A kit for diagnosing sepsis

Abstract

The present application belongs to the field of biomedicine technology, and specifically relates to a kit for diagnosing sepsis. The kit for diagnosing sepsis comprises a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody; the first anti-human CD14 monoclonal antibody is 3B7 antibody; and the second anti-human CD14 monoclonal antibody is 13C2 antibody. The beneficial effects of the present application include: the kit for diagnosing sepsis described in the present application utilizes plasma diagnostic technology, and has the advantages of simple operation, non-invasiveness, high throughput, and low cost in the diagnosis of sepsis; the kit for diagnosing sepsis described in the present application provides a new target for early intervention and diagnosis and treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a kit for diagnosing sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, commonly seen in patients with severe trauma or infectious diseases. Causes of the disease include infections caused by bacteria, fungi, viruses, and parasites, which lead to an imbalance in the body's inflammatory response and immune regulation. Currently, patients suspected of sepsis are diagnosed according to the Sepsis-3 sepsis diagnostic criteria. The treatment principles for patients with sepsis include anti-infection, fluid resuscitation, and multi-organ function support. With the development of the national economy and the improvement of medical standards, attention to the diagnosis and treatment of sepsis continues to increase, but the cause and pathogenesis of sepsis in intensive care units are not yet fully understood. Currently, sepsis is considered to be a complex disease based on infection, mediated by disorders of the innate and adaptive immune systems, and involving multiple factors such as coagulation disorders and decreased hemoglobin.

[0003] The CD14 molecule is a glycoprotein with a molecular weight of 53-55 kDa and is composed of 356 amino acids. It is anchored to the membranes of macrophages, monocytes, Kupffer cells, neutrophils, and some B cells through glycosylphosphatidylinositol (GPI). CD14 is a co-receptor for the Toll-like receptor TLR-4 and plays a key role in the innate immune response.

[0004] CD14 can recognize bacterial lipopolysaccharides, pathogens, and damage-associated molecular patterns, thereby promoting inflammatory immune responses. Recent studies have shown that plasma soluble CD14 has certain value in the diagnosis and prevention of neonatal sepsis, but CD14 as a diagnostic marker has not been reported in the diagnosis of sepsis. Summary of the Invention

[0005] The present application provides a kit for diagnosing sepsis that is simple to operate, non-invasive, high-throughput, and low-cost.

[0006] The present application provides a kit for diagnosing sepsis, the kit comprising a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody;

[0007] The first anti-human CD14 monoclonal antibody is the 3B7 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 3B7 antibody is shown in SEQ ID NO.1:

[0008] The sequence of SEQ ID NO.1 is:

[0009] EVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTGYNPSLKSRVSVTRDTSKNRFFLQLNSVTPEDTATYYCATVRYWGQGTTVTVSS;

[0010] The amino acid sequence of the light chain variable region (mVL) of the 3B7 antibody is shown in SEQ ID NO.2:

[0011] The sequence of SEQ ID NO.2 is:

[0012] DIVLTQSPASLAVSLGQRATISCRASESVDSFGKNFIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLKIK;

[0013] The second anti-human CD14 monoclonal antibody is the 13C2 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 13C2 antibody is shown in SEQ ID NO.3:

[0014] The sequence of SEQ ID NO.3 is:

[0015] EVQLVESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWVDGNTNYNSALMSRLTITKDNSQSQVFFKMNSLQTDDTAMYYCARDRGNWDVWFTYWGQGTLVTVSA;

[0016] The amino acid sequence of the light chain variable region (mVL) is shown in SEQ ID NO.4:

[0017] The sequence of SEQ ID NO.4 is:

[0018] DIVLTQSQKFMSTSVGDRVSVTCKASQNVGTHVAWYQQQPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNGYPYTFGGGTKLEIK.

[0019] According to some embodiments of the kit for diagnosing sepsis described herein, when detected by the kit, the level of sCD14 molecules in the plasma of sepsis patients is elevated.

[0020] According to some embodiments of the kit for diagnosing sepsis described in the present application, the test sample of the kit is venous blood.

[0021] According to some embodiments of the kit for diagnosing sepsis described herein, the kit is an ELISA detection kit.

[0022] According to some embodiments of the kit for diagnosing sepsis described in the present application, the kit further comprises a microwell ELISA plate, a sample diluent, a horseradish peroxidase-labeled antibody, a washing solution, a substrate, a stop solution, and a sealing film.

[0023] According to some embodiments of the kit for diagnosing sepsis described herein, the sample diluent includes PBS buffer.

