New anti-soluble CD14 subtype antibodies and their applications

Abstract

An anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42. Also disclosed are a kit for detecting soluble CD14 subtypes, a method for detecting soluble CD14 subtypes, and corresponding applications.

Description

Technical Field

[0001] The present application relates to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, a kit for detecting soluble CD14 subtypes, a method for detecting soluble CD14 subtypes and corresponding applications. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Literature reports that the incidence of sepsis is increasing annually, and the mortality rate remains high, posing a serious threat to human health. According to sepsis treatment guidelines, early diagnosis is an effective way to improve the survival rate of patients with sepsis. Blood culture is the "gold standard" for the diagnosis of sepsis, but its positive rate is low and the incubation time is long. There is an urgent need to identify highly specific and sensitive biomarkers for the early diagnosis and accurate prediction of sepsis.

[0003] The diagnostic criteria for sepsis are constantly being updated. The latest diagnostic criteria for sepsis (Sepsis 3.0) emphasize infection and the resulting uncontrolled body response and organ damage. Although Sepsis 3.0 remains controversial, it reflects advances in clinical research and diagnosis, the complexity of sepsis, and the importance of clinical diagnosis. Currently, widely used biomarkers for the diagnosis of sepsis include interleukin-6 (IL-6), C-reactive protein (CRP), and procalcitonin (PCT). CRP is an acute phase protein synthesized by the liver and has a strong sensitivity for bacterial infections, but its specificity is low, its half-life is long, and it has no clear correlation with the progression of sepsis. IL-6 peaks early and can rise before clinical symptoms appear, facilitating the early diagnosis of sepsis, but its sensitivity is low. PCT is widely used in the diagnosis of infection and sepsis. PCT is often not elevated in non-bacterial infections. PCT levels are often not significantly elevated in certain types of infection, such as skin and soft tissue infections. PCT is not suitable for predicting the prognosis of perioperative intra-abdominal infections and septic shock. Furthermore, some non-infectious diseases can be accompanied by elevated PCT levels. Although PCT has been shown to be clearly associated with infection, false positive diagnoses are common in conditions such as trauma, surgery, and burns. Therefore, PCT is not a perfect marker for the diagnosis of infection or sepsis, and using PCT alone is unreliable.

[0004] CD14 is a high-affinity receptor for the lipopolysaccharide-lipopolysaccharide binding protein complex, anchored to the cell surface by glycosylphosphatidylinositol. CD14 is primarily present on the membranes of monocytes and macrophages. CD14 exists in two forms: membrane-bound CD14 (mCD14) and soluble CD14 (sCD14). Membrane-bound CD14 is primarily expressed on the surface of monocytes and macrophages. Soluble CD14 is distributed in plasma and is the portion of the LPS-LBF-CD14 complex released into the human blood after CD14 detaches from the cell membrane. It primarily mediates the response of endothelial and epithelial cells to LPS.

[0005] Soluble CD14 subtype (sCD14-ST or presepsin) is a new biomarker of sepsis, with potential application in the diagnosis, prognosis, and guidance of antimicrobial therapy. It has been a hot topic in sepsis biomarker research in recent years. Shirakawa et al. established a rabbit model of endotoxic shock and a cecal ligation and puncture (CLP) model and found that the endotoxic shock model did not elevate presepsin concentrations, while the CLP model significantly elevated presepsin levels. This suggests that the elevation of presepsin is caused by phagocytosis of the bacterial infection rather than physiological anti-inflammatory effects. Presepsin is the N-terminal fragment of sCD14, which is cleaved in plasma by proteases such as cathepsin D. Its molecular weight is approximately 13 kDa. Currently, presepsin is considered highly sensitive and specific for assessing sepsis severity and prognosis, and can accurately guide the use of antimicrobial agents in sepsis.

[0006] So far, attempts have been made to detect, diagnose and accurately predict sepsis with high specificity and sensitivity. Summary of the Invention

[0007] Therefore, one object of the present application is to provide an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which is suitable for detecting soluble CD14 subtypes in a sample. In addition, another object of the present application is to provide a kit for detecting soluble CD14 subtypes, a method for detecting soluble CD14 subtypes, and corresponding applications.

[0008] The first aspect of the present application relates to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, and the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof comprises:

[0009] (i) Heavy chain variable region (VH) complementarity determining region (CDR):

[0010] VH CDR1: X1X2X3MX4;

[0011] VH CDR2: YIX5X6ADX7;

[0012] VH CDR3: X8X9X 10 AX 11 ;

[0013] and

[0014] (ii) Light chain variable region (VL) complementarity determining region (CDR):

[0015] VL CDR1:KX 12 X 13 X 14 N;

[0016] VL CDR2:LX 15 X 16 ;

[0017] VL CDR3: VX 17 X 18 X 19 ;

[0018] Where X1 to X 19 One or more amino acid sequences listed below as options:

[0019] X1 = any amino acid;

[0020] X2=F or V; X3=A, E or K; X4=A or L;

[0021] X5=SYMGS, SSGSS, AYTMY, TYSGS or SSKSS;

[0022] X6=GAYY, TIYY, AKYY, TKAS or SNLA;

[0023] X7=AKLG, TVKG, SYTA, MTKG or YGNT;

[0024] X8=A or Q;X9=G or Q;X 10 =Q or F; X 11 =M, Y, or V;

[0025] X 12 =YYAS, SSAT, SSQS or SGSS;

[0026] X 13 =AAKL, LLAT, LLYS or KAAS;

[0027] X 14=YRNIKL, TWAKGN, NGKTYL, or YTNGAL;

[0028] X 15 =VQS, KTS, QTS or VSK;

[0029] X 16 =LAS, LDS, LTA or DSK;

[0030] X 17 =G, A, S or Q;

[0031] X 18 =GTH, GNT, GTA or TAI;

[0032] X 19 =FITA, FPRT, YGHV or NYGH.

[0033] The second aspect of the present application relates to a kit for detecting soluble CD14 subtypes, which kit at least comprises the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application.

