining the concentration of the analyte solution by using a quantitative method for the analyte known in the field; preparing test strips having different contents/concentrations of detection molecules specifically binding to the analyte; the test strip having the desired cutoff value is then determined to have the test molecule content/concentration based on the test results.

In one aspect, the invention provides a kit for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis patient comprising means for detecting H-NGAL protein (e.g., a molecule that specifically binds to H-NGAL protein, such as an antibody or antigen-binding fragment thereof), and optionally means for detecting IL-6 protein (e.g., a molecule that specifically binds to IL-6 protein, such as an antibody or antigen-binding fragment thereof), and optionally a reference colorimetric card (which can be used for automated readings), the result being negative or positive based on a comparison of the test result to the reference colorimetric card.

In one embodiment, the kit comprises a test strip of the present invention.

In one aspect, the present invention provides a device for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis patient, comprising: means for detecting H-NGAL protein and optionally IL-6 protein in the peritoneal dialysis dialysate of a peritoneal dialysis subject, and optionally means for displaying the results of the detection.

In one embodiment, the device comprises means for alerting the patient to have peritonitis or to be at risk of having peritonitis when the H-NGAL protein in the peritoneal dialysis permeate is greater than or equal to a cut-off value (e.g., a concentration of greater than or equal to 500pg/mL as described herein), e.g., means for providing a specific signal (e.g., a sound, visual message, etc.), e.g., an electronic display screen, etc.

In one embodiment, the apparatus may be connected to a digital processing device (e.g., a personal data assistant or smart phone, a notebook computer, a desktop computer, a tablet computer or device, a remote web server, etc.) to alert the patient or associated person of infection or risk of infection.

In one embodiment, the device includes a means for indicating that the patient has, or is at risk for having, peritonitis when the H-NGAL protein in the peritoneal dialysis dialysate is at or above a cut-off value (e.g., a concentration at or above 500pg/mL cut-off value as described herein) and the IL-6 protein is at or above a cut-off value (e.g., a concentration at or above 200pg/mL cut-off value as described herein).

As used herein, the means for detecting H-NGAL protein and/or IL-6 protein may be any suitable unit or device capable of detecting, preferably quantifying, the protein, including, for example, a test strip, a protein array, a mass spectrometer, and the like.

In one embodiment, the device comprises a test strip and/or kit as described herein.

As used herein, a means for presenting a test result can communicate the test result (e.g., negative or positive, or a particular numerical value, etc.) to the subject or associated medical personnel in any suitable manner (e.g., a prompt tone, digital signal, networked message, etc.), including, for example, a display screen, etc.

The apparatus may be interconnected with other systems including, but not limited to, smartphones, tablets, laptops, and combinations of computing devices and cloud computing resources.

Embodiments of the apparatus and methods described herein may be implemented in various systems including, but not limited to, smart phones, tablets, laptops, and combinations of computing devices and cloud computing resources. For example, some operations may occur in one device, while other operations may occur at a remote location, such as one or more remote servers. For example, the collection of data may be performed at a smartphone and the data analysis may be performed at a server or cloud computing resource. Any single computing device or combination of computing devices may perform the methods.

In another aspect, the invention provides the use of a molecule specifically binding to H-NGAL protein as described herein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL protein in the peritoneal dialysis permeate of the peritoneal dialysis patient.

In another aspect, the invention provides the use of a molecule that specifically binds to H-NGAL protein as described herein in combination with a molecule that specifically binds to IL-6 protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL and IL-6 proteins in the peritoneal dialysis dialysate of the peritoneal dialysis patient.

In another aspect, the invention provides the use of a molecule specifically binding to H-NGAL protein as described herein for the preparation of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL protein in the peritoneal dialysis permeate of the peritoneal dialysis patient.

In another aspect, the invention provides the use of a combination of a molecule that specifically binds to H-NGAL protein and a molecule that specifically binds to IL-6 protein as described herein for the preparation of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL and IL-6 protein in the peritoneal dialysis permeate of the peritoneal dialysis patient.

