Specific detection of homodimeric hnl

Abstract

The present disclosure relates to a method for detecting the presence or absence of homodimer human neutrophil lipocalin (HNL) in a sample. Methods, kits, and devices for establishing the presence of or excluding a bacterial infection are also provided. The present invention allows for accurate and reliable detection of bacterial infections and monitoring of bacterial infections as well as monitoring of the efficacy of antibacterial treatments by specifically detecting homodimers HNL using a specific combination of specific monoclonal antibodies.

Description

O 2 0 2 4 / 2 4 6 0 4 8 A 1 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 05 December 2024 (05.12.2024) (10) International Publication Number WO 2024 / 246048 A1 WIPO PCT (51) International Patent Classification: G01N 33 / 577 (2006.01) G01N 33 / 68 (2006.01) G01N 33 / 92 (2006.01) (74) Agent: BRANN AB; Box 3690, 103 59 Stockholm (SE). (21) International Application Number: PCT / EP2024 / 064616 (22) International Filing Date: 28 May 2024 (28.05.2024) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 2350677-7 01 June 2023 (01,06.2023) SE (71) Applicant: P & M VENGE AB [SE / SE]; Skolgatan 23, 75312 UPPSALA (SE). (72) Inventors: VENGE, Per; Skolgatan 23, 75312 UPPSALA (SE). XU, Shengyuan; Svartbäcksgatan 121, 75334 UP- PSALA (SE). (81) Designated States (unless otherwise indicated, for every kind of national protectionavailable): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN. BR. BW, BY, BZ. CA, CH, CL, CN, CO, CR, CU, CV, CZ, DE, DJ, DK, DM. Do, DZ, EC, EE, EG, ES, Fi, GB, GD, GE, GH, GM, GT, HN, HR, Hu, ID, IL, IN, IO, IR, IS, IT, JM, Jo, JP, KE, KG. KH, KN, KP, KR, KW. KZ, LA, LC. LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, SV, RW, SG, SDK, SA, SC, SE SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), K AZJ, KZY, BAM ( TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, ME, MK, MT: NL, NO, PL, RSIF, Ti, RO DETECTION OF HOMODIMERIC HNL 0.0 0.5 1.0 1.5 2.0 0 0 7 8 3.0 Figure 1 MAV3 / MAVZ ELISA.2.5 5 6 3 1 2 0 6 2 5 5 Dimer Monomer HNL, ng / ml (57) Abstract: The present disclosure relates to a method for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample. A method, a kit-of-parts and a device for establishing the presence of a bacterial infection or ruling out thereof are also provided. The present invention allows for accurate and reliable detection of a bacterial infection and monitoring thereof as well as monitoring efficiency of an antibacterial treatment by specific detection of homodimeric HNL using a specific combination of specific monoclonal antibodies. [Continued on next page] WO 2024 / 246048 A1 SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ. GW, KM, ML, MR, NE, SN, TD, TG). Published: - with international search report (Art. 21(3)) - with sequence listing part of description (Rule 5.2(a)) - in black and white; the international application as filed contained color or greyscale and is available for download fromPATENTSCOPЕ WO 2024 / 246048 PCT / EP2024 / 064616 SPECIFIC DETECTION OF HOMODIMERIC HNL 5 10 TECHNICAL FIELD The present invention relates to the methods and means for detecting and monitoring bacterial infections as well as monitoring efficiency of an antibacterial treatment. More specifically, the present invention relates to a method for detecting a presence or an absence of homodimeric human neutrophil lipocalin (HNL) in a sample as well as to a method, a kit-of-parts and a device for establishing a presence or an absence of a bacterial infection. BACKGROUND Bacteria can cause a number of conditions and diseases which are a major burden to society. Among such conditions and diseases are sepsis, gastrointestinal tract infections, urinary tract infections, tuberculosis, pneumonia and gonorrhoea. The treatment of 15 bacterial infections largely relies on administration of antibiotics. Incorrect administration of antibiotics, e. g. administration of antibiotics when treating infectionswhich are not caused by bacteria, may lead to that at least some of the bacteria present in the body become drug resistant. These bacteria may spread into the environment and ultimately become multidrug resistant. 20 25 30 Bacterial antimicrobial resistance (AMR) is currently one of the leading threats to public health, i. e. AMR threatens our ability to treat common infectious diseases. The death rate of patients with infections caused by common drug-resistant bacteria treated in hospitals can be much higher than that of patients with infections caused by the same non- drug-resistant bacteria. For example, patients with infections caused by methicillin-resistant Staphylococcus aureus, a common source of severe infections in the community and in hospitals, are estimated to be 64% more likely to die than patients with infections caused by Staphylococcus aureus that is not methicillin-resistant. Another serious burden to the society and the health care system is sepsis with about 11million deaths per year worldwide according to the estimations of WHO. Currently, early diagnosis of sepsis relies mainly on detecting bacterial growth and partly on detecting higher than normal concentrations of biomarkers, such as CRP, procalcitonin and heparin- binding protein. In the majority of cases sepsis is caused by bacterial agents and is treated 1 WO 2024 / 246048 PCT / EP2024 / 064616 5 with antibiotics. To opt for antibiotic treatment, it is important to be able to distinguish sepsis from other conditions, such as systemic inflammatory disease (SIRS). Up to now it has been proven to be difficult with the known in the art methods and means to discriminate between sepsis and SIRS. Sensitivity and specificity for such methods are in the range 70-80 % at most. Therefore, there is an increasing need in the field to reliably detect bacterial infections and to administer antibiotics only to patients who have bacterial infections. One of the proteins which has been used as a biomarkerfor detecting a bacterial infection is human neutrophil lipocalin (HNL). HNL is secreted by neutrophils upon their 10 activation and plays a well-established role during an extracellular bacterial infection. The secreted HNL can be detected in bodily fluids. 15 20 25 30 HNL is a ubiquitous glycoprotein localised in specific granules of neutrophils. HNL can occur as a 25-kD monomer or a 45-kD disulphide-linked homodimer. Furthermore, a part of HNL can be covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulphide bridge forming a 135-kD complex. The amino acid sequence of HNL is shown in SEQ ID NO: 1 corresponding to the protein sequence found in www.uniprot.org / uniprot / P80188, in particular referring to isoform 1. Sensitivity and specificity for the known in the art serum-based assays which employ measurement of the amount of HNL are in the range 75-94 %. The lower figures of the range are obviously not satisfactory for the clinical setting. EP0756708 B1 relates to the use of HNL for diagnosing inflammation caused by a bacterial infection. Disclosed immunoassays comprise bringing a sample suspected of containing an abnormal level of HNL in contact with an antibody specific for HNL (anti- HNL antibody). The term anti-HNL antibody means an antibody preparation reacting specifically with HNL, such as in monomeric forms, HNL as included in di-, multi- or heteromeric forms and / or fragments of monomeric HNL exhibiting HNL unique determinants and epitopes. WO 2016 / 079219 A1 relates to a binding agent capable of specifically binding to a polypeptide epitope of HNL as well as to the means and methods for detecting a bacterial infection and discriminating between a bacterial infection and a viral infection. Despite the existing methods, there is still a need for methods and means that would allow for more accurate and reliable detection and monitoring of a bacterial 2 (19) State Intellectual Property Office of the People's Republic of China (12) Patent Application for Invention (10) Publication Number of Application (43) Publication Date of Application (21) Application Number 202480035579.7(22) Application date 2024.05.28 (30) Priority data 2350677-7 2023.06.01 SE (85) PCT international application enters national phase date 2025.11.27 (86) PCT international application application data PCT / EP2024 / 064616 2024.05.28 (87) PCT international application publication data WO2024 / 246048 EN 2024.12.05 (71) Applicant Swedish Diagnostics Development GmbH Address Uppsala, Sweden (72) Inventors P. Weng and S. Xu (74) Patent agency China Patent Agency (Hong Kong) Limited 72001 Patent Attorneys Luo Wenfeng and Yang Sijie (51) Int.Cl. G01N 33 / 577 (2006.01) G01N 33 / 92 (2006.01) G01N 33 / 68(2006.01) (54) Invention Title: Specific Detection of Homodimer HNL (57) Abstract: This disclosure relates to a method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample. A method, kit, and apparatus for establishing or ruling out the presence of bacterial infection are also provided. This invention allows for accurate and reliable detection and monitoring of bacterial infection, as well as monitoring the efficacy of antimicrobial therapy, by using a specific combination of specific monoclonal antibodies to specifically detect homodimer HNL. Claims (3 pages), Description (16 pages), Sequence Listing (electronic publication), Drawings (5 pages) CN 121219586 A 2025.12.26 CN 1 21 21 95 86 A 1. An in vitro method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample, wherein the method comprises: providing a sample; providing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each of the homodimers comprising a first monomer and a second monomer, the first monoclonal antibody configured to bind to the first monomer, the second monoclonal antibody configured to bind to the second monomer; contacting the sample with the first monoclonal antibody; contacting the sample with the second monoclonal antibody; detecting the presence of homodimer HNL, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first group of mouse complementarity-determining regions (CDRs), the first group of mouse CDRs comprising: The first heavy chain CDR (CDRH1), where CDRH1 is SEQ ID NO: 6, the second heavy chain CDR (CDRH2), where CDRH2 is SEQ ID NO: 7, and the third heavy chain CDR(CDRH3), wherein CDRH3 is SEQ ID NO: 8, wherein the light chain variable region comprises a second group of mouse complementarity-determining regions (CDRs), the second group of mouse CDRs comprising: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9, a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10, and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11. 2. The in vitro method according to claim 1, wherein the sample is derived from at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid, another bodily fluid, feces, and / or liquid formulations thereof. 3. The in vitro method according to any of the preceding claims, wherein the first monoclonal antibody is immobilized on a solid carrier. 4. The in vitro method according to any of the preceding claims, wherein each of the CDRs is configured to bind to an epitope formed between amino acids 145 to 154 of HNL as defined in SEQ ID NO: 1. 5. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises an amino acid sequence as illustrated in SEQ ID NO: 5 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 5. 6. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises an amino acid sequence as illustrated in SEQ ID NO: 4 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 4. 7. The in vitro method according to any one of claims 1-4, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises an amino acid sequence as illustrated in SEQ ID NO: 3 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 3. 8. The in vitro method according to any one of claims 1-4, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises an amino acid sequence as illustrated in SEQ ID NO: 3.Claim 1 / 3 page 2 CN 121219586 A 2 The sequence shown in CN 121219586 A 2 or the amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with SEQ ID NO: 2. 9. The in vitro method according to any one of claims 1-4, wherein each of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a heavy chain variable region sequence as illustrated in SEQ ID NO: 5 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 5, and wherein each of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a light chain variable region sequence as illustrated in SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 4. 10. The in vitro method according to any one of claims 1-4, wherein each of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a heavy chain variable region sequence as illustrated in SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 3, and wherein each of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a light chain variable region sequence as illustrated in SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 2. 11. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody is a humanized antibody. 12. An in vitro method for determining the presence of a bacterial infection, the method comprising: providing a sample; providing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each of the homodimers comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer.The sample is contacted with the first monoclonal antibody, and the sample is contacted with the second monoclonal antibody. A value corresponding to the amount of homodimer HNL in the sample is determined, and the value is compared with a control value. When the value is significantly higher than the control value, a bacterial infection is established, or when the value is significantly lower than the control value, a bacterial infection is established, wherein the first monoclonal antibody and / or the second monoclonal antibody are as defined in any one of claims 1-11. 13. A kit for detecting the presence of homodimer HNL in a sample according to any one of claims 1-11 and / or for establishing the presence of bacterial infection according to claim 12, wherein the kit comprises one or more of the monoclonal antibodies according to claims 1-11 and one or more of instructions for use, buffer solutions, and devices, wherein the devices are arranged for analyzing the sample. 14. An apparatus for determining the presence of a bacterial infection, the apparatus comprising: a first compartment containing a monoclonal antibody for detecting a homodimer of HNL: a first monoclonal antibody and a second monoclonal antibody, each of the homodimers comprising a first monomer and a second monomer, the first monoclonal antibody configured to bind to the first monomer, the second monoclonal antibody configured to bind to the second monomer, wherein the first monoclonal antibody and / or the second monoclonal antibody are as defined in any one of claims 1-11, wherein the first compartment is adapted to contact a sample, the sample being suspected of containing or not containing a homodimer of HNL; a second compartment containing a substrate for activating neutrophils present in the sample, wherein the activation is performed prior to determining the presence of a bacterial infection, wherein the second compartment includes an opening connecting it to the first compartment. A first connector, wherein the first connector is arranged for connecting the device to another device for retrieving a sample from a subject suspected of having at least one bacterial infection and viral infection; and a second connector, wherein the second connector is arranged for connecting the device to a computer and / or device for analyzing the interaction between the homodimer of HNL, the first monoclonal antibody, and the second monoclonal antibody, the analysis allowing determination of the amount of homodimer HNL in the sample. 15. The device of claim 14, further comprising a binding agent for one or more determinants selected from: ABTB1, ADIPOR1, ARHGDIB, ARPC2, ATP6V0B, Clorf83, CD15, CES1, CORO1A, CSDA, EIF4B, EPSTI1, GAS7. HERC5, IFI6, KIAA0082, IFIT1, IFIT3, IFITM1, IFITM3, LIPT1, IL7R, ISG20, LOC26010, LY6E, LRDD, LTA4H, MAN1C1, MBOAT2, NPM1, OAS2, PARP12, PARP9, QARS, RAB13, RAB31, RAC2, RPL34, PDIA6, PTEN, RSAD2, SART3, SDCBP, SMAD9, SOCS3, TRIM22, SART3, UBE2N, XAF1, ZBP1, CRP and MX1. 16. The apparatus according to claim 14 or 15, wherein the apparatus is any one of: a point-of-care device, a test strip, an apparatus for radioimmunoassay, an apparatus for ELISA, an apparatus for sandwich immunoassay, an apparatus for immunoradioassay, an apparatus for gel diffusion precipitation assay, an apparatus for immunodiffusion assay, an apparatus for in situ immunoassay using colloidal gold, enzyme, or radioisotope labeling, an apparatus for Western blotting, an apparatus for precipitation assay, an apparatus for gel agglutination assay, an apparatus for hemagglutination assay, an apparatus for immunofluorescence assay, an apparatus for protein A assay, an apparatus for immunoelectrophoresis assay, and an apparatus for analyte detection involving magnetic separation of analytes. Claims 3 / 3 Page 4 CN 121219586 A Specific Detection Technology of Homodimer HNL