[0034] The third aspect of the present application relates to a method for detecting soluble CD14 subtypes, the method comprising:

[0035] Mixing the sample to be tested and a solid support coated with a capture antibody, so that the capture antibody coated on the solid support binds to the soluble CD14 subtype in the sample to be tested, wherein the capture antibody is an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof according to the first aspect of the present application;

[0036] The mixture is washed to remove unbound substances;

[0037] adding a labeled detection antibody to the washed mixture and mixing the mixture, so that the detection antibody binds to the soluble CD14 subtype bound to the capture antibody to form a sandwich complex;

[0038] washing the sandwich complex to remove unbound substances;

[0039] A detection substrate is added to the washed sandwich complex to detect the concentration of soluble CD14 isoforms in the sample.

[0040] The fourth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof of the first aspect of the present application in the preparation of an analytical reagent for assessing whether a patient has sepsis, wherein the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof of the first aspect of the present application is used to detect the concentration of soluble CD14 subtypes in a patient's blood sample, and an increase in the concentration of the soluble CD14 subtype relative to a reference value is correlated with an increased likelihood that the patient has sepsis.

[0041] The fifth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof of the first aspect of the present application in the preparation of a diagnostic reagent for evaluating the prognosis of a patient suspected of sepsis, wherein the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof of the first aspect of the present application is used to detect the concentration of soluble CD14 subtypes in a blood sample of a patient suspected of sepsis, and an increased concentration of the soluble CD14 subtype relative to a reference value is associated with an increased risk of death in the patient suspected of sepsis.

[0042] The present application also relates to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, comprising:

[0043] (a) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 17; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 32, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33;

[0044] (b) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 37;

[0045] (c) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 3, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 16; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 38;

[0046] (d) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 18, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 39;

[0047] (e) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 16, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33; or

[0048] (f) A VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 38.

[0049] The embodiments of the present application also relate to an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, wherein the soluble CD14 subtype capture antibody is an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof secreted by a hybridoma cell line with a deposit number of CGMCC NO. 18536 (deposit unit: General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: October 23, 2019).

[0050] The anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments thereof provided in the embodiments of the present application have high affinity for soluble CD14 subtypes and are therefore capable of accurately and quantitatively detecting the concentration of soluble CD14 subtypes in a sample. In addition, the anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments thereof provided in the embodiments of the present application have high sensitivity for determining the concentration of soluble CD14 subtypes. In addition, the embodiments of the present application are capable of performing highly specific determinations in a double antibody sandwich method, that is, the anti-soluble CD14 subtype monoclonal antibodies and antigen-binding antibody fragments thereof provided in the embodiments of the present application specifically bind only to soluble CD14 subtypes present in human blood, and do not specifically bind to the high molecular weight sCD14 therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Hereinafter, some embodiments of the present application will be described with reference to the accompanying drawings. The objects and advantages of the present application will be further understood according to the following detailed description and the accompanying drawings. The accompanying drawings show:

[0052] Figure 1 Shown are SDS-PAGE and Western Blot of prokaryotic expressed proteins;

[0053] Figure 2 Shown are SDS-PAGE and Western Blot of eukaryotic expressed proteins;

[0054] Figure 3 Anti-soluble CD14 isoform polyclonal antibody SDS-PAGE and Western Blot are shown;

[0055] Figure 4 The standard curve of the kit for detecting soluble CD14 isoforms is shown;

[0056] Figure 5 A ROC curve diagram showing PCT and the kit of an embodiment of the present application for assessing whether a patient has sepsis;

[0057] Figure 6 A survival rate curve showing the use of the kit according to an embodiment of the present application for evaluating the prognosis of suspected sepsis;

[0058] Figure 7 The figure shows the ROC curve of PCT and the kit according to the embodiment of the present application for evaluating the prognosis of suspected sepsis. DETAILED DESCRIPTION

[0059] Hereinafter, the present application will be described in more detail through specific examples. However, these examples are only representative and the present application will in no way be understood to be limited by these examples.

[0060] When these expressions, features, values ​​or ranges are mentioned in conjunction with expressions such as "about, substantially, generally, at least, at least" etc. in the context of this application, this application also includes the exact or precise expressions, features, values ​​or ranges etc.

[0061] In the context of the present application, the phrase "specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42" means that the antibody claimed in the present application specifically recognizes a sequence corresponding to the amino acid sequence of SEQ ID No. 42 among the sequences of soluble CD14 subtypes as an epitope.

[0062] In the scope of this application, an antibody may also refer to an antibody or an antigen-binding antibody fragment thereof, and an antigen-binding antibody fragment refers to a fragment of an antibody that specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42 and has the same antigen-binding ability as the original antibody.

[0063] In the scope of this application, unless otherwise specified, soluble CD14 refers to human soluble CD14.

[0064] In the context of this application, the term detection can be used interchangeably with terms such as "determination", "quantification", and "analysis", and is intended to include both quantitative and qualitative determinations. Detection in this application is preferably performed in vitro.

[0065] In the scope of this application, the term ROC (receiver operating characteristic) curve refers to a curve obtained by dividing the diagnostic test results into several critical points, with the sensitivity corresponding to each critical point as the ordinate and the specificity as the abscissa. The ROC curve is an effective tool for comprehensive and accurate evaluation of diagnostic tests. Another function of the ROC curve is to determine the optimal threshold for detection. In most cases, the ROC curve method determines the critical point. The point on the curve that is as close to the upper left as possible is selected, and the critical point is determined to be the best in combination with professional conditions. In application, according to the ROC curve, combined with the sensitivity and specificity results of each cut-off point, the cut-off point on the curve that is as close to the upper left as possible with the largest Youden index is selected as the optimal critical point, so that the sensitivity and specificity of the test are both high, while the misdiagnosis rate and missed diagnosis rate are small.