In another aspect, the invention provides the use of a molecule specifically binding to IL-6 protein as described herein for the preparation of a composition, test strip or kit for the detection of peritoneal dialysis dialysate from a peritoneal dialysis patient in combination with a molecule specifically binding to H-NGAL protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient.

In another aspect, the invention provides the use of a molecule that specifically binds to H-NGAL protein as described herein in the manufacture of a composition, test strip or kit for use in the detection of peritoneal dialysis dialysate from a peritoneal dialysis patient in combination with a molecule that specifically binds to IL-6 protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient.

In another aspect, the present invention provides the use of a molecule that specifically binds to H-NGAL protein as described herein in the preparation of a test strip for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL protein in a peritoneal dialysis permeate of the peritoneal dialysis patient, wherein the test strip has an H-NGAL cutoff value selected from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 pg/mL. In one embodiment, the test strip has an H-NGAL cutoff of 500pg/mL.

In another aspect, the invention provides the use of a combination of a molecule that specifically binds to H-NGAL protein and a molecule that specifically binds to IL-6 protein as described herein in the manufacture of a test strip for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis patient by detecting H-NGAL and IL-6 proteins in the peritoneal dialysis permeate of the peritoneal dialysis patient, wherein the test strip has an H-NGAL cutoff value selected from 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000pg/mL and an IL-6 cutoff value selected from 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000pg/mL. In one embodiment, the test strip has an H-NGAL cutoff of 500pg/mL and an IL-6 cutoff of 200pg/mL.

In certain embodiments, the molecule that specifically binds to H-NGAL protein is selected from the group consisting of antibodies N457, N308, and N432, and any combination thereof.

Unless the context clearly dictates otherwise, the word "or" is intended to include "and"; the word "comprising" or "comprises" does not exclude other elements or steps.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally included step refers to the presence or absence of that step.

As used herein, the term "about" refers to a range of values that includes the particular value, which one of skill in the art can reasonably consider similar to the particular value. In embodiments, the term "about" means within standard error using measurements commonly accepted in the art. In certain embodiments, about refers to +/-10% or 5% of the particular value.

Those skilled in the art will appreciate that any modifications or changes can be made to the technical solution of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and such modifications and changes are also included in the scope of the present invention. The above embodiments are merely illustrative of the present invention and do not limit the scope of the invention. The scope of the invention is defined by the appended claims, and the scope defined by the claims will be clearly understood by those skilled in the art from this description and the common general knowledge in the art.

Examples

The invention is further illustrated by the following examples, but any examples or combination thereof should not be construed as limiting the scope or embodiment of the invention.

Example 1: test strip manufacture

1. Material

Quality control line: goat anti-rabbit IgG antibody, available from Shandong Shuojing organisms, inc. (Cat.No: P1303)

And (3) detection line: streptavidin, available from Hangzhou Kuige science and technology Co., ltd (Cat. No: KYSA-003)

Antibody:

H-NGAL antibodies (Cat. No: N316 and N457) from Hai peptide Biotechnology (Shanghai) Ltd

IL-6 antibodies (Cat. No: DA011 and DA 012), purchased from Novoprotein

NGAL antibodies (Cat. No:4C10A7 and 5A9D 12) purchased from Nanking GenScript

Biotin, available from Thermo Fisher Scientific (Cat. No: 21312)

2. Test strip preparation

2.1 sample pad

50mL of a glass fiber treatment solution (24 g of tris (hydroxymethyl) aminomethane, 50g of sucrose, 5g of trehalose, 15g of casein, 5g of polyvinylpyrrolidone, 3.5g of anhydrous sodium carbonate, and NaN) was weighed ₃ 1g, added in sequence, stirred to be completely dissolved, triton X-100 mL, stirred to be completely mixed, added with a proper amount of hydrochloric acid to adjust the pH value to 8.5, placed at room temperature for storage, poured into a stainless steel plate, and a piece of glass fiber (Ahlstrom, cat.No:8964 Placed in the glass fiber bag, and taken out after the glass fiber is completely saturated. Placing the glass fiber on a net frame, and keeping the glass fiber at ambient humidity<The whole sample pad was obtained after fully drying at 20% condition at 30-40 ℃ for 12 hours. And cutting according to the product requirements.