[0001] The present invention relates to methods and means for detecting and monitoring bacterial infections and monitoring the efficacy of antimicrobial therapy. More specifically, the present invention relates to a method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample and a method, kit, and apparatus for determining the presence of a bacterial infection. Background Art

[0002] Bacteria can cause a variety of conditions and diseases, which are a significant burden on society. Among such conditions and diseases are sepsis, gastrointestinal infections, urinary tract infections, tuberculosis, pneumonia, and gonorrhea. Treatment of bacterial infections relies heavily on the administration of antibiotics. Inappropriate administration of antibiotics, such as when treating infections not caused by bacteria, can lead to at least some of the bacteria present in the body becoming resistant. These bacteria may spread into the environment and eventually become multidrug resistant.

[0003] Antimicrobial resistance (AMR) is currently one of the major threats to public health, meaning that AMR threatens our ability to treat common infectious diseases. The mortality rate of patients treated in hospitals with infections caused by common drug-resistant bacteria can be far higher than that of patients with infections caused by the same non-drug-resistant bacteria. For example, the mortality rate of patients with infections caused by methicillin-resistant bacteria is high.The risk of death for patients infected with Staphylococcus aureus (a common and serious source of infection in communities and hospitals) is estimated to be 64% higher than for those infected with methicillin-resistant Staphylococcus aureus.

[0004] Another serious burden on social and healthcare systems is sepsis, which, according to WHO estimates, kills approximately 11 million people globally each year. Currently, early diagnosis of sepsis relies primarily on detecting bacterial growth and, in part, on detecting biomarkers such as CRP, procalcitonin, and heparin-binding protein at elevated levels. In most cases, sepsis is caused by bacterial factors and treated with antibiotics. To select antibiotic treatment, it is important to be able to differentiate sepsis from other conditions such as systemic inflammatory diseases [SIRS]. To date, it has been shown that it is difficult to differentiate sepsis from SIRS using known existing techniques and methods. The sensitivity and specificity of such methods are at most in the range of 70-80%.

[0005] Therefore, there is a growing need in the field for reliable detection of bacterial infections and for administering antibiotics only to patients with bacterial infections.

[0006] One of the proteins that has been used as biomarkers for detecting bacterial infection is human neutrophil lipid carrier protein (HNL). HNL is secreted by neutrophils upon activation and plays a well-established role during extracellular bacterial infection. Secreted HNL can be detected in body fluids.

[0007] HNL is a ubiquitous glycoprotein located in specific granules of neutrophils. HNL can exist as a 25-kD monomer or a 45-kD disulfide-linked homodimer. Furthermore, a portion of HNL can be covalently conjugated to gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge, thereby forming a 135-kD complex. The amino acid sequence of HNL is shown in SEQ ID NO: 1, corresponding to the protein sequence present at www.uniprot.org / uniprot / P80188, specifically isotype 1.

[0008] Known prior art serum-based assays for measuring the amount of HNL have a sensitivity and specificity in the range of 75-94%. This lower range of figures is clearly unsatisfactory for a clinical setting.