[0066] In a first aspect, the present application claims protection for an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, and the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof comprises:

[0067] (i) Heavy chain variable region (VH) complementarity determining region (CDR):

[0068] VH CDR1: X1X2X3MX4;

[0069] VH CDR2: YIX5X6ADX7;

[0070] VH CDR3: X8X9X 10 AX 11 ;

[0071] and

[0072] (ii) Light chain variable region (VL) complementarity determining region (CDR):

[0073] VL CDR1:KX 12 X 13 X 14 N;

[0074] VL CDR2:LX 15 X 16 ;

[0075] VL CDR3: VX 17 X 18 X 19 ;

[0076] Where X1 to X 19 It is one or more amino acid sequences listed below as options (see Tables 1 to 6).

[0077] Table 1

[0078]

[0079] Table 2

[0080]

[0081]

[0082] Table 3

[0083]

[0084] Table 4

[0085]

[0086] Table 5

[0087]

[0088] Table 6

[0089]

[0090] In some embodiments of the first aspect of the present application, the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences listed in Table 7 (see Table 7).

[0091] Table 7

[0092]

[0093]

[0094] In some embodiments of the first aspect of the present application, the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 are selected from the amino acid sequences listed in Table 8 (see Table 8).

[0095] Table 8

[0096]

[0097]

[0098] In some embodiments of the first aspect of the present application, the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3, and VL CDR1, VL CDR2 and VL CDR3 are any one of the following 1) to 62):

[0099] 1) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 1, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of SEQ ID NO: 34;

[0100] 2) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 1, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of SEQ ID NO: 41;

[0101] 3) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 1, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of SEQ ID NO: 33;

[0102] 4) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 1, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of SEQ ID NO: 39;

[0103] 5) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 1, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 20, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 40;

[0104] 6) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 35;

[0105] 7) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of SEQ ID NO: 38;

[0106] 8) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 38;

[0107] 9) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of SEQ ID NO: 40;

[0108] 10) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0109] 11) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 9, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0110] 12) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 35;

[0111] 13) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 36;

[0112] 14) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of SEQ ID NO: 34;

[0113] 15) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 41;

[0114] 16) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 20, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 37;

[0115] 17) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 35;

[0116] 18) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 40;

[0117] 19) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 34;

[0118] 20) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of SEQ ID NO: 34;

[0119] 21) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 34;

[0120] 22) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 2, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 36;

[0121] 23) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 41;

[0122] 24) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of SEQ ID NO: 34;

[0123] 25) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0124] 26) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0125] 27) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0126] 28) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 9, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 36;

[0127] 29) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 9, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0128] 30) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 41;

[0129] 31) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 3, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0130] 32) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0131] 33) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of SEQ ID NO: 33;

[0132] 34) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 40;

[0133] 35) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0134] 36) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 33;

[0135] 37) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 34;

[0136] 38) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 4, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 40;

[0137] 39) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 40;

[0138] 40) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 34;

[0139] 41) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 20, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 39;

[0140] 42) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 8, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 33;

[0141] 43) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 33;

[0142] 44) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0143] 45) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 40;

[0144] 46) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 33;

[0145] 47) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 36;

[0146] 48) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0147] 49) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0148] 50) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 17, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 37;

[0149] 51) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0150] 52) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 16, VL CDR1 consists of SEQ ID NO: 23, VL CDR2 consists of SEQ ID NO: 27, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 34;

[0151] 53) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 12, VH CDR3 consists of SEQ ID NO: 19, VL CDR1 consists of SEQ ID NO: 24, VL CDR2 consists of SEQ ID NO: 31, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 38;

[0152] 54) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0153] 55) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 15, VL CDR1 consists of SEQ ID NO: 26, VL CDR2 consists of SEQ ID NO: 28, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 33;

[0154] 56) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 21, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0155] 57) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 5, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 20, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0156] 58) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 6, VH CDR2 consists of SEQ ID NO: 7, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0157] 59) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 6, VH CDR2 consists of SEQ ID NO: 10, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 25, VL CDR2 consists of SEQ ID NO: 32, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0158] 60) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 6, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 18, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 29, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39;

[0159] 61) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 6, VH CDR2 consists of SEQ ID NO: 11, VH CDR3 consists of SEQ ID NO: 14, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of the amino acid sequences of SEQ ID NO: 39; or

[0160] 62) VH CDR1 consists of the amino acid sequences of SEQ ID NO: 6, VH CDR2 consists of SEQ ID NO: 13, VH CDR3 consists of SEQ ID NO: 20, VL CDR1 consists of SEQ ID NO: 22, VL CDR2 consists of SEQ ID NO: 30, and VL CDR3 consists of SEQ ID NO: 38.

[0161] In some embodiments of the first aspect of the present application, an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof is provided, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, comprising:

[0162] (a) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 17; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 32, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33;

[0163] (b) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 37;

[0164] (c) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 3, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 16; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 38;

[0165] (d) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 18, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 39;

[0166] (e) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 16, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33; or

[0167] (f) A VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 38.

[0168] In some embodiments of the first aspect of the present application, the soluble CD14 monoclonal antibody or antigen-binding antibody fragment thereof is an anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof secreted and produced by the hybridoma cell line with a deposit number of CGMCC NO.18536.

[0169] In some embodiments of the first aspect of the present application, the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof does not specifically bind to high molecular weight soluble CD14.

[0170] In some embodiments of the first aspect of the present application, the antigen-binding antibody fragment can be selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFV), dimerized V region (dimeric antibody), disulfide bond stabilized V region (dsFv), sc(Fv)2, a polypeptide comprising CDR, a polypeptide comprising a heavy chain variable region, and an antigen-binding antibody fragment comprising a polypeptide comprising a light chain variable region.

[0171] The second aspect of the present application claims protection for a kit for detecting soluble CD14 subtypes, which kit at least comprises the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the first aspect of the present application.