2.2 coating and drying of the nitrocellulose Membrane

Quality control line: the loading was fixed (1. Mu.L/cm) and the nitrocellulose membrane was coated with 1.5mg/mL goat anti-rabbit IgG antibody.

And (3) detection line: the amount of the solution to be added was fixed (1. Mu.L/cm), and the nitrocellulose membrane was coated with 1.0mg/mL of streptavidin.

Wherein the quality control line and the detection line are separated by about 4mm.

And (3) a drying process: drying at 30-40 deg.C for 2 hr.

2.3 gold label pad

Adding 1%K mass concentration into 1mL colloidal gold solution (four ten-thousandths of nano-gold, particle size 35 nm) ₂ CO ₃ 10 μ L of the solution, and 20 μ g of antibody (H-NGAL antibody (Cat. No: N316), IL-6 antibody (Cat. No: DA 011), NGAL antibody (Cat. No:4C10A 7)). Carrying out centrifugal concentration on the gold-labeled antibody according to a ratio of 10 to 1, and diluting a gold-labeled antibody solution subjected to centrifugal concentration according to a ratio of 10 to 12 percent; then, the mixture was uniformly applied to a glass fiber in a solid phase, dried at 30 to 40 ℃ for 12 hours, and the dried gold-labeled antibody reaction membrane was cut into a width of 5mm for use.

2.4 biotinylation pad

The coating antibody (H-NGAL antibody (Cat. No: N457), IL-6 antibody (Cat. No: DA 012), NGAL antibody (Cat. No:5A9D 12)) and biotin were mixed in a molar ratio of 1: biotinylation is carried out at 20 ℃, and after 2-8 ℃ photophobic reaction for 2h, unreacted biotin is removed by dialysis. Diluting the biotinylation antibody solution according to the proportion of 0.35-0.5%, uniformly and solidly fixing on glass fiber, drying for 12 hours at the temperature of 30-40 ℃, and cutting the dried biotin antibody reaction film into a width of 5mm for use.

2.5 Assembly

Fixedly placing on a bottom plate: the nitrocellulose membrane prepared, then the upper absorbent paper (which had a partial overlap (about 1.0-1.5 mm) with the nitrocellulose membrane and was located thereon), followed by the gold-labeled pad (which had a partial overlap (about 1.0-1.5 mm) with the nitrocellulose membrane and was located thereon), then the biotinylated pad (which had a partial overlap (about 1.0-1.5 mm) with the gold-labeled pad and was located thereon), and finally the 2.1 sample pad obtained (which had a partial overlap (about 1.5 mm) with the biotinylated pad and was located thereon). The test strip produced was, in order from one end to the other: the upper end of the kit is provided with absorbent paper, a nitrocellulose membrane (a quality control line and a detection line in sequence), a gold label pad, a biotinylation pad and a sample pad (a sample adding hole is arranged on the sample adding pad). The assembled test strip is shown in fig. 3.

Finally, a handle paste and MAX adhesive tapes (strips) and white pastes (cards and pens) are pasted, cut into the required width of the reagent, and stored in a dark place at 4-30 ℃.