[0009] EP 0756708 B1 relates to the use of HNL for the diagnosis of inflammation caused by bacterial infection. The disclosed immunoassay involves contacting a sample suspected of containing abnormal levels of HNL with an antibody specific to HNL (anti-HNL antibody). Terminology Specification 1 / 16 page 5 CN 121219586 A “anti-HNL antibody” refers to an antibody preparation that reacts specifically with HNL, such as HNL in monomeric form, including HNL in dimer, multimer or heteromeric form, and / or monomeric HNL fragments having unique HNL determinants and epitopes.

[0010] WO2016 / 079219 A1 relates to a binding agent capable of specifically binding to a polypeptide epitope of HNL, and means and methods for detecting bacterial infections and distinguishing bacterial infections from viral infections.

[0011] Despite existing methods, there is a need for methods and means that allow for more accurate and reliable detection and monitoring of bacterial infections and monitoring of the efficacy of antimicrobial therapy. There is also a need for more accurate and reliable exclusion of bacterial infections. Summary of the Invention

[0012] In view of the foregoing, as defined in the appended claims, one object of the present invention is to provide a method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample, and a method, kit, and apparatus for determining the presence of bacterial infection. The present invention at least partially alleviates the problems discussed above and at least partially addresses one or more of the needs mentioned above. Further details and advantages of the invention will emerge from the following detailed disclosure and dependent claims.

[0013] According to a first aspect of the present invention, an in vitro method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample is provided, wherein the method comprises: i) providing a sample; ii) providing monoclonal antibodies for detecting the homodimer of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer and the second monoclonal antibody being configured to bind to the second monomer; iii) contacting the sample with the first monoclonal antibody; iv) contacting the sample with the second monoclonal antibody; v) detecting the presence of the homodimer HNL, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first set of mouse complementarity-determining regions (CDRs), the first set of mouse CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6; and a second heavy chain CDR (CDRH2), wherein CDRH2 is SEQ ID NO: 6. 7, and a third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 8, wherein the light chain variable region includes a second group of mouse complementary determinant regions (CDRs), the second group of mouse CDRs including: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9, a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10, and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11.

[0014] According to another aspect of the present invention, an in vitro method for determining the presence of bacterial infection is provided, wherein the method includes: i) providing a sample, ii)Provided are monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer consisting of a first monomer and a second monomer, the first monoclonal antibody configured to bind to the first monomer, and the second monoclonal antibody configured to bind to the second monomer. (Instructions for use, page 2 / 16, CN 121219586 A) iii) Contact the sample with the first monoclonal antibody; iv) Contact the sample with the second monoclonal antibody; v) Determine a value corresponding to the amount of homodimer HNL in the sample; vi) Compare this value to a control value; vii) When the value is significantly higher than the control value, establish the presence of bacterial infection, or when the value is significantly lower than the control value, establish the absence of bacterial infection. Each of the first and second monoclonal antibodies contains a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains a first group of mouse complementarity-determining regions (CDRs), the first group of mouse CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6, and a second heavy chain CDR. (CDRH2), wherein CDRH2 is SEQ ID NO: 7, and the third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 8, wherein the light chain variable region includes a second group of mouse complementarity-determining regions (CDRs), the second group of mouse CDRs including: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9, a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10, and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11.

[0015] In the method of this document, the steps of providing the sample and providing the monoclonal antibody can be performed in any order. Furthermore, the steps of contacting the sample with the first monoclonal antibody and the second monoclonal antibody respectively can be performed in any order, but it is preferred that the sample be contacted with the first antibody first.

[0016] According to another aspect of the invention, a kit is provided for detecting the presence of homodimer HNL in a sample and / or establishing the presence of bacterial infection, wherein the kit comprises one or more monoclonal antibodies as described herein and at least one of instructions for use, buffer, and device, wherein the device is arranged for performing sample analysis.

[0017] According to another aspect of the invention, an device is provided for establishing the presence of bacterial infection, wherein the device comprises: a first compartment containing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer.Each of the first and second monoclonal antibodies comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a first set of mouse complementarity-determining regions (CDRs), which includes: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6; a second heavy chain CDR (CDRH2), wherein CDRH2 is SEQ ID NO: 7; and a third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 8. The light chain variable region comprises a second set of mouse complementarity-determining regions (CDRs), which includes: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9; a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10; and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11. The first compartment is adapted to contact the sample, which is suspected to contain or not contain HNL homodimers. (Instructions 3 / 16, page 7 CN) 121219586 A body; a second compartment, wherein the second compartment contains a substrate for activating neutrophils present in the sample, wherein the activation is performed prior to establishing the presence of a bacterial infection, wherein the second compartment contains an opening connecting it to the first compartment; a first connector, wherein the first connector is arranged for connecting the device to another device for removing a sample from a subject suspected of having at least one of a bacterial infection and a viral infection; and a second connector, wherein the second connector is arranged for combining the device with a computer and / or device for analyzing the interaction between a homodimer of HNL, a first monoclonal antibody, and a second monoclonal antibody, the analysis allowing determination of the amount of homodimer HNL in the sample.

[0018] These and other aspects of the invention are apparent from and elucidated with reference to embodiments described below. Brief Description of the Drawings

[0019] For illustrative purposes, the invention will be described in more detail below with reference to embodiments thereof illustrated in the accompanying drawings.

[0020] Figure 1 illustrates, in a diagrammatic representation, that the specific binding and detection of the homodimer of HNL can be achieved using the method according to the invention (exemplified by an enzyme-linked immunosorbent assay (ELISA) using a first monoclonal antibody MAB3 and a second monoclonal antibody MAB3).

[0021] Figure 2 illustrates, in a diagrammatic representation, that the specific binding and detection of the homodimer of HNL can be achieved using the method according to the invention (exemplified by an ELISA using a first monoclonal antibody MAB2 and a second monoclonal antibody MAB2).

[0022] Figure 3 illustrates, in a diagrammatic representation, the method according to the invention using the same antibody as both the capture and detection reagents.The method cannot be performed using any monoclonal antibody specific to HNL. Figure 3 illustrates that HNL dimers are undetectable when antibody MAB5 is used as the first and second monoclonal antibodies in the method of this document.

[0023] Figure 4 schematically illustrates the level of HNL homodimers in plasma samples obtained from patients suffering from acute infections such as acute bacterial infections and acute viral infections. The level of HNL homodimers is detected by means of the method according to the invention using the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3.

[0024] Figure 5 schematically illustrates the level of HNL homodimers in plasma samples obtained from patients suffering from acute infections such as viral infections, bacterial pneumonia, mycoplasma infections, tonsillitis, urinary tract infections (UTI), gastrointestinal (GI) infections, erysipelas, and sepsis. The level of HNL homodimers is detected by means of the method according to the invention using the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3. Detailed Description

[0025] In the following detailed description, technical terms and expressions are defined, and embodiments of the invention are described.

[0026] Unless otherwise expressly defined herein, all terms and expressions used in this application should be interpreted according to their meaning as commonly applied in the relevant prior art.

[0027] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context explicitly specifies otherwise. Thus, for example, “first monoclonal antibody” includes one or more first monoclonal antibodies, “second monoclonal antibody” includes one or more second monoclonal antibodies, “first monomer” includes one or more first monomers, and “second monomer” includes one or more second monomers.

[0028] The terms “first,” “second,” etc., are used to distinguish between similar elements and are not necessarily used to describe the order of events or chronological order. It should be understood that each term thus used may be replaced by another appropriate term.

[0029] The term “comprising” should be considered to include the term “consisting of.” If a group is defined below as containing at least a certain number of elements, this is intended to also cover groups consisting only of those elements.

[0030] It should be understood that the present invention is not limited to the specific methods, schemes, proteins, bacteria, vectors, reagents, etc., described herein.

[0031] Within the scope of the present invention, the term "antibody" should be understood to mean any protein that can be produced by plasma cells in response to an antigen (i.e., a foreign substance that induces an immune response). Antibodies, also known as immunoglobulins, can be exemplified by peptides comprising natural and / or modified amino acids that allow the peptide to bind to the sequence of the HNL region described herein.

[0032] Within the scope of this invention, the term "monoclonal antibody" should be understood to mean an antibody obtained from a substantially homogeneous population of antibodies (as defined herein), i.e., the individual antibodies constituting the population are identical except for possibly present naturally occurring mutations or alternative post-translational modifications. Monoclonal antibodies can be produced by hybridoma cells or by means of recombinant DNA technology. Non-limiting examples of monoclonal antibodies include mouse, rabbit, rat, chicken, chimeric, humanized, or human monoclonal antibodies and their functional fragments, i.e., fragments having the desired biological activity. Human monoclonal antibodies can be produced using transgenic animals that are free of endogenous immunoglobulins and engineered to contain human immunoglobulin loci.

[0033] According to the invention, a monoclonal antibody or its functional fragment can be linked to, for example, a fluorescent portion, a radioactive portion, a chromogenic substrate, etc.