[0172] The kit according to the second aspect of the present application may also include other reagent components, including but not limited to primary antibodies, secondary antibodies, labeled antibodies, labeling enzymes and other labeling substances; colorimetric substrates, fluorescent substrates, chemiluminescent substrates, biotin-streptavidin and other specific binding substances, insoluble carriers, blocking agents, diluents, washing solutions, standard substances, etc.

[0173] For example, a kit for chemiluminescent enzyme immunoassay (CLEIA) may include an antibody coated on a solid phase, an enzyme-labeled antibody, a chemiluminescent substrate, a diluent, a washing solution, etc. Of course, in other embodiments, the antibodies of the present invention may also be used to prepare enzyme immunoassay kits, electrochemiluminescent immunoassay kits, immunochromatography kits, etc. based on double antibody sandwich methods or competitive methods.

[0174] In some embodiments of the second aspect of the present application, the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to the present application is used as a capture antibody, which is coated on a solid phase carrier and used to capture the soluble CD14 subtype in the sample to be tested.

[0175] In some embodiments of the second aspect of the present application, the solid phase carrier can be selected from magnetic microspheres, chips, test papers, etc., preferably magnetic microspheres, and more preferably superparamagnetic magnetic beads.

[0176] In some embodiments of the second aspect of the present application, the kit for detecting soluble CD14 subtypes further comprises a labeled soluble CD14 subtype detection antibody, which may be an anti-soluble CD14 subtype polyclonal antibody.

[0177] In some embodiments of the second aspect of the present application, the signal marker of the soluble CD14 subtype detection antibody can be, for example, a chemiluminescent marker (such as alkaline phosphatase, luminol, isoluminol, acridinium ester, horseradish peroxidase), an electrochemiluminescent marker (such as terpyridine ruthenium), quantum dots (such as gold quantum dots, CdSe quantum dots, ZnCdSe quantum dots, etc.), fluorescent microspheres, etc., or a combination thereof.

[0178] The third aspect of the present application is to claim a method for detecting soluble CD14 subtypes, which comprises contacting the anti-soluble CD14 subtype monoclonal antibody of the first aspect of the present application with a sample to be tested.

[0179] The method for immunologically determining the soluble CD14 subtype in a sample using the antibody or antigen-binding antibody fragment of the first aspect of the present application can be, for example, enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), fluorescent antibody method (FAT), fluorescent enzyme immunoassay (FEIA), electrochemiluminescent immunoassay (ECLIA), radioimmunoassay (RIA), immunochromatography, agglutination method, competitive method, etc., but is not limited to these methods. The embodiments of the present application are particularly suitable for application to chemiluminescent enzyme immunoassay.

[0180] ELISA (enzyme-linked immunosorbent assay) is an immunoassay method that uses enzyme-labeled antibodies. It includes direct and indirect methods, such as sandwich ELISA. Sandwich ELISA uses antibodies with different antigen recognition sites. One antibody is coated on a solid phase, while the other antibody sandwiches the antigen to be detected, forming an antibody-antigen-antibody complex.

[0181] The principle of chemiluminescent enzyme immunoassay is to allow the antigen in the sample to react with the antibody fixed on the solid phase, then react with the enzyme-labeled antibody, and after washing, add the chemiluminescent substrate for enzyme reaction, and then measure the luminescence intensity.

[0182] In some embodiments of the third aspect of the present application, the method includes:

[0183] Mixing the sample to be tested and a solid support coated with a capture antibody, so that the capture antibody coated on the solid support binds to the soluble CD14 subtype in the sample to be tested, wherein the capture antibody is an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof according to the first aspect of the present application;

[0184] The mixture is washed to remove unbound substances;

[0185] adding a labeled detection antibody to the washed mixture and mixing the mixture, so that the detection antibody binds to the soluble CD14 subtype bound to the capture antibody to form a sandwich complex;

[0186] washing the sandwich complex to remove unbound substances;

[0187] A detection substrate is added to the washed sandwich complex to detect the concentration of soluble CD14 isoforms in the sample. For example, a chemiluminescent substrate is added to the washed sandwich complex and the number of photons generated by the reaction is detected to obtain a chemiluminescent signal value of the sample, which is proportional to the concentration of the soluble CD14 isoforms.

[0188] In some embodiments of the third aspect of the present application, the sample to be tested can be blood, a blood component such as whole blood, serum, or plasma, preferably a plasma sample. However, the sample of the present application is not limited thereto and can also be body fluids such as urine, tissue fluid, lymph, joint fluid, breast milk, cerebrospinal fluid, pus, saliva, tears, mucus, nasal discharge, sputum, ascites, water, and semen.

[0189] The fourth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof according to the first aspect of the present application in the preparation of an analytical reagent for assessing whether a patient has sepsis, wherein the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof according to the first aspect of the present application is used to detect the concentration of soluble CD14 subtypes in a patient's blood sample, and an increase in the concentration of the soluble CD14 subtype relative to a reference value is correlated with an increased likelihood that the patient has sepsis.

[0190] In some embodiments of the fourth aspect of the present application, the concentration of the soluble CD14 isoform is compared with a cutoff value to determine whether the concentration of the soluble CD14 isoform is above the cutoff value, thereby assessing whether the patient has sepsis. An increase in the concentration of the soluble CD14 isoform relative to the reference value is positively correlated with an increased likelihood that the patient has sepsis.

[0191] In some embodiments of the fourth aspect of the present application, the concentration of soluble CD14 subtypes in a blood sample of the patient is detected within 72 hours after suspicion of sepsis is raised in the patient.

[0192] The fifth aspect of the present application relates to the use of the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof according to the first aspect of the present application in the preparation of an analytical reagent for evaluating the prognosis of a patient suspected of sepsis, wherein the anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof according to the first aspect of the present application is used to detect the concentration of soluble CD14 subtype in a blood sample of a patient suspected of sepsis, and an increased concentration of the soluble CD14 subtype relative to a reference value is associated with an increased risk of death in the patient suspected of sepsis.

[0193] In some embodiments of the fifth aspect of the present application, an increase in the concentration of the soluble CD14 subtype relative to the reference value is positively correlated with an increased risk of death in the patient suspected of sepsis.