[0024] According to some embodiments of the kit for diagnosing sepsis described herein, the washing solution comprises PBS buffer.

[0025] According to some embodiments of the kit for diagnosing sepsis described herein, the stop solution comprises 2M H2SO4.

[0026] The beneficial effects of the present application include: the kit for diagnosing sepsis described in the present application utilizes plasma diagnostic technology, and has the advantages of simple operation, non-intervention, high throughput, and low cost in the diagnosis of sepsis; the kit for diagnosing sepsis described in the present application provides a new target for early intervention and diagnosis and treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the standard curve of the kit for diagnosing sepsis described in this application;

[0028] Figure 2 This is a schematic diagram of the test results of the sepsis diagnosis kit described in this application for detecting the soluble sCD14 levels in the venous blood of different populations;

[0029] Figure 3 This is a schematic diagram of the changes in the levels of soluble sCD14 in the venous blood of sepsis patients on the 1st, 3rd, and 7th days after admission to the hospital using the sepsis diagnosis kit described in this application. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described in detail below. The examples of the embodiments are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0031] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0032] The present application embodiment provides a kit for diagnosing sepsis, the kit comprising a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody;

[0033] The first anti-human CD14 monoclonal antibody is the 3B7 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 3B7 antibody is shown in SEQ ID NO.1:

[0034] The sequence of SEQ ID NO.1 is:

[0035] EVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTGYNPSLKSRVSVTRDTSKNRFFLQLNSVTPEDTATYYCATVRYWGQGTTVTVSS;

[0036] The amino acid sequence of the light chain variable region (mVL) of the 3B7 antibody is shown in SEQ ID NO.2:

[0037] The sequence of SEQ ID NO.2 is:

[0038] DIVLTQSPASLAVSLGQRATISCRASESVDSFGKNFIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLKIK;

[0039] The second anti-human CD14 monoclonal antibody is the 13C2 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 13C2 antibody is shown in SEQ ID NO.3:

[0040] The sequence of SEQ ID NO.3 is:

[0041] EVQLVESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWVDGNTNYNSALMSRLTITKDNSQSQVFFKMNSLQTDDTAMYYCARDRGNWDVWFTYWGQGTLVTVSA;

[0042] The amino acid sequence of the light chain variable region (mVL) is shown in SEQ ID NO.4:

[0043] The sequence of SEQ ID NO.4 is:

[0044] DIVLTQSQKFMSTSVGDRVSVTCKASQNVGTHVAWYQQQPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNGYPYTFGGGTKLEIK.

[0045] The technical solution of this application is further described below with reference to specific embodiments.

[0046] Example 1

[0047] Whole blood samples were collected from 8 healthy individuals, 7 patients with sepsis, 8 patients with pancreatitis, and 8 patients with cervical cancer in anticoagulant tubes. Venous blood samples were centrifuged within 30 minutes at 1000 × g for 10 minutes. Plasma samples were obtained and immediately tested.

[0048] The CD14 levels in the plasma samples were detected using the sepsis diagnosis kit described in the present application, and the average value of each group was calculated.

[0049] The kit for diagnosing sepsis described in the embodiment of the present application includes: a microwell ELISA plate, a standard corresponding to the sCD14 molecule, a sample diluent, a horseradish peroxidase-labeled antibody, a washing solution, a substrate 3,3',5,5'-tetramethylbenzidine, a stop solution, and a sealing film.

[0050] The specific detection method includes the following steps:

[0051] (1) Dilution and use of sCD14 standard: Prepare the standard within 2 hours before use.

[0052] Prepare 5000 pg / ml sCD14 standard: Take 1000 μl PBS diluent and add it to the tube containing sCD14 standard. Cover the tube and invert / shake repeatedly to help dissolve it, then let it stand for more than 10 minutes.

[0053] Prepare 8 concentration gradients of sCD14 standards: prepare 8 centrifuge tubes (Eppendorf tubes), add 500μl of diluent to each tube, and label them as 2500pg / ml, 1250pg / ml, 625pg / ml, 312.5pg / ml, 156.25pg / ml, 78.125pg / ml, 39.0625pg / ml, and 0pg / ml respectively. Take 500μl of the 5000pg / ml standard and add it to the tube marked with 2500pg / ml. After mixing, take out 500μl and add it to the next tube, and so on.

[0054] (2) Sample addition: Add 100 μl of CD14 standard (each concentration is added in sequence) and plasma sample to each well. Incubate at 37°C for 2 h, then discard the liquid in the wells. Add 300 μl of cleaning solution to each well, wash the plate three times, discard the liquid in the wells, and absorb the water with filter paper / absorbent paper.