[0034] According to this document, the first monoclonal antibody may alternatively be referred to as a "capturing agent," "capture reagent," "capture agent," etc. Similarly, the second monoclonal antibody may be referred to as a “detecting reagent”, “detection reagent”, etc.

[0035] It is known that typical immunoglobulin (antibody) structural units comprise tetramers. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a “light” chain (about 25 kD) and a “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The term “variable light chain” (VL) refers to the variable region of the light chain. The term “variable heavy chain” (VH) refers to the variable region of the heavy chain. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for interacting with other components of the immune system.

[0036] Within the scope of the invention, the term “complementarity-determining region (CDR)” should be understood to refer to each of the three hypervariable regions of the variable region of each antibody chain. The variable region consists of a seven-amino acid region, of which four are framework regions and three are hypervariable regions. CDRs are primarily responsible for the binding of antibodies to antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3. CDRs are numbered sequentially starting from the N-terminus and are usually also identified by the chain in which a particular CDR is located. Thus, for example, VH CDR3 is located in the variable domain of the heavy chain of the antibody, while VL CDR1 is located in the variable domain of the light chain of the antibody. The sequence of the frame region is relatively conserved within the species.

[0037] Within the scope of this invention, the term "epitope" should be understood to refer to the portion or region of the antigen to which the antibody binds. Epitopes typically contain 5-10 amino acids with a unique spatial conformation. When an antibody recognizes a sequential amino acid sequence of an antigen (antigen...), it binds to the antigen...When an antibody recognizes a non-continuous amino acid folded together by the tertiary fold of an antigen, it is said to have a linear epitope. When an antibody recognizes a non-continuous amino acid folded together by the tertiary fold of an antigen, it is said to have a conformational epitope, also known as a discontinuous epitope. Linear epitopes are generally retained upon exposure to denaturing solvents, while conformational epitopes are generally lost upon treatment with denaturing solvents. Methods for determining the spatial conformation of epitopes include X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0038] Within the scope of this invention, the terms “specifically binding,” “specifically binding,” “bind specifically,” etc., should be understood to refer to the ability of a molecule to recognize and bind to a specific target molecule, such as when distinguishing other molecules that are structurally and / or compositionally similar. The first and second monoclonal antibodies of this document are capable of specifically binding to HNL in monomeric and dimer forms. Specification 5 / 16 pages 9 CN 121219586 A

[0039] In order to mathematically describe the accuracy of tests that report the presence or absence of a condition, the terms “sensitivity” and “specificity” are used. If individuals with the condition are considered "positive" and those without the condition are considered "negative," then sensitivity measures how well a test can identify true positives, while specificity measures how well a test can identify true negatives. Sensitivity is calculated as TP / (TP+FN), where TP is a "true positive" and FN is a "false negative." Specificity is calculated as TN / (TN+FP), where TN is a "true negative" and FP is a "false positive."

[0040] The illustrative embodiments of the present invention are described below.

[0041] According to a first aspect of the present invention, a method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample is provided, wherein the method comprises: providing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, contacting the sample with the first monoclonal antibody, contacting the sample with the second monoclonal antibody, detecting the presence of homodimer HNL, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first group of mouse complementarity-determining regions (CDRs), the first group of mouse CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6, a second heavy chain CDR (CDRH2), wherein CDRH2 is SEQ ID NO: 7, and a third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 6. NO: 8,The light chain variable region includes a second set of mouse complementarity-determining regions (CDRs), which include: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9; a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10; and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11.

[0042] The sample may be a bodily fluid. The sample may be derived from bodily fluids. Bodily fluids may be at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid, and another bodily fluid. The sample may also include liquid preparations obtained during various steps of purifying HNL from its natural source or recombinant production system. The sample may include at least one of feces and its liquid preparations. Advantageously, plasma samples may be used. The sample may be obtained from animals, such as mammals, such as humans. The sample may be obtained from a subject suspected of having at least one of bacterial and viral infections.

[0043] The sample may be contacted first with the second monoclonal antibody, and then the sample may be contacted with the first monoclonal antibody.

[0044] The sample may be incubated in a designated space in the presence of the first monoclonal antibody, and then the second monoclonal antibody may be added to the designated space. Incubation of the sample in the presence of the first monoclonal antibody may be performed under conditions that allow the first monoclonal antibody to bind to the first monomer of the homodimer of HNL. Similarly, after the addition of the second monoclonal antibody, time may be allowed for the second monoclonal antibody to bind to the second monomer of the homodimer of HNL, which is a homodimer monomer to which the first monoclonal antibody has not yet bound.

[0045] The method according to the invention, using a combination of the first and second monoclonal antibodies, allows not only the specific detection of HNL, but also the specific detection of the homodimer HNL. Since it is the homodimer HNL that is associated with the presence of bacterial infection, the method according to the invention allows for a more accurate and reliable detection of the presence of bacterial infection compared to what can be provided by known prior art methods. Instructions for Use, Page 6 / 16, CN 121219586 A

[0046] The amino acids of the first monomer to which the first monoclonal antibody binds and the amino acids of the second monomer to which the second monoclonal antibody binds can be similar. The above-described CDR can be configured to bind to epitopes of amino acids 145 to 154 of the first or second monomer as defined in SEQ ID NO: 1. The amino acids of the first and second monomers to which the above-described CDR can bind can be determined by X-ray crystallography.

[0047] “Binding” can be understood as about 75%, greater than about 80%, greater than about 81%, 82%, 83%, 84%, 85%, 86%, or 87%.88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% affinity binding, the affinity being assessed by, for example, competitive ELISA, SPR (Biacore), and any other suitable method known in the art. The affinity of the binding refers to the strength of the interaction between the two molecules, which is determined by factors such as the shape, charge, and hydrophobicity of the binding site.

[0048] The amino acid sequence of the heavy chain variable region may comprise the sequence shown in SEQ ID NO: 5, and / or the amino acid sequence of the light chain variable region may comprise the sequence shown in SEQ ID NO: 4.

[0049] The amino acid sequence of the heavy chain variable region may comprise the sequence shown in SEQ ID NO: 3, and / or the amino acid sequence of the light chain variable region may comprise the sequence shown in SEQ ID NO: 2.

[0050] The amino acid sequence of at least one of the heavy chain variable region and the light chain variable region of at least one of the first monoclonal antibody and the second monoclonal antibody may be mutated at a single position or multiple positions. Examples of mutable amino acid residues are those within the frame region, with at least 10% surface exposure, and within 4.5 Å of the CDR residue. The amino acid residue to be mutated can be selected by visually examining the three-dimensional structural model of the amino acids adjacent to one or more selected amino acid residues in the CDR and / or frame region.

[0051] At least one amino acid residue of the CDR sequence or the heavy chain and / or light chain variable region of the above-described monoclonal antibody (i.e., the first monoclonal antibody and / or the second monoclonal antibody) may be substituted, deleted, or added to the amino acid sequence by another amino acid residue. Substitution, addition, and / or deletion may be performed anywhere in the amino acid sequence, provided that the specific binding activity with HNL is maintained. For each variable region / CDR sequence, at least one, two, three, four, five, six, or more substitutions, additions, and / or deletions may be performed, provided that the specific binding activity of the above-described monoclonal antibody with HNL is maintained. In this context, specific binding activity refers to the ability of an antibody to bind specifically to an HNL molecule with high selectivity and affinity.

[0052] At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a sequence as illustrated in SEQ ID NO: 5. At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 5.

[0053] At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises the sequence illustrated in SEQ ID NO: 3. At least one of the first monoclonal antibody and the second monoclonal antibody may have a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 3.

[0054] At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising a second group of mouse CDRs, wherein the light chain comprises the sequence illustrated in SEQ ID NO: 4. At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 4.

[0055] At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2. At least one of the first monoclonal antibody and the second monoclonal antibody may have a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0056] Therefore, monoclonal antibodies as defined in this document also encompass monoclonal antibodies having the following: the heavy chain of each of the first monoclonal antibody and the second monoclonal antibody may contain the heavy chain variable region sequence as shown in SEQ ID NO: 5, and the light chain of each of the first monoclonal antibody and the second monoclonal antibody may contain the light chain variable region sequence as shown in SEQ ID NO: 4.

[0057] The heavy chain of each of the first monoclonal antibody and the second monoclonal antibody may contain the heavy chain variable region sequence as shown in SEQ ID NO: 3, and the light chain of each of the first monoclonal antibody and the second monoclonal antibody may contain the light chain variable region sequence as shown in SEQ ID NO: 2.

[0058] The amino acid sequence of at least one of the heavy chain constant region and the light chain constant region of the above-described monoclonal antibody may be mutated at a single or multiple positions as described above, provided that the specific binding activity of the above-described monoclonal antibody is maintained.

[0059] The amino acid sequence of the first monoclonal antibody may be similar to the amino acid sequence of anti-HNL MAB2 or anti-HNL MAB3.The amino acid sequence of the second monoclonal antibody may be identical to that of MAB2 or anti-HNL MAB3.