[0194] In some embodiments of the fifth aspect of the present application, the concentration of soluble CD14 subtypes in a blood sample of the patient is detected within 72 hours after suspicion of sepsis is raised in the patient.

[0195] Some embodiments of the present application are described below. These embodiments are not intended to limit the present application but to better illustrate the present application.

[0196] Example 1: Preparation of anti-soluble CD14 subtype antibodies

[0197] Step 1.1 Expression of soluble CD14 subtype immunogens

[0198] Soluble CD14 subtype immunogens were expressed using prokaryotic expression systems and eukaryotic expression systems, respectively. The following describes the corresponding processes in detail:

[0199] (1) Using a prokaryotic expression system to express soluble CD14 subtype immunogens

[0200] After synthesizing the target DNA sequence, the pET30a expression vector was selected to construct the plasmid and transformed into Escherichia coli BL21star (DE3) to obtain a prokaryotic expression engineering strain. The recombinant plasmid was induced to express efficiently in the Escherichia coli expression system by IPTG (isopropylthiogalactoside). The target protein was mainly present in the inclusion body, and Ni was used to express the target protein. 2+ The target protein was purified by NTA affinity column. The target protein sequence was Thr Thr Pro Glu Pro Cys Glu Leu Asp AspGlu Asp Phe Arg Cys Val Cys Asn Phe Ser Glu Pro Gln Pro Asp Trp Ser Glu AlaPhe Gln Cys Val Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu Asn Leu GluPro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg Gln Tyr Ala (SEQ ID No. 43). Figure 1 As shown, the concentration of the target protein was determined to be 5.42 mg / mL by Bradford assay, and the purity and molecular weight of the target protein were determined by SDS-PAGE and Western Blot.

[0201] (2) Using eukaryotic expression system to express soluble CD14 subtype immunogens

[0202] After synthesizing the target DNA sequence, the pcDNA3.4 expression vector was selected to construct the plasmid. The HEK293-6E expression system was used, and the culture conditions were: serum-free FreeStyle TM Culture was performed in 293 Expression Medium (Thermo Fisher Scientific) at 37°C and 5% CO2. The constructed plasmid was transfected into the 293-6E expression system using transient transfection. After 6 days of culture, the cell culture supernatant was collected. The supernatant was filtered and affinity purified. The purified protein was enzymatically digested using optimized experimental conditions and purified to obtain the target protein. Figure 2 As shown, the concentration of the target protein was determined to be 1.55 mg / mL by Bradford assay, and the purity and molecular weight of the target protein were determined by SDS-PAGE and Western Blot.

[0203] Step 1.2 Preparation of anti-soluble CD14 isoform polyclonal antibodies

[0204] The purified proteins expressed by the prokaryotic system and the eukaryotic system obtained in step 1.1, i.e., the antigens, are mixed in a certain proportion and coupled with the carrier protein keyhole limpet hemocyanin (KLH for short), so as to be used as an immunogen for immunization of New Zealand white rabbits according to the following steps: dilute the immunogen with physiological saline, and then mix it with an adjuvant such as Freund's incomplete adjuvant (Sigma-Aldrich) at a ratio of 1:1. The antigen and adjuvant are completely mixed to form a stable emulsion, which is injected subcutaneously under the skin around the shoulders of the New Zealand white rabbits and intramuscularly in the hind thighs. Approximately 1 / 4 of the immunogen is used in each area, and the amount of antigen for each immunization is about 100 μg to 500 μg. A total of four immunizations are performed, with an interval of 2 weeks between each immunization. After the fourth immunization, the antiserum of the New Zealand white rabbits is collected and antigen affinity purified to obtain soluble CD14 subtype polyclonal antibodies. Figure 3 The results of SDS-PAGE and Western Blot using anti-soluble CD14 subtype polyclonal antibodies are shown in FIG. Figure 3 In the SDS-PAGE on the left, lane M is the protein marker, the first lane is the polyclonal antibody, and the second lane is the rabbit IgG; Figure 3 In the Western Blot on the right, lane M is the protein marker, the first lane is the immunogen (100 ng) + purified antibody (1 μg / mL) + goat anti-rabbit IgG [IRDye800cw] (0.125 μg / mL), the second lane is the immunogen (50 ng) + purified antibody (1 μg / mL) + goat anti-rabbit IgG [IRDye800cw] (0.125 μg / mL), the third lane is the immunogen (100 ng) + non-immune serum + goat anti-rabbit IgG [IRDye800cw] (0.125 μg / mL), and the fourth lane is the immunogen (100 ng) + goat anti-rabbit IgG [IRDye800cw] (0.125 μg / mL).

[0205] Step 1.3 Preparation of anti-soluble CD14 isoform monoclonal antibodies

[0206] The purified proteins expressed and purified by the prokaryotic and eukaryotic systems obtained in step 1.1, i.e., antigens, are mixed in a certain ratio and coupled with the carrier protein keyhole limpet hemocyanin (KLH). Mice are then immunized as immunogens according to the following steps: For the initial immunization, the antigen (10 μg to 50 μg) is mixed with an adjuvant, such as Freund's complete adjuvant (Sigma-Aldrich), at a ratio of 1:1 and then injected subcutaneously at multiple sites. Three weeks later, a second immunization is performed, using the same dose as the initial immunization, supplemented with incomplete Freund's adjuvant (Sigma-Aldrich) and injected subcutaneously. Three weeks later, a third immunization is performed, using the same dose as the initial immunization, but without the addition of an adjuvant. Three weeks later, a booster immunization is performed at a dose of 100 μg to 500 μg. Three days after the booster immunization, the spleen is harvested for fusion. After the third immunization, the potency of the mouse antiserum was assessed using ELISA. Splenocytes from the animal with the highest potency were selected and fused with myeloma cells to obtain a parent clone cell line, deposited under CGMCC No. 18536. Within the scope of this application, various known myeloma cells can be used, such as mouse-derived P3, NS-1, P3U1, and SP2 / 0, rat-derived YB2 / 0 and Y3-Ag1, human-derived SKO-007, and human-mouse hybrid myeloma cells SHM-D33. Mouse SP2 / 0 is preferred for this application.