3. Preparation of NGAL, IL-6 and H-NGAL standard

NGAL (neutrophil gelatinase-associated lipocalin recombinant antigen (monomer)): qingdao Shuiching Biotechnology Co., ltd, batch number: 190703

H-NGAL: (neutrophil gelatinase-associated lipocalin recombinant antigen (homodimer)): nanjing Ai De general Biotechnology, inc., cat No.: AEP0061

IL-6: (interleukin 6): NIBSC, cargo number: 89-548

And carrying out gradient dilution on the corresponding standard substance by using a peritoneal dialysis dialysate negative sample to obtain NGAL, H-NGAL and IL-6 solutions with different concentrations. Detecting the solutions with different concentrations by using test strips with different cut-off values; the detection was repeated not less than 3 times at each concentration.

Example 2: peritonitis diagnosis and detection method

1. Patients with at least 2 symptoms as follows are diagnosed as having PERITONITIS according to the ISPD GUIDELINES (ISPD GUIDELINES/RECOMMENDINDIONS, ISPD PERITONITIS RECOMMENDINDIONS: 2016UPDATE ON PREVENTION AND TREATMENT, peritoneal analysis International, vol.36, pp.481-508): (1) Clinical features consistent with peritonitis, i.e., abdominal pain and/or dialysis dialysate turbidity; (2) Dialysis dialysate leukocyte count>100/. Mu.L or>0.1×10 ⁹ L (dwell time of at least 2 hours) (dwell time) and (3) positive dialysis permeate culture.

Peritonitis in peritoneal dialysis patients was diagnosed according to the above criteria. Peritoneal dialysis includes both continuous ambulatory peritoneal dialysis (CAPD for short) and automated peritoneal dialysis (APD for short). The subject dialyzes every several hours using a volume of dialysate prescribed by the prescription, according to the doctor's judgment and the prescribed prescription. Peritoneal Dialysis was performed according to the International GUIDELINES of the Peritoneal Dialysis society (ISPD GUIDELLINES/RECOMMENDATION, peritoneal analysis International, vol.36, pp.481-508), as described above.

2. Detection method

2.1 obtaining peritoneal dialysis dialysate samples from peritoneal dialysis patients

2.2 Add 70-80. Mu.L (2 drops) of peritoneal dialysis permeate sample to the well of the test strip prepared in example 1 and react for 10-15 minutes

2.3 comparing the test strip with a reference color comparison card (shown in figure 2), and reading a color development result, wherein the color development intensity is greater than or equal to 0.5, and the test strip is positive. Negative result = positive quality control line + negative detection line; positive result = quality control line positive + detection line positive. Partial test results are shown in FIGS. 6-8.

3. Detection and results

3.1 detection of IL-6 and NGAL protein (total NGAL protein, including the monomeric, homodimeric and heterodimeric forms described) by blinding in peritoneal dialysis transudates of peritoneal dialysis patients with peritonitis and clinical diagnosis without peritonitis, information as shown in Table 1 below.

TABLE 1

The results of the sample measurements shown in Table 1 are shown in Table 2.

According to the results in table 2, detection of NGAL at a cutoff of 120ng/mL showed better specificity (87.5%) and sensitivity of 90.0%. The sensitivity for IL-6 detection at a cut-off of 200pg/mL was 100% better than the other two cut-offs, and the specificity was 96.1% slightly lower than the other two cut-offs. In contrast, however, NGAL is less specific and less sensitive than IL 6.

3.2 detection of H-NGAL, IL-6 and NGAL (total NGAL protein, including the monomeric, homodimeric and heterodimeric forms described) by blinding in peritoneal dialysis transudates of peritoneal dialysis patients with peritonitis and clinical diagnosis without peritonitis, information as shown in Table 3 below.

TABLE 3

The results of sample measurements shown in Table 3 are shown in Table 4.

The detection of H-NGAL (500 pg/mL) (sensitivity 98.18%, specificity 96.08%) AND simultaneous detection ("AND") of H-NGAL (500 pg/mL) AND IL-6 (200 pg/mL) (sensitivity 98.18%, specificity 97.87%) showed higher sensitivity AND specificity.