[0060] The amino acid sequence of the first monoclonal antibody may be identical to that of the second monoclonal antibody. The first monoclonal antibody may have the same amino acid sequence as anti-HNL MAB2, while the second monoclonal antibody may have the same amino acid sequence as anti-HNL MAB2. The first monoclonal antibody may have the same amino acid sequence as anti-HNL MAB3, while the second monoclonal antibody may have the same amino acid sequence as anti-HNL MAB3.

[0061] The amino acid sequence of the light chain variable region of anti-HNL MAB2 is as follows: DIVLTQSTSSLSVSLGDRVTINCRASQDISNYLNWYQEKPDGTVKLLIYFTSRLHSGVPSRFSGSGSG TDYSLTITNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRADAAPTV (see also WO2016 / 079219 A1).

[0062] The above amino acid sequence is the same as that shown in SEQ ID NO: 2.

[0063] The amino acid sequence of CDRL1 of anti-HNL MAB2 is as follows: RASQDISNYLN.

[0064] The amino acid sequence of CDRL2 of anti-HNL MAB2 is as follows: FTSRLHS.

[0065] The amino acid sequence of CDRL3 of anti-HNL MAB2 is as follows: QQGNTLPWT.

[0066] The amino acid sequences of CDRL1, CDRL2, and CDRL3 of anti-HNL MAB2 are identical to those shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0067] The amino acid sequence of the heavy chain variable region of anti-HNL MAB2 is as follows: EVQLEESGPGLVAPSQSLSITCTISGFSLTSYGIHWLRQPPGKDLEWLVVIWGDGSTTSNSALKSRLS ISKDNSKSQVFFKMSGLQTDDTAIYYCARHRYSDYHAMDYWGPGTSVTVS (see also WO2016 / 079219 A1).

[0068] The above amino acid sequence is identical to that shown in SEQ ID NO: 3.

[0069] The amino acid sequence of CDRH1 of anti-HNL MAB2 is as follows: GFSLTSYGIH.

[0070] The amino acid sequence of CDRH2 for anti-HNL MAB2 is as follows: VIWGDGSTTSNSALKS.

[0071] Anti-HNLThe amino acid sequence of CDRH3 of MAB2 is as follows: HRYSDYHAMDY.

[0072] The amino acid sequences of CDRH1, CDRH2, and CDRH3 of anti-HNL MAB2 are the same as those shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0073] The amino acid sequence of the light chain variable region of anti-HNL MAB3 is as follows: DIVLTQTTSSLSVSLGDRVTINCRASQDISNYLNWYQEKPDGTVKLLIYFTSRLHSGVPSRFSGSGSG TDYSLTITNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRADAAPTV (see also WO2016 / 079219 A1).

[0074] The above amino acid sequence is the same as that shown in SEQ ID NO: 4.

[0075] The amino acid sequence of CDRL1 of anti-HNL MAB3 is as follows: RASQDISNYLN.

[0076] The amino acid sequence of CDRL2 of anti-HNL MAB3 is as follows: FTSRLHS.

[0077] The amino acid sequence of CDRL3 of anti-HNL MAB3 is as follows: QQGNTLPWT.

[0078] The amino acid sequences of CDRL1, CDRL2, and CDRL3 of anti-HNL MAB3 are identical to those shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0079] The amino acid sequence of the heavy chain variable region of anti-HNL MAB3 is as follows (see also WO 2016 / 079219 A1): EVKLQESGPGLVAPSQSLSITCTISGFSLTSYGIHWLRQPPGKDLEWLVVIWGDGSTTSNSALKSRLS ISKDNSKSQVFFKMSGLQTDDTAIYYCARHRYSDYHAMDYWGPGTSVTVSS.

[0080] The above amino acid sequence is the same as that shown in SEQ ID NO: 5.

[0081] The amino acid sequence of CDRH1 of anti-HNL MAB3 is as follows: GFSLTSYGIH.

[0082] The amino acid sequence of CDRH2 of anti-HNL MAB3 is as follows: VIWGDGSTTSNSALKS.

[0083] The amino acid sequence of CDRH3 of anti-HNL MAB3 is as follows: HRYSDYHAMDY.

[0084] The amino acid sequences of CDRH1, CDRH2, and CDRH3 of anti-HNL MAB3 are respectively as shown in SEQ ID.The same as those illustrated in NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0085] At least one of the first monoclonal antibody and the second monoclonal antibody may be a humanized antibody.

[0086] The first monoclonal antibody may be immobilized on a solid support and thus configured to bind to a first monomer (the homodimer of HNL), while the second monoclonal antibody may be in solution and thus configured to bind to a second monomer (the homodimer of HNL) (i.e., a monomer not yet bound by the first monoclonal antibody immobilized on the solid support). Thus, the first monoclonal antibody may act as a capture reagent, while the second monoclonal antibody may act as a detection reagent. Those skilled in the art will understand that, alternatively, the first monoclonal antibody may be configured to bind to the first monomer in a manner different from that described above. Similarly, those skilled in the art will understand that, alternatively, the second monoclonal antibody may be configured to bind to the second monomer in a manner different from that described above.

[0087] The above method may also include determining the amount of homodimer HNL in the sample. The amount of homodimer HNL in the sample can be determined by measuring the amount of immune complexes formed between the monoclonal antibody and the homodimer HNL present in the sample. The detection of the formed immune complexes can be accomplished using a variety of known techniques, such as radioimmunoassay (RIA) and ELISA (more specifically, double monoclonal antibody sandwich immunoassay), as well as immunoradioassay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay (using, for example, colloidal gold, enzyme, or radioisotope labeling), Western blotting, precipitation reaction, agglutination assays (e.g., gel agglutination assay and hemagglutination assay), immunofluorescence assay, protein A assay, and immunoelectrophoresis.

[0088] The monoclonal antibody described above may be labeled or unlabeled. If the antibody is labeled, the reporter group may be any suitable reporter group known in the art, including radioisotopes, fluorescent groups (e.g., fluorescein or rhodamine), luminescent groups, enzymes, biotin, and dye particles. Labels that can be indirectly detected by the detectable reaction products they produce include various enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, xanthine oxidase, sugar oxidase (e.g., glucose oxidase), and luciferase. Enzymes can cleave suitable substrates to form colored reaction products or fluorescent reaction products. Alternatively, monoclonal antibodies can be detected by a third antibody capable of binding to a first antibody and / or a second antibody, which is then labeled as described herein. Typically, when using a third antibody for detection, the third antibody is configured to bind to a monoclonal antibody used as a detection agent (see elsewhere herein). Typically, only one of the first and second antibodies is labeled.

[0089] Unlabeled monoclonal antibodies can be immobilized on a solid support to serve as a “capture agent” (or “capture reagent”) for capturing homodimers (HNLs) contained within a sample. The solid support can be any material (known to those skilled in the art) to which antibodies can be attached. For example, the solid support can be a test well in a microtiter plate or a nitrocellulose membrane or another suitable membrane. Alternatively, the support can be a tube, bead, particle, or disc. The support can be made of glass, glass fiber, latex, or plastic materials (such as polyethylene, polypropylene, polystyrene, polyvinyl chloride) or a porous matrix. Other materials that can be used include agarose, dextran, polyacrylamide, nylon, Sephadex, cellulose, and polysaccharides. The support can also be magnetic particles or fiber optic sensors. In the context of this invention, the term “immobilization” refers both to non-covalent association (such as adsorption) and covalent linkage (which can be a direct link between the antibody and functional groups on the support or a link via a cross-linking agent). Immobilization by adsorption into the wells of a microtiter plate or immobilization to a membrane is considered. In this case, adsorption can be achieved by contacting the monoclonal antibody with the solid carrier in a suitable buffer for an appropriate amount of time. The appropriate contact duration depends on the temperature but is typically between about 1 hour and about 1 day. Generally, contacting the wells of a plastic microtiter plate (including polystyrene or polyvinyl chloride) with an amount of anti-HNL antibody ranging from about 10 ng to about 10 μg, or from about 100 ng to about 1 μg, is sufficient to immobilize a sufficient amount of monoclonal antibody. After immobilization, the remaining protein binding sites on the carrier can be blocked. Any suitable blocking agent known to those skilled in the art can be used, including bovine serum albumin, Tween™ 20 (Sigma Chemical Co., St. Louis, Missouri), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffer solution of skim milk powder containing preservatives, salts, and an antifoaming agent). The carrier can then be incubated together with the sample (i.e., the sample suspected of containing HNL homodimers). The sample can be applied directly or advantageously diluted, for example, in a buffer solution containing a small amount (0.1 wt%–5.0 wt%) of protein (such as BSA, NGS, or BLOTTO).

[0090] According to this document, a first monoclonal antibody can be used as a capture reagent and immobilized on a surface, while a second monoclonal antibody is used as a detection reagent and labeled with a marker or alternatively, a labeled third antibody is used for detection.