[0207] Then, the mother clones were subjected to positive screening and reverse screening by ELISA (both positive screening and reverse screening experiments used indirect ELISA method).

[0208] For the positive screening experiment, the antigen was diluted with coating buffer (PBS buffer, pH = 7.4) to prepare a concentration of 1 μg / mL of the antigen to be coated. 100 μL of the antigen to be coated was added to a 96-well plate and incubated at room temperature for 1 hour. PBS buffer (pH = 7.4) was used as a washing solution to remove unreacted antigen. 100 μL of blocking solution was added to the 96-well plate and incubated at room temperature for 1 hour to block the unreacted solid phase surface. Excess blocking solution was removed again with washing solution (PBS buffer, pH = 7.4). Then, 100 μL of the supernatant of the cells to be screened was added and incubated at room temperature for 30 minutes. After washing with washing solution (PBS buffer, pH = 7.4) to remove the antibodies that were not bound to the solid phase coated antigen, 100 μL of peroxidase-labeled goat anti-mouse antibody was added. The peroxidase-labeled goat anti-mouse antibody binds to the mouse antibody that recognizes the solid phase antigen and catalyzes the luminescence of the substrate solution. The intensity of luminescence is used to determine whether there are antibodies that can bind to the antigen in the cell supernatant.

[0209] During the antibody screening process, a counter-screening assay using sCD14 was performed to confirm that the selected clones had no cross-reactivity with sCD14. For the counter-screening assay, the His-tag protein and sCD14 protein were diluted in coating buffer (PBS, pH 7.4) to a concentration of 1 μg / mL. Subsequent experimental steps were identical to those for the positive screening assay. The final luminescence intensity was used to determine whether the cell supernatant antibodies were reactive with the counter-screening agent.

[0210] After screening the mother clone, subsequent subcloning was performed. The subclones were affinity-ranked and epitope-classified. The subclone supernatants were paired with the polyclonal antibodies prepared in step 1.2 to detect the antigen. Tables 9-11 summarize the affinity ranking, epitope classification, and paired antibody screening results for anti-soluble CD14 isotype monoclonal antibodies. Based on the affinity ranking and antibody pairing results, the antibody clone number 16D5B2 was selected for subsequent production and purification, thereby obtaining a murine monoclonal antibody.

[0211] Table 9

[0212]

[0213] Table 10

[0214]

[0215]

[0216] Table 11

[0217]

[0218] Step 1.4: Determination of amino acid sequence of anti-soluble CD14 isoform monoclonal antibody

[0219] The variable regions of the anti-soluble CD14 subtype monoclonal antibodies obtained by screening in step 1.3 were sequenced. The total RNA of hybridoma cells was extracted according to the kit operation manual, and then the PrimeScript TMAccording to the operating manual of the 1st Strand cDNA Synthesis Kit, total RNA is reverse transcribed into cDNA using an epitope-specific antisense primer or a universal primer. The cDNA sequence synthesized by reverse transcription can be used to obtain the amino acid sequence of the corresponding anti-soluble CD14 subtype monoclonal antibody. The anti-soluble CD14 subtype monoclonal antibody, for example, includes a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID No. 3, a VH CDR2 consisting of the amino acid sequence of SEQ ID No. 9, and a VH CDR3 consisting of the amino acid sequence of SEQ ID No. 16, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID No. 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID No. 29, and a VL CDR3 consisting of the amino acid sequence of SEQ ID No. 38. The anti-soluble CD14 subtype monoclonal antibody specifically recognizes the epitope consisting of the amino acid sequence of SEQ ID No. 42.

[0220] Example 2: Determination of specific epitopes of anti-soluble CD14 subtype monoclonal antibodies

[0221] In the present application, there is no particular limitation on the method for determining the epitope, and for example, it can be carried out in the following manner: first, a polypeptide is synthesized by moving three amino acids from the N-terminus to the C-terminus of the target protein sequence, and each peptide chain consists of 15 amino acids; then, the polypeptides are respectively labeled with biotin and coated on a 96-well plate containing streptavidin through biotin-streptavidin binding; then, the antibody to be tested and peroxidase-labeled goat anti-mouse IgG are respectively added to the 96-well plate; and the amino acid sequence recognized by the antibody to be tested is determined by the signal value of the final reaction.

[0222] Example 3: Preparation of a kit for detecting soluble CD14 subtypes

[0223] The monoclonal antibody with clone number 16D5B2 screened in Example 1 was used as a capture antibody and coated on superparamagnetic magnetic beads (Merck, The surface of superparamagnetic magnetic beads was modified with carboxyl groups. The carboxyl groups on the surface of the superparamagnetic magnetic beads were coupled to the amino groups of the monoclonal antibody clone 16D5B2 under EDC / NHS catalysis to prepare a magnetic bead coating. The magnetic bead coating was diluted in 50 mM MES (2-morpholinoethanesulfonic acid) buffer (0.5 M NaCl, 0.5% BSA, 0.05% Tween 20, pH = 6.0) to a concentration of 1.0 mg / mL. The anti-soluble CD14 subtype polyclonal antibody prepared in step 1.2 of Example 1 was used as a detection antibody. The detection antibody was labeled with alkaline phosphatase (Roche Life Science). The enzyme-labeled marker was diluted in 50 mM MES buffer (0.5 M NaCl, 0.5% BSA, 0.05% Tween 20, pH = 6.0) to a concentration of 1 μg / mL.

[0224] In the current example, the detection reaction is based on a sandwich format: the plasma sample (40 μL), capture antibody (50 μL), and detection antibody (50 μL) are added to a reaction tube and incubated. The antigens, namely soluble CD14 isoforms, within the sample bind to the capture and detection antibodies, forming a sandwich complex. After incubation in the reaction tube, the magnetic beads are attracted by a magnetic field, thereby washing away unbound material.