4. Conclusion

a) According to the results of table 2 and table 4: the total NGAL cut-off was determined to be 120ng/mL, the IL6 cut-off was determined to be 200pg/mL, and the H-NGAL cut-off was determined to be 500pg/mL.

b) The results in table 4 show that detecting H-NGAL shows higher specificity and sensitivity than detecting total NGAL; detection of H-NGAL showed higher specificity than detection of IL-6.

c) The detection of H-NGAL (500 pg/mL) (sensitivity 98.18%, specificity 96.08%) AND the simultaneous detection of H-NGAL (500 pg/mL) AND IL-6 (200 pg/mL) ("AND") (sensitivity 98.18%, specificity 97.87%) showed higher sensitivity AND specificity.

Example 3: further experiments on the method of diagnosis and detection of peritonitis

Further diagnosis and detection of peritonitis was performed according to the method of example 2. The results are shown in the following table.

From the results in Table 5, it can be seen that the specificity of the H-NGAL detection is improved due to the expansion of the sample size, while the specificity and sensitivity, especially specificity, of the IL-6 detection is reduced due to more false positive and false negative samples.

TABLE 5

Example 4: clinical diagnostic result comparison and analysis of different methods and reagents

To compare the performance of different methods for diagnosing peritonitis, the clinical diagnosis results of the following three methods or reagents were comparatively analyzed.

WBC count

IL-6 (200 pg/ml) POCT (point of care testing) prototype

3.H-NGAL (500 pg/ml) POCT prototype

Patients with at least 2 symptoms are diagnosed as having PERITONITIS according to ISPD GUIDELINES (ISPD guidielines/RECOMMENDIMENTS, ISPD PERITONITIS RECOMMENDIMENTS: 2016UPDATE ON PREVENTION AND TREATIME, peritoneal analysis International, vol.36, pp.481-508) as follows: (1) Clinical features consistent with peritonitis, i.e., abdominal pain and/or dialysis dialysate turbidity; (2) Dialysis dialysate leukocyte count>100/. Mu.L or>0.1×10 ⁹ L (time to stay in the abdomen for at least 2 hours) with 50% polymorphonuclear leukocytes (PMNs); and (3) culturing the positive dialysis dialysate.

After a dwell time of at least 2 hours, the WBC count was considered positive if the dialysis exudate white cell count ≧ 100 cells/μ Ι _ has >50% polymorphonuclear leukocytes (PMNs), and negative if the dialysis exudate white cell count <100 cells/μ Ι.

IL-6 detection and H-NGAL detection as described in the examples.

The 4-table analysis of table 6 was used for qualitative product statistical analysis, and the sensitivity and specificity were calculated as follows. According to the results shown in the table below, the WBC count and the H-NGAL detection are very close in performance, with the possibility of replacing the WBC detection with the H-NGAL detection.

TABLE 6

Sensitivity = a/(a + c) × 100%,95% confidence interval

Specificity = d/(b + d). Times.100%, 95% confidence interval

PPV = a/(a + b) × 100%,95% confidence interval

NPV = d/(c + d) 100%,95% confidence interval

Table 7: WBC counting and clinical diagnostic analysis

Sensitivity =74/76 × 100% =97.37%,95% ci (90.90-99.28%)

Specificity =128/129 × 100% =99.22%,95% ci (95.74-99.86%)

PPV＝74/75×100％＝98.67％

NPV＝128/130×100％＝98.46％

Table 8: H-NGAL (500 pg/ml) POCT prototype and clinical diagnostic analysis

Sensitivity =75/76 × 100% =98.68%,95% ci (92.92-99.77%)

Specificity =127/129 × 100% =98.45%,95% ci (94.52-99.57%)

PPV＝75/77×100％＝97.40％

NPV＝127/128×100％＝99.22％

Table 9: IL-6 (200 pg/ml) POCT prototype and clinical diagnostic assays

Sensitivity =72/76 × 100% =94.74%,95% ci (87.23-97.93%)

Specificity =111/129 × 100% =86.05%,95% ci (79.02-90.99%)

PPV＝72/90×100％＝80.00％

NPV＝111/115×100％＝96.52％

Table 10: comparing the performance of clinical diagnostic results for WBC, IL-6 and H-NGAL.