[0091] An appropriate contact duration (i.e., incubation time) between the sample and the monoclonal antibody is sufficient to detect the presence of an immune complex formed between the monoclonal antibody and the homodimer HNL present in the sample, the monoclonal antibody being immune-specific to the HNL homodimer. The incubation time can be sufficient to achieve the effect of detecting the presence of the bound monoclonal antibody in the sample.The binding level achieved when the monoclonal antibody and the unbound monoclonal antibody are in equilibrium is at least about 95%. Those skilled in the art will understand that the time required to reach equilibrium can be readily determined by monitoring the level of binding occurring over a period of time. An incubation time of about 10 to 30 minutes at room temperature is generally sufficient. Therefore, after contacting the sample with the first and second monoclonal antibodies, respectively, the sample is incubated in the presence of the respective antibodies.

[0092] Unbound components of the sample can be removed by washing with a suitable buffer (such as PBS containing 0.1% Tween™ 20). Therefore, if a solid carrier is used to immobilize the first monoclonal antibody, the solid carrier can be washed after contact with the sample. Then, a "detection reagent" (i.e., a second monoclonal antibody bound to a second monomer of the homodimer of HNL) can be added to the space containing the immune complex formed by the "capture reagent" (i.e., the first monoclonal antibody) and the homodimer HNL contained in the sample. The detection reagent can be directly labeled and contains at least a first detectable marker or "reporter" molecule. Alternatively, the detection reagent (i.e., the second monoclonal antibody) may be an unlabeled anti-HNL antibody. This unlabeled second monoclonal antibody can then be detected by its binding to a third labeled antibody or a labeled reagent. For example, if the second monoclonal antibody is mouse immunoglobulin, the third antibody may be a labeled anti-mouse immunoglobulin antibody. Similarly, if the monoclonal antibody is rabbit immunoglobulin, the third antibody may be a labeled anti-rabbit immunoglobulin antibody.

[0093] The detection reagent (i.e., the second monoclonal antibody) may be incubated with the immune complex formed by the "capture reagent" (i.e., the first monoclonal antibody) and the homodimer HNL contained in the sample for a suitable time sufficient to detect the bound detection reagent. The appropriate incubation time may be determined by monitoring the level of binding occurring over a period of time. Unbound detection reagent may be removed, and the amount of immune complex formed between the homodimer HNL, the capture reagent, and the detection reagent may then be measured using a suitable assay and suitable analytical instrument. Those skilled in the art will be able to select this assay and this analytical instrument. To indicate the presence of a condition associated with homodimer HNL, the value corresponding to the amount of immune complex formed between the homodimer HNL, the capture reagent, and the detection reagent should be at least twice the control value. The control value can be obtained by measuring the value of a sample obtained from an individual with normal amounts of homodimer HNL.

[0094] Alternatively, the sample and detection reagent may be contacted simultaneously with the capture agent, rather than added sequentially. In yet another alternative, the sample and detection reagent may be pre-incubated together in a designated space before the capture reagent is added to that designated space.

[0095] The determination of the amount of homodimer HNL in a sample can be used in a variety of diagnostic, prognostic and monitoring methods, including methods for diagnosing homodimer HNL-related conditions, methods for distinguishing inflammatory diseases from non-inflammatory diseases, and methods for monitoring the efficacy of antimicrobial therapy (e.g., for the purpose of customizing further administration of antimicrobial therapy).

[0096] According to a second aspect of the present invention, a method for determining the presence of a bacterial infection is provided, wherein the method comprises: providing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, the second monoclonal antibody being configured to bind to the second monomer, contacting a sample with the first monoclonal antibody, contacting the sample with the second monoclonal antibody, determining a value corresponding to the amount of homodimer HNL in the sample, comparing the value with a control value, and establishing the presence of a bacterial infection when the value is significantly higher than the control value, or establishing the absence of a bacterial infection when the value is significantly lower than the control value, wherein each of the first monoclonal antibody and the second monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first group of mouse complementarity-determining regions (CDRs), the first group of mouse CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6, and a second heavy chain CDR (CDRH2), wherein CDRH2 is SEQ ID NO: 6. 7, and a third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 8, wherein the light chain variable region comprises a second group of mouse complementarity-determining regions (CDRs), the second group of mouse CDRs comprising: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9, a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10, and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11.

[0097] More specifically, a method is provided for determining the presence of inflammation caused by and / or associated with a bacterial infection. Detailed information regarding the first and second monoclonal antibodies and the contact and incubation of the sample with the monoclonal antibodies is given elsewhere herein.

[0098] Homodimer HNL levels in samples above a certain threshold or control value are associated with the presence of homodimer HNL-related diseases (see page 11 / 16 of CN 121219586 A), while homodimer HNL levels in samples below the threshold or control value indicate a low probability of HNL-related diseases. Similarly, homodimer HNL levels above a certain threshold (control value) are associated with the presence of bacterial infection.Relatedly, homodimer HNL levels below the threshold (or control value) indicate a low probability of bacterial infection.

[0099] Any known method in the art can be used to determine the threshold that distinguishes the majority of normal populations from the majority of diseased populations. Alternatively, endpoint values ​​for negative (no disease), indeterminate, and positive (disease present) results can be determined from data. For example, a normal range (indicating a negative result) can be determined, which includes values ​​of the majority of normal populations but excludes values ​​of almost all diseased populations. Accordingly, a range indicating a positive result can be determined, which includes values ​​of the majority of diseased populations but excludes values ​​of almost all normal populations.

[0100] The above-described methods for establishing the presence of bacterial infection may further include comparing the concentration of at least one determinant (such as a peptide, protein, or isoform thereof) in the sample with a statistical validation threshold for said at least one determinant. A “determinant” can be understood as a polypeptide whose amount differs in a disease state from its amount in a normal state. Exemplary determinants may include TRAIL, IL1RA, IP10, Mac-2BP, B2M, BCA-1, CHI3L1, eosinophil activation chemokine, IL1a, MCP, CD62L, VEGFR2, CHP, CMPK2, COROIC, EIF2AK2, ISG15, RPL22L1, RTN3, CD112, CD134, CD182, CD231, CD235A, CD335, CD337, CD45, CD49D, CD66A / C / D / E, CD73, CD84, EGFR, GPR162, HLA-A / B / C, ITGAM, NRG1, RAP IB, SELI, SPINT2, SSEA1, IgG nonspecific binding molecules, IL1, 1-TAC, TNFR1. ABTB1, ADIPOR1, ARHGDIB, ARPC2, ATP6V0B, Clorf83, CD15, CES1, COROIA, CRP, CSDA, EIF4B, EPSTI1, GAS 7, HERC5, IFI6, KIAA0082, IFI1, IFI3, IFITM1, IFITM3, LIPT1, IL7R, ISG20, LOC26010, LY6E, LRDD, LTA4H, MAN1C1, MBOAT2, MX1, NPM1, OAS2, PARP12, PARP9, QARS, RAB13, RAB31, RAC2, RPL34, PDIA6, PTEN, RSAD2, SART3, SDCBP, TRIM 22, SMAD9, SOCS3, UBE2N, XAF1, or ZBP1. These determinants can be used individually or in any combination.

[0101] Where available, and unless otherwise described herein, determinants for the expression products of genes are identified based on official letter abbreviations or gene symbols designated by the Human Genome Organization Nomenclature Committee (HGNC) and listed on the National Center for Biotechnology Information (NCBI) website (also known as Entrez Gene).

[0102] Determinants may also encompass non-peptide factors, non-blood-borne factors, or non-analytical physiological markers of health status, such as clinical parameters and conventional laboratory risk factors. For example, as used herein, determinants may include non-peptide characteristics (i.e., non-peptide determinants) such as neutrophil percentage (abbreviated Neu (%)), lymphocyte percentage (abbreviated Lym (%)), monocyte percentage (abbreviated Mon (%)), absolute neutrophil count (abbreviated ANC) and absolute lymphocyte count (abbreviated ALC), white blood cell count (abbreviated WBC), age, sex, and highest temperature (i.e., the highest core body temperature since the initial onset of symptoms). Determinants may also include any combination of any calculated index or any of the aforementioned measurements (including time trends and variability) generated mathematically.

[0103] Advantageously, the determinants used in the above methods may include at least one determinant of a polypeptide and at least one non-polypeptide feature.

[0104] The above methods can be used to monitor the effectiveness or ineffectiveness of treatments using antimicrobial substances such as antibiotics. Such treatments can be administered to treat bacterial infections, for example, in cases of sepsis. This use may include repeating the above methods at least twice, wherein samples can be obtained at a first time point before or during treatment, and at a second time point after the first time point at the end of treatment or during treatment. A lower amount of homodimer HNL in the sample at the second time point compared to the first time point should indicate the effectiveness of the treatment.