[0225] The chemiluminescent substrate is added to the reaction tube, and the luminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoyl)-phenyl-1,2-dioxetane, AMPPD) is decomposed by alkaline phosphatase, removing a phosphate group to generate an unstable intermediate product, which produces methyl m-oxybenzoate anion through intramolecular electron transfer. When the excited methyl m-oxybenzoate anion returns to the ground state from the excited state, chemiluminescence is generated. Then, the number of photons generated by the intermediate reaction is measured by a photomultiplier tube. The number of photons generated is proportional to the concentration of the soluble CD14 subtype in the sample. Finally, a series of samples containing different concentrations of soluble CD14 subtypes are tested to obtain their corresponding luminescence values, and then a standard curve is established, such as Figure 4 shown.

[0226] Example 4: Use of anti-soluble CD14 subtype monoclonal antibodies or antigen-binding antibody fragments thereof in the preparation of analytical reagents for assessing whether a patient has sepsis

[0227] The kit prepared in Example 3 was used to detect soluble CD14 subtypes in samples from 141 patients suspected of having sepsis. PCT levels were also measured, and the diagnostic efficacy of soluble CD14 subtypes and PCT for sepsis was compared. The threshold for soluble CD14 subtype detection was 500 pg / mL, and the sensitivity and specificity for diagnosing sepsis were 0.76 and 0.91, respectively. Figure 5 As shown in Figure 3, the AUCs of the ROC curves for soluble CD14 isoforms and PCT were 0.91 and 0.84, respectively, indicating that soluble CD14 isoforms have a higher diagnostic efficacy.

[0228] Example 5: Use of anti-soluble CD14 subtype monoclonal antibodies or antigen-binding antibody fragments thereof in the preparation of analytical reagents for assessing the prognosis of patients suspected of sepsis

[0229] The kit prepared in Example 3 was used to detect the concentration of soluble CD14 subtypes in plasma samples of 88 suspected sepsis patients within 72 hours of admission, and the prognosis of the suspected sepsis patients within 30 days was tracked. The Kaplan-Meier analysis method was used to obtain the relationship curve of the hospital survival rate within 30 days (see Figure 6 The survival rates of patients in different groups were significantly different. For group III (soluble CD14 subtype concentration ≥ 1003.97 pg / mL), the survival probability of patients was < 40% (p < 0.01).

[0230] The 88 patients suspected of sepsis were selected as subjects and the ROC curve was used to compare the prognostic value of PCT and soluble CD14 subtype concentration within 72 hours of admission and the survival rate within 30 days of admission. Figure 7 As shown in the figure, the AUCs of PCT detection and soluble CD14 subtype detection were 0.72 and 0.83, respectively. Therefore, soluble CD14 subtype can more accurately predict the patient's survival status.

[0231] It can be concluded from the various embodiments described above that the anti-soluble CD14 subtype monoclonal antibodies or antigen-binding antibody fragments thereof or the kits of the embodiments of the present application are highly specific and sensitive and are suitable for detecting soluble CD14 subtypes in the sample to be tested, thereby improving the quality and accuracy of soluble CD14 subtype detection.

[0232] The present application is not restricted to the exemplary embodiments by the description thereof. Rather, the present application includes every novel feature and every combination of features, which in particular includes every combination of features in the patent claims, even if this feature or this combination itself is not specified in detail in the patent claims or exemplary embodiments. SEQUENCE LISTING <110> Wuhan Panorama Biotechnology Co., Ltd., Shenzhen Mindray Bio-Medical Electronics Co., Ltd. <120> New anti-soluble CD14 subtype antibodies and their applications <130> JCP1960996P-CN-D1 <160> 43 <170> PatentIn version 3.3 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Val Ala Met Leu 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <400> 2 Ser Val Glu Met Leu 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Ser Phe Ala Met Leu 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Ser Phe Ala Met Ala 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <400> 5 Ser Val Lys Met Leu 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <400> 6 Ser Phe Lys Met Ala 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <400> 7 Tyr Ile Ser Tyr Met Gly Ser Thr Ile Tyr Tyr Ala Asp Ala Lys Leu 1 5 10 15 Gly <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <400> 8 Tyr Ile Ser Ser Lys Ser Ser Ser Asn Leu Ala Ala Asp Ala Lys Leu 1 5 10 15 Gly <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <400> 9 Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <400> 10 Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Met Thr Lys 1 5 10 15 Gly <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <400> 11 Tyr Ile Thr Tyr Ser Gly Ser Gly Ala Tyr Tyr Ala Asp Tyr Gly Asn 1 5 10 15 Thr <210> 12 <211> 34 <212> PRT <213> Artificial Sequence <400> 12 Tyr Ile Ser Tyr Met Gly Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly Tyr Ile Ser Tyr Met Gly Ser Thr Ile Tyr Tyr Ala Asp Thr Val 20 25 30 Light Gly <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <400> 13 Tyr Ile Ser Ser Lys Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <400> 14 Ala Gly Phe Ala Tyr 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <400> 15 Gln Gln Phe Ala Tyr 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial Sequence <400> 16 Gln Gly Phe Ala Tyr 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <400> 17 Gln Gly Phe Ala Met 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <400> 18 Gln Gln Val Ala Tyr 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <400> 19 Ala Gly Phe Ala Met 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <400> 20 Ala Gly Phe Ala Tyr 1 5 <210> twenty one <211> 16 <212> PRT <213> Artificial Sequence <400> twenty one Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> twenty two <211> 16 <212> PRT <213> Artificial Sequence <400> twenty two Lys Tyr Tyr Ala Ser Ala Ala Lys Leu Tyr Arg Asn Ile Lys Leu Asn 1 5 10 15 <210> twenty three <211> 16 <212> PRT <213> Artificial Sequence <400> twenty three Lys Tyr Tyr Ala Ser Leu Leu Ala Thr Tyr Arg Asn Ile Lys Leu Asn 1 5 10 15 <210> twenty four <211> 16 <212> PRT <213> Artificial Sequence <400> twenty four Lys Tyr Tyr Ala Ser Leu Leu Ala Thr Thr Trp Ala Lys Gly Asn Asn 1 5 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <400> 25 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr Trp Ala Lys Gly Asn Asn 1 5 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <400> 26 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Tyr Arg Asn Ile Lys Leu Asn 1 5 10 15 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <400> 27 Leu Val Gln Ser Leu Asp Ser 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <400> 28 Leu Val Ser Lys Leu Thr Ala 1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <400> 29 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <400> 30 Leu Val Gln Ser Asp Ser Lys 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Leu Gln Thr Ser Asp Ser Lys 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <400> 32 Leu Val Ser Lys Leu Ala Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <400> 33 Val Gln Gly Thr His Asn Tyr Gly His 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <400> 34 Val Gly Gly Thr His Phe Pro Arg Thr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <400> 35 Val Gly Thr Ala Ile Phe Pro Arg Thr 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <400> 36 Val Gly Gly Thr His Phe Ile Thr Ala 1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <400> 37 Val Ser Gly Thr His Phe Pro Arg Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <400> 38 Val Gln Gly Thr His Phe Pro Arg Thr 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <400> 39 Val Gln Thr Ala Ile Phe Pro Arg Thr 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <400> 40 Val Gln Gly Thr His Tyr Gly His Val 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <400> 41 Val Gln Gly Asn Thr Phe Ile Thr Ala 1 5 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <400> 42 Pro Cys Glu Leu Asp Asp Glu Asp Phe Arg Cys Val 1 5 10 <210> 43 <211> 64 <212> PRT <213> Artificial Sequence <400> 43 Thr Thr Pro Glu Pro Cys Glu Leu Asp Asp Glu Asp Phe Arg Cys Val 1 5 10 15 Cys Asn Phe Ser Glu Pro Gln Pro Asp Tyr Ser Glu Ala Phe Gln Cys 20 25 30 Val Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu Asn Leu Glu 35 40 45 Pro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg Gln Tyr Ala 50 55 60