Claims

1. Use of a molecule that specifically binds to homodimeric neutrophil gelatinase-associated lipocalin (H-NGAL) for the preparation of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject by detecting H-NGAL protein in the peritoneal dialysis permeate of the peritoneal dialysis subject.

2. Use of a combination of a molecule specifically binding to H-NGAL protein and a molecule specifically binding to interleukin-6 (IL-6) protein for the preparation of a composition, test strip or kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject by detecting H-NGAL and IL-6 proteins in the peritoneal dialysis permeate of the subject.

3. Use of a molecule specifically binding to IL-6 protein for the preparation of a composition, test strip or kit for the detection of peritoneal dialysis dialysate of a peritoneal dialysis subject in combination with a molecule specifically binding to H-NGAL protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject.

4. Use of a molecule specifically binding to H-NGAL protein for the preparation of a composition, test strip or kit for the detection of peritoneal dialysis permeate from a peritoneal dialysis subject in combination with a molecule specifically binding to IL-6 protein for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject.

5. Molecule specifically binding to H-NGAL protein for use in the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject by detecting H-NGAL protein in the peritoneal dialysis permeate of the subject.

6. The use of any one of claims 1-5, wherein the molecule is selected from the group consisting of an antibody or an antigen binding fragment thereof, a ligand, an aptamer, preferably from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody and an antigen binding fragment thereof, more preferably from the group consisting of a monoclonal antibody and an antigen binding fragment thereof.

7. Use of a molecule that specifically binds to H-NGAL protein for the preparation of a test strip for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject by detecting H-NGAL protein in the peritoneal dialysis permeate of the subject, preferably wherein the test strip has an H-NGAL cut-off value of 500pg/mL.

8. Use of a combination of a molecule specifically binding to H-NGAL protein and a molecule specifically binding to IL-6 protein for the preparation of a test strip for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject by detecting H-NGAL and IL-6 proteins in the peritoneal dialysis permeate of the subject, wherein the test strip preferably has an H-NGAL cut-off of 500pg/mL and an IL-6 cut-off of 200pg/mL.

9. A method for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising: detecting H-NGAL protein in the peritoneal dialysis dialysate of the subject, if H-NGAL protein is greater than or equal to the cut-off value, indicating that the subject has, or is at risk of having, peritonitis; optionally, the method further comprises detecting IL-6 protein in the peritoneal dialysis permeate, if IL-6 protein is greater than or equal to the cut-off value, indicating that the subject has, or is at risk of having, peritonitis.

10. The method of claim 9, wherein detecting H-NGAL comprises:

(a) Detecting H-NGAL protein in the peritoneal dialysis permeate; or

(b) Detecting total NGAL, including monomeric, H-NGAL and heterodimeric forms, in the peritoneal dialysis permeate and detecting monomeric and heterodimeric forms, thereby determining the amount of H-NGAL.

11. The method of claim 9 or 10, wherein the cut-off value for H-NGAL protein is 500pg/mL, and optionally, the cut-off value for IL-6 protein is 200pg/mL.

12. The method of any one of claims 9-11, comprising:

-obtaining a peritoneal dialysis permeate sample:

-contacting a first molecule, such as an antibody, that specifically binds to H-NGAL protein with the sample, optionally the first molecule being immobilized on a solid support; and

-detecting the formed complex.

13. The method of any one of claims 9-12, comprising:

-obtaining a peritoneal dialysis permeate sample:

-contacting a first molecule, such as an antibody, that specifically binds to H-NGAL protein with the sample, optionally the first molecule being immobilized on a solid support;

-adding a second molecule, such as an antibody, which specifically binds to the H-NGAL protein, optionally labelled, wherein the second molecule binds to a different epitope on the H-NGAL protein than the first molecule;

optionally adding a substance that specifically binds to the second molecule, such as an antibody against the second molecule, optionally labeled, and

-detecting the formed complex.