[0105] To improve the sensitivity of the methods disclosed herein, a pre-activator substance can be used as a neutrophil activator, i.e., a substance used to activate neutrophils. The pre-activator substance may be an N-formyl peptide, preferably a tripeptide fMLP. See specification 12 / 16 pages 16 CN 121219586 A. Advantageously, protein A can be used. In addition, other neutrophil activators can be used. At least one of lipopolysaccharide (LPS), platelet-activating factor, unmethylated CpG oligonucleotides, and tumor necrosis factor (TNF) can be used. Advantageously, fMLP can be used alone or in combination with one or more of the other neutrophil activators described above.

[0106] The methods described above according to the first and second aspects can be used to distinguish between bacterial infections and non-bacterial infections (such as viral infections), mixed infections (i.e., co-infection with bacteria and viruses), and viral infections, respectively. Those skilled in the art should be able to employ...The above objectives are achieved according to the method of the present invention. Bacterial infection can be any of sepsis, gastrointestinal infection, urinary tract infection, tuberculosis, pneumonia, and gonorrhea.

[0107] In the method of this document, the sample may be contacted first with a second monoclonal antibody and then with a first monoclonal antibody. The method of this document may include the step of providing a sample to be contacted with the first or second monoclonal antibody. The method of this document is performed in vitro.

[0108] According to another aspect of the present invention, a kit is provided for detecting the presence of homodimer HNL in a sample and / or establishing the presence of a bacterial infection, wherein the kit includes one or more monoclonal antibodies as defined herein and one or more of instructions for use, buffers, and devices arranged for analysis of the sample. Preferably, the kit includes a first monoclonal antibody and a second monoclonal antibody as defined herein. However, since the first and second monoclonal antibodies can be the same, the kit may contain only one antibody that can be used as the first and second monoclonal antibodies in the method of this document.

[0109] The sample may be derived from at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid, another bodily fluid, feces, and their liquid formulations.

[0110] The kit may also include an enzyme, or a second monoclonal antibody may be enzyme-labeled for use as a signal generation means. The kit may also include a substrate and substrate precursor of the enzyme, which provide a detectable chromophore or fluorophore.

[0111] The buffers described above in the kit may include at least one of a blocking buffer and a lysis buffer.

[0112] At least one of the reagents in the kit may be provided in powder form, for example, as a lyophilized reagent.

[0113] At least a portion of the components of the kit may be pre-attached to a solid carrier, or may be applied to the surface of a solid carrier while the kit is being used.

[0114] The signal generation means may be pre-associated with the monoclonal antibody described herein. The signal generation means may need to be combined with at least one of a buffer, antibody-enzyme conjugate, enzyme substrate, etc., before use. The solid surface may be in the form of a tube, bead, microtiter plate, microsphere, or another form suitable for immobilizing at least one of proteins, peptides, and polypeptides. The kit may also include at least one reagent for detecting and measuring other biological parameters, such as reagents for measuring the expression and / or amount of any of procalcitonin, C-reactive protein, CD64, and any of the determinants (peptide determinants) mentioned herein, and reagents for determining the number of white blood cells (such as neutrophils, T cells, B cells, monocytes, eosinophils, and basophils).

[0115] Containers used in the kit may generally include at least one of vials, test tubes, flasks, bottles, syringes, and another suitable container.

[0116] The apparatus for arranging the analysis of the sample may be as described below.

[0117] According to another aspect of the present invention, an apparatus for determining the presence of a bacterial infection is provided, wherein the apparatus comprises: a first compartment containing monoclonal antibodies for detecting homodimers of HNL: a first monoclonal antibody and a second monoclonal antibody, each homodimer consisting of a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer. Each of the first and second monoclonal antibodies comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a first set of mouse complementarity-determining regions (CDRs), the first set of mouse CDRs comprising: a first heavy chain CDR (CDRH1), wherein CDRH1 is SEQ ID NO: 6; a second heavy chain CDR (CDRH2), wherein CDRH2 is SEQ ID NO: 7; and a third heavy chain CDR (CDRH3), wherein CDRH3 is SEQ ID NO: 8. The light chain variable region includes a second set of mouse complementarity-determining regions (CDRs), the second set of mouse CDRs comprising: a first light chain CDR (CDRL1), wherein CDRL1 is SEQ ID NO: 9; a second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10; and a third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO: 11; wherein the first compartment is adapted to contact a sample, the sample being suspected of containing or not containing a homodimer of HNL; and a second compartment, wherein the second compartment contains a substrate for activating neutrophils present in the sample, wherein the activation is performed prior to establishing the presence of bacterial infection, wherein the second compartment contains an opening connecting it to the first compartment; A first connector, wherein the first connector is arranged for connecting the device to another device for retrieving a sample from a subject suspected of having at least one bacterial infection and a viral infection; and a second connector, wherein the second connector is arranged for combining the device with a computer and / or device for analyzing the interaction between a homodimer of HNL, a first monoclonal antibody, and a second monoclonal antibody, the analysis allowing determination of the amount of homodimer HNL in the sample. Detailed information regarding the first and second monoclonal antibodies and the substrate for neutrophil activation is disclosed elsewhere herein.

[0118] The device may be a test strip with a monoclonal antibody immobilized on at least a portion of its surface, or a particle carrying the immobilized monoclonal antibody on at least a portion of its surface. The surface may be three-dimensional; for example, particles having a partially porous surface (layer) may carry the aforementioned monoclonal antibody. Importantly, the device includes a contact area that can be exposed to the sample.The device can be connected to a computer or device, thereby allowing the analysis and measurement of the characteristics of the interaction between the monoclonal antibody disclosed herein and the HNL homodimer present in a sample. This interaction can be characterized using a signal specifically generated when the monoclonal antibody disclosed herein binds to the HNL homodimer (no such signal is generated in a control setting where the above interaction does not occur). This signal can be at least one of physical, chemical, and biological signals. Examples of signals include colorimetric signals, fluorescence signals, spectroscopically measurable signals, ionization changes, conductivity changes, etc.

[0119] The device may also include a binder or antibody for detecting one or more of the above determinants. The device may involve magnetic separation of the analyte of interest prior to detection.

[0120] The apparatus may be any of the following: a point-of-care device, a test strip, an apparatus for radioimmunoassay, an apparatus for ELISA, an apparatus for sandwich immunoassay, an apparatus for immunoradioassay, an apparatus for gel diffusion precipitation assay, an apparatus for immunodiffusion assay, an apparatus for in situ immunoassay using colloidal gold, enzyme, or radioisotope labeling, an apparatus for Western blotting, an apparatus for precipitation assay, an apparatus for gel agglutination assay, an apparatus for hemagglutination assay, an apparatus for immunofluorescence assay, an apparatus for protein A assay, an apparatus for immunoelectrophoresis assay, and an apparatus for analyte detection involving magnetic separation of analytes.

[0121] It should be understood that, even if not explicitly mentioned, the above-described embodiments of the first aspect of the invention are also applicable to other aspects of this specification, page 14 / 16 of CN 121219586 A.

[0122] The invention has been described above primarily with reference to some embodiments. However, as will be readily apparent to those skilled in the art, other embodiments besides those disclosed above are also possible within the scope of the invention as defined by the appended claims.

[0123] Experimental Procedures The embodiments of the present invention are provided for illustrative purposes only and should not be construed as limiting the invention as defined by the appended claims. All references provided below and elsewhere in this application are hereby incorporated by reference.

[0124] Preparation of Antibodies Monoclonal antibodies anti-HNL MAB2 and anti-HNL MAB3 were prepared by standard hybridoma technology via fusion of spleen cells from HNL dimer-immunized Balb / c mice with Sp2 / 0 mouse myeloma cells, followed by cloning. The activity of the supernatant against the purified HNL was screened by ELISA. Selected hybridomas were amplified, from which the purified antibodies had the IgG1 subtype.

[0125] Characterization of Antibodies Chemical crosslinking, high-quality MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry were used to characterize the antigen HNL and the monoclonal antibodies.The interaction interface between each of anti-HNL MAB2 and anti-HNL MAB3 was characterized (see WO 2016 / 079219 A1).

[0126] The epitopes of each of anti-HNL MAB2 and anti-HNL MAB3 include amino acids 145-154 as shown in SEQ ID No: 26 of WO 2016 / 079219. In addition, the epitope of anti-HNL MAB2 includes amino acids 83-88 as shown in SEQ ID No: 26 of WO 2016 / 079219.

[0127] Proof of concept was performed using the method described in this document for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample using ELISA with the first monoclonal antibody MAB3 and the second monoclonal antibody MAB3, obtaining specific binding to the homodimer of HNL and specific detection of the homodimer of HNL (Figure 1).

[0128] Using the method described herein with the first monoclonal antibody MAB2 and the second monoclonal antibody MAB2, specific binding to the homodimer of HNL and specific detection of the homodimer of HNL were obtained (Figure 2).