Claims

1. An anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof, which specifically recognizes an epitope consisting of the amino acid sequence of SEQ ID No. 42, It includes: (a) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 17; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 32, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33; (b) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 37; (c) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 18, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 21, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; (d) a VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 16, and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 33; or (e) A VH comprising a VH CDR1 consisting of the amino acid sequence of SEQ ID NO: 6, a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; and a VL comprising a VL CDR1 consisting of the amino acid sequence of SEQ ID NO: 22, a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 consisting of the amino acid sequence of SEQ ID NO:

38.

2. The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to claim 1, wherein The anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof is an anti-soluble CD14 subtype monoclonal antibody or the antigen-binding antibody fragment thereof secreted and produced by the hybridoma cell line with the deposit number of CGMCCNO.18536.

3. The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to claim 1, The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof does not specifically bind to high molecular weight soluble CD14.

4. The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to claim 1, The antigen-binding antibody fragment is an antigen-binding antibody fragment selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFV), dimerized V region (dimeric antibody), disulfide bond-stabilized V region (dsFv) and sc(Fv)2.

5. A kit for detecting soluble CD14 subtypes, wherein: The kit for detecting soluble CD14 subtypes comprises at least the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 4.

6. The kit for detecting soluble CD14 subtypes according to claim 5, The anti-soluble CD14 subtype monoclonal antibody or its antigen-binding fragment is used as a capture antibody, which is coated on a solid phase carrier and used to capture the soluble CD14 subtype in the sample to be tested.

7. The kit for detecting soluble CD14 subtypes according to claim 6, The capture antibody is coated on the surface of magnetic beads.

8. The kit for detecting soluble CD14 subtypes according to claim 6, The capture antibody is coated on the surface of superparamagnetic magnetic beads.

9. The kit for detecting soluble CD14 subtypes according to claim 6, The kit for detecting soluble CD14 subtypes further comprises a labeled detection antibody, which is an anti-soluble CD14 subtype polyclonal antibody.

10. A non-diagnostic method for detecting soluble CD14 isoforms, comprising: Mixing a sample to be tested and a solid support coated with a capture antibody, so that the capture antibody coated on the solid support binds to the soluble CD14 subtype in the sample to be tested, wherein the capture antibody is an anti-soluble CD14 subtype monoclonal antibody or an antigen-binding antibody fragment thereof according to any one of claims 1 to 4; The mixture is washed to remove unbound substances; adding a labeled detection antibody to the washed mixture and mixing the mixture, so that the detection antibody binds to the soluble CD14 subtype bound to the capture antibody to form a sandwich complex; washing the sandwich complex to remove unbound substances; A detection substrate is added to the washed sandwich complex to detect the concentration of soluble CD14 isoforms in the sample. The method according to claim 10 , wherein the sample to be tested is a blood sample. The method according to claim 10 , wherein the sample to be tested is a plasma sample.

13. Use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 4 in the preparation of an analytical reagent for assessing whether a patient has sepsis, wherein: The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof is used to detect the concentration of soluble CD14 subtypes in a patient's blood sample. An increased concentration of the soluble CD14 subtype relative to a reference value is associated with an increased likelihood that the patient has sepsis.

14. The use according to claim 13, in, The concentration of soluble CD14 isoforms is measured in blood samples from patients within 72 hours of suspicion of sepsis.

15. Use of the anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 4 in the preparation of an analytical reagent for evaluating the prognosis of a patient suspected of sepsis, wherein: The anti-soluble CD14 subtype monoclonal antibody or antigen-binding antibody fragment thereof is used to detect the concentration of soluble CD14 subtypes in a blood sample of a patient suspected of sepsis. An elevated concentration of the soluble CD14 subtype relative to a reference value is associated with an increased risk of death in the patient suspected of sepsis.

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