[0055] (3) Add detection antibody: Add 100 μl / well of diluted detection antibody, cover with sealing film, incubate at 37°C for 1 hour, shake off the liquid in the well, add 300 μl of cleaning solution to each well, wash the plate 3 times, discard the liquid in the well, and absorb the moisture with filter paper / absorbent paper.

[0056] (4) Add enzyme-labeled antibody: Add 100 μl / well of horseradish peroxidase-labeled antibody, cover with sealing film, incubate at 37°C for 1 hour, shake off the liquid in the well, add 300 μl of cleaning solution to each well, wash the plate 6 times, discard the liquid in the well, and absorb the moisture with filter paper / absorbent paper.

[0057] (5) Color development: Add 3,3',5,5'-tetramethylbenzidine, a color development substrate for the enzyme-labeled antibody, 100 μl / well, incubate at 37°C in the dark for 15 min, and then add 2M H2SO4 stop solution 50 μl / well.

[0058] (6) Plate reading: Thermo Multiskan™ Fc enzyme label reader was used to measure the OD value at a wavelength of 450 nm.

[0059] Draw the standard curve: the horizontal axis is the concentration of the standard, the vertical axis is the OD value, and the linear regression curve of the standard is drawn as follows: Figure 1 As shown, the concentration value of each sample was calculated according to the curve equation.

[0060] The linear regression correlation coefficient of the standard curve is R2≥0.99, the accuracy is 95% of the addition rate, and the repeatability is <5% of the coefficient of variation.

[0061] The levels of sCD14 in the plasma of 7 patients with sepsis, 8 healthy subjects, 8 patients with pancreatitis, and 8 patients with cervical cancer were analyzed by enzyme-linked immunosorbent assay (the average value of each group was calculated). Figure 2 shown.

[0062] from Figure 2 It can be seen that the expression level of sCD14 in the venous blood of healthy people is low, while the expression level of sCD14 in patients with sepsis is significantly higher than that in healthy people. The expression of sCD14 in the venous blood of patients with pancreatitis is slightly higher than that in healthy people, and the expression of sCD14 in the venous blood of patients with cervical cancer is no different from that in healthy people.

[0063] Example 2

[0064] Whole blood was collected from 15 patients with sepsis on the first, third, and seventh days of hospitalization. The specimens were required to be venous blood and placed in anticoagulant tubes. Blood samples were centrifuged within 30 minutes at 1000 × g for 10 minutes. Plasma samples were obtained and tested immediately.

[0065] The levels of sCD14 in the plasma of 15 patients with sepsis were analyzed by enzyme-linked immunosorbent assay on the 1st, 3rd and 7th day after admission (the average value of each group was calculated). Figure 3 shown.

[0066] from Figure 3 Comparison of sCD14 expression in sepsis patients on day 1, day 3, and day 7 after admission revealed no difference in soluble sCD14 levels between day 1 and day 3. However, as the disease progressed to day 7, sCD14 expression was generally higher than on days 1 and 3. This suggests that excessive inflammatory responses and immune metabolic changes occur during the pathogenesis of sepsis, leading to the release of numerous metabolites and immune-related biomarkers. Exploring the expression and dynamic changes of plasma sCD14 at different stages in sepsis patients may provide new targets for early intervention, diagnosis, and treatment of sepsis.

[0067] Although the above embodiments have been shown and described, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. Changes, modifications, substitutions and variations of the above embodiments by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. A kit for diagnosing sepsis, characterized in that: The kit includes a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody; The first anti-human CD14 monoclonal antibody is the 3B7 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 3B7 antibody is shown in SEQ ID NO.1: The sequence of SEQ ID NO.1 is: EVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTGYNPSLKSRVSVTRDTSKNRFFLQLNSVTPEDTATYYCATVRYWGQGTTVTVSS; The amino acid sequence of the light chain variable region (mVL) of the 3B7 antibody is shown in SEQ ID NO.2: The sequence of SEQ ID NO.2 is: DIVLTQSPASLAVSLGQRATISCRASESVDSFGKNFIHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLKIK; The second anti-human CD14 monoclonal antibody is the 13C2 antibody, and the amino acid sequence of the heavy chain variable region (mVH) of the 13C2 antibody is shown in SEQ ID NO.3: The sequence of SEQ ID NO.3 is: EVQLVESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWVDGNTNYNSALMSRLTITKDNSQSQVFFKMNSLQTDDTAMYYCARDRGNWDVWFTYWGQGTLVTVSA; The amino