[0129] For comparison, Figure 3 shows the results of using the first and second monoclonal antibodies (MAB5) without the CDR sequences of the first and second monoclonal antibodies as defined herein. As can be seen in Figure 3, the dimeric HNL could not be detected using MAB5 as the first and second monoclonal antibodies. However, when MAB5 was used with another anti-HNL monoclonal antibody (MAB1), the dimeric HNL could be detected (thus demonstrating that MAB5 can bind to HNL in its dimeric form). Therefore, any combination of monoclonal antibodies that are not specific to HNL (where the first and second monoclonal antibodies have the same CDR sequence) can be used for the detection of the homodimer of human neutrophil lipid carrier protein (HNL) in a sample according to the method described herein.

[0130] The clinical sample patient group analyzed using anti-HNL MAB3 consisted of 206 patients, each with signs of infection and a body temperature exceeding 38°C. The etiology of the infection was determined through routine clinical procedures. None of the patients were taking immunosuppressive drugs. For each patient, the infection was either bacterial or viral. Bacterial infections included in the study were bacterial pneumonia, mycoplasma infection, tonsillitis, urinary tract infection (UTI), gastrointestinal (GI) infection, erysipelas, and sepsis. In most cases, the bacterial infection was an acute respiratory infection, such as pneumonia and tonsillitis.

[0131] Blood samples were drawn before the start of antimicrobial therapy, anticoagulated with EDTA, and centrifuged at 3000 g. See page 15 / 16 of the blood sample manual (19 CN).After centrifugation of sample 121219586 A, plasma was harvested and stored at -70°C before analysis. The concentration of HNL MAB3 / MAB3 (an immune complex between the homodimer HNL and the monoclonal antibody) in the plasma was determined using ELISA as described herein. It was demonstrated that the above method can reliably and accurately detect the presence or absence of acute bacterial infection (e.g., acute viral infection only, no bacterial infection) (Figures 4 and 5). [Instruction manual 16 / 16 pages 20 CN 121219586 A Figure 1 Instruction manual Figure 1 / 5 pages 21 CN 121219586 A Figure 2 Instruction manual Figure 2 / 5 pages 22 CN 121219586 A Figure 3 Instruction manual Figure 3 / 5 pages 23 CN 121219586 A Figure 4 Instruction manual Figure 4 / 5 pages 24 CN 121219586 A Figure 5 Instruction manual Figure 5 / 5 pages 25 CN 121219586 A]

Claims

1. An in vitro method for detecting the presence of homodimer human neutrophil lipid carrier protein (HNL) in a sample, wherein the method comprises: Provide samples, A monoclonal antibody for detecting homodimers of HNL is provided: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer. The sample is brought into contact with the first monoclonal antibody. Contact the sample with the second monoclonal antibody. Detect the presence of homodimer HNL. Each of the first monoclonal antibody and the second monoclonal antibody contains a heavy chain variable region and a light chain variable region. The heavy chain variable region includes a first set of mouse complementarity determination regions (CDRs), which include: The first heavy chain CDR (CDRH1), where CDRH1 is SEQ ID NO: 6, The second heavy chain CDR (CDRH2), where CDRH2 is SEQ ID NO: 7, and The third chain CDR (CDRH3), where CDRH3 is SEQ ID NO: 8, The light chain variable region includes a second set of mouse complementarity determination regions (CDRs), which include: The first light chain CDR (CDRL1), where CDRL1 is SEQ ID NO: 9, The second light chain CDR (CDRL2), wherein CDRL2 is SEQ ID NO: 10, and The third light chain CDR (CDRL3), wherein CDRL3 is SEQ ID NO:

11.

2. The in vitro method according to claim 1, wherein the sample is derived from at least one of blood, plasma, serum, urine, CSF, bone marrow, saliva, sputum, intestinal fluid, another bodily fluid, feces, and / or liquid preparations thereof.

3. The in vitro method according to any one of the preceding claims, wherein the first monoclonal antibody is immobilized on a solid support.

4. The in vitro method according to any one of the preceding claims, wherein each of the CDRs is configured to bind to an epitope formed between amino acids 145 to 154 of HNL as defined in SEQ ID NO:

1.

5. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises an amino acid sequence as illustrated in SEQ ID NO: 5 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

5.

6. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises an amino acid sequence as illustrated in SEQ ID NO: 4 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

4.

7. The in vitro method according to any one of claims 1-4, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises an amino acid sequence as illustrated in SEQ ID NO:3 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

3.

8. The in vitro method according to any one of claims 1-4, wherein at least one of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises an amino acid sequence as illustrated in SEQ ID NO:2 or having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

2.

9. The in vitro method according to any one of claims 1-4, wherein each of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a heavy chain variable region sequence as illustrated in SEQ ID NO: 5 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 5, and wherein each of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a light chain variable region sequence as illustrated in SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

4.

10. The in vitro method according to any one of claims 1-4, wherein each of the first monoclonal antibody and the second monoclonal antibody has a heavy chain comprising a first group of mouse CDRs, wherein the heavy chain comprises a heavy chain variable region sequence as illustrated in SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO: 3, and wherein each of the first monoclonal antibody and the second monoclonal antibody has a light chain comprising a second group of mouse CDRs, wherein the light chain comprises a light chain variable region sequence as illustrated in SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, the same as SEQ ID NO:

2.

11. The in vitro method according to any one of the preceding claims, wherein at least one of the first monoclonal antibody and the second monoclonal antibody is a humanized antibody.

12. An in vitro method for determining the presence of a bacterial infection, the method comprising: Provide samples, A monoclonal antibody for detecting homodimers of HNL is provided: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer. The sample is brought into contact with the first monoclonal antibody. Contact the sample with the second monoclonal antibody. Determine the value corresponding to the amount of homodimer HNL in the sample. Compare the value with the control value. A bacterial infection is confirmed when the value is significantly higher than the control value, or a bacterial infection is confirmed when the value is significantly lower than the control value. The first monoclonal antibody and / or the second monoclonal antibody are as defined in any one of claims 1-11.

13. A kit for detecting the presence of homodimer HNL in a sample according to any one of claims 1-11 and / or for establishing the presence of bacterial infection according to claim 12, wherein the kit comprises one or more monoclonal antibodies according to claims 1-11 and one or more instructions for use, buffer solutions, and devices, wherein the devices are configured for analyzing the sample.

14. An apparatus for determining the presence of a bacterial infection, the apparatus comprising: A first compartment, wherein the first compartment contains monoclonal antibodies for detecting HNL homodimers: a first monoclonal antibody and a second monoclonal antibody, each homodimer comprising a first monomer and a second monomer, the first monoclonal antibody being configured to bind to the first monomer, and the second monoclonal antibody being configured to bind to the second monomer. The first monoclonal antibody and / or the second monoclonal antibody are as defined in any one of claims 1-11. The first compartment is adapted to contact the sample, which is suspected to contain or not contain HNL homodimers; A second compartment, wherein the second compartment contains a substrate for activating neutrophils present in the sample, wherein the activation is performed prior to establishing the presence of a bacterial infection, wherein the second compartment contains an opening connecting it to the first compartment; A first connector, wherein the first connector is arranged to connect the device to another device for retrieving samples from a subject suspected of having at least one of bacterial and viral infections; as well as A second connector, wherein the second connector is arranged to combine the device with a computer and / or equipment for analyzing the interaction between the homodimer of HNL, the first monoclonal antibody and the second monoclonal antibody, the analysis allowing determination of the amount of homodimer HNL in the sample.

15. The apparatus of claim 14, further comprising a binding agent for one or more determinants selected from: ABTB1, ADIPOR1, ARHGDIB, ARPC2, ATP6VOB, Clorf83, CD15, CES1, CORO1A, CSDA, EIF4B, EPSTI1, GAS 7. HERC5, IFI6, KIAA0082, IFIT1, IFIT3, IFITM1, IFITM3, LIPT1, IL7R, ISG20, LOC26010, LY6E, LRDD, LTA4H, MAN1C1, MBOAT2, NPM1, OAS2, PA RP12, PARP9, QARS, RAB13, RAB31, RAC2, RPL34, PDIA6, PTEN, RSAD2, SART3, SDCBP, SMAD9, SOCS3, TRIM22, SART3, UBE2N, XAF1, ZBP1, CRP, and MX1.

16. The apparatus of claim 14 or 15, wherein the apparatus is any one of: a point-of-care device, a test strip, an apparatus for radioimmunoassay, an apparatus for ELISA, an apparatus for sandwich immunoassay, an apparatus for immunoradioassay, an apparatus for gel diffusion precipitation assay, an apparatus for immunodiffusion assay, an apparatus for in situ immunoassay using colloidal gold, enzyme, or radioisotope labeling, an apparatus for Western blotting, an apparatus for precipitation assay, an apparatus for gel agglutination assay, an apparatus for hemagglutination assay, an apparatus for immunofluorescence assay, an apparatus for protein A assay, an apparatus for immunoelectrophoresis assay, and an apparatus for analyte detection involving magnetic separation of analytes.

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