14. The method of any one of claims 9-13, comprising:

-obtaining a peritoneal dialysis permeate sample:

-contacting a first antibody molecule specifically binding to H-NGAL protein with the sample, wherein the first antibody molecule is labeled with biotin;

-adding a second antibody molecule that specifically binds to H-NGAL protein, wherein the second antibody molecule is labeled with colloidal gold and the second antibody molecule is a different antibody that binds to a different epitope on H-NGAL protein than the first antibody molecule;

adding streptavidin, optionally wherein the streptavidin is immobilized on a solid support, and

-detecting the complex formed.

15. A method of treating peritonitis in a peritoneal dialysis subject comprising:

(a) Detecting H-NGAL protein in the peritoneal dialysis permeate of the subject;

(b) Determining the level of H-NGAL;

(c) Comparing the H-NGAL level to a cutoff value; and

(d) Administering an antibiotic treatment if the H-NGAL level exceeds a cutoff value.

16. The method of claim 15, wherein the cut-off value for H-NGAL is 500pg/mL.

17. The method of claim 15 or 16, wherein the antibiotic therapy is selected from the group consisting of ertapenem, cefoxitin, doripenem, imipenem, cilastatin, meropenem, moxifloxacin, piperacillin, tazobactam, ticarcillin, clavulanate, and tigecycline; or cefepime, cefotaxime, ceftazidime or ceftriaxone in combination with metronidazole; or ceftizolin, cefotaxime, ceftriaxone, ciprofloxacin, levofloxacin in combination with metronidazole; or cefepime, ceftazidime, ciprofloxacin and levofloxacin are combined with metronidazole; or gentamicin or tobramycin in combination with clindamycin or metronidazole.

18. A test strip for use in the diagnosis, in particular the rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end and comprising, in succession starting from the first end, a sample pad, a binding pad, a nitrocellulose membrane and optionally an absorbent pad, wherein the nitrocellulose membrane comprises a detection line and a quality control line, wherein:

the binding pad comprises a first molecule that specifically binds to H-NGAL protein, optionally labelled, and optionally further comprises a second molecule that specifically binds to H-NGAL protein, optionally labelled, optionally wherein the first and second molecules are not the same;

the detection line comprises an immobilized substance that detects complexes formed by the H-NGAL protein and the first and/or second molecule, and

the control line comprises an immobilized species that specifically binds to the first molecule and/or the second molecule.

19. The test strip of claim 18, wherein:

the binding pad further comprises a third molecule that specifically binds to IL-6 protein, optionally labeled, and optionally further comprises a fourth molecule that specifically binds to IL-6 protein, optionally labeled, optionally wherein the third and fourth molecules are not the same;

the nitrocellulose membrane further comprises a detection line for detecting an immobilized substance of a complex formed by the IL-6 protein and the third molecule and/or the fourth molecule, and

optionally, the control line comprises an immobilized substance that specifically binds to the third and/or fourth molecule.

20. A test strip for the diagnosis, in particular the rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end and comprising, in succession starting from the first end, a sample pad, a binding pad, a nitrocellulose membrane and optionally an absorbent pad, wherein the nitrocellulose membrane comprises a detection line and a quality control line, wherein:

the conjugate pad comprises:

(a) A first molecule that specifically binds to H-NGAL protein, optionally labelled, and optionally further comprising a second molecule that specifically binds to H-NGAL protein, optionally labelled, optionally wherein the first and second molecules are not identical;

(b) A third molecule that specifically binds to an IL-6 protein, optionally labeled, and optionally further comprising a fourth molecule that specifically binds to an IL-6 protein, optionally labeled, optionally wherein the third and fourth molecules are not the same;

the detection line comprises a detection line comprising an immobilized substance detecting complexes formed by the H-NGAL protein and the first and/or second molecules and a detection line comprising an immobilized substance detecting complexes formed by the IL-6 protein and the third and/or fourth molecules, and

21. The test strip of any one of claims 18-20, wherein the molecule is labeled with a label selected from the group consisting of biotin, fluorescent, radioactive, luminescent, chemical, enzymatic, or particulate labels, preferably colloidal gold labels.

22. A test strip for the diagnosis, in particular the rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising a solid support having a first and a second end, comprising successively, starting from said first end, a sample pad, a biotinylated pad, a gold-labeled pad, a nitrocellulose membrane and an absorbent pad, wherein said nitrocellulose membrane comprises a detection line and a quality control line, wherein:

the biotinylated pad comprises a biotin-labeled first molecule that specifically binds to H-NGAL protein,

the gold-labeled pad comprises a second molecule labeled by colloidal gold and specifically binding to H-NGAL protein, the detection line comprises immobilized streptavidin,

the control line comprises an immobilized species that specifically binds to the first molecule and/or the second molecule, optionally wherein the first molecule and the second molecule are not the same.

23. The test strip of any one of claims 18-22, wherein the molecule and/or substance is selected from an antibody or an antigen-binding fragment thereof, a ligand, an aptamer, preferably from a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody or an antigen-binding fragment thereof, more preferably a monoclonal antibody or an antigen-binding fragment thereof.

24. The test strip of any one of claims 18-23, wherein the molecule is a primary antibody or antigen-binding fragment thereof and the control line comprises a secondary antibody or antigen-binding fragment thereof that binds the first, second, third, and/or fourth antibody, such as an anti-IgG antibody molecule.

25. The test strip of any one of claims 18-24, which has at least one of the following characteristics:

(a) The sample pad and the conjugate pad have overlapping portions, wherein the sample pad is above the conjugate pad;

(b) The conjugate pad and the nitrocellulose membrane have an overlapping portion, wherein the conjugate pad is on top of the nitrocellulose membrane;

(c) The biotinylation pad and the gold-labeled pad have overlapping portions, wherein the biotinylation pad is on top of the gold-labeled pad,

(d) The gold-labeled pad and the nitrocellulose membrane have an overlapping portion, wherein the gold-labeled pad is on the nitrocellulose membrane; and

(e) The absorbent pad and the nitrocellulose membrane have an overlapping portion, wherein the absorbent pad is on top of the nitrocellulose membrane.

26. The test strip of any one of claims 18-25, having an H-NGAL protein cutoff of 500pg/mL and/or having an IL-6 protein cutoff of 200pg/mL.

27. A kit for the diagnosis, preferably rapid diagnosis, of peritonitis or the risk of peritonitis in a peritoneal dialysis patient comprising means for detecting H-NGAL protein, and optionally means for detecting IL-6 protein, and optionally a reference color chip, wherein the result is determined to be negative or positive by comparing the result of the detection to the reference color chip.

28. The kit of claim 27, comprising the test strip of any one of claims 18-26.

29. A device for diagnosing, preferably rapidly diagnosing, peritonitis or the risk of peritonitis in a peritoneal dialysis subject, comprising: means for detecting H-NGAL protein in the peritoneal dialysis dialysate of a peritoneal dialysis subject and optionally means for detecting IL-6 protein, and optionally means for displaying the results of the detection.

30. The device of claim 29, comprising means for indicating to a subject that a peritonitis has occurred or is at risk of having a peritonitis when H-NGAL protein in the peritoneal dialysis dialysate is greater than or equal to a cut-off value, such as 500pg/mL, and optionally when IL-6 protein in the peritoneal dialysis dialysate is greater than or equal to a cut-off value, such as 200pg/mL.

31. A test strip according to any one of claims 18 to 26, a kit according to claim 27 or 28 and/or a device according to claim 29 or 30 for use in a method according to any one of claims 9 to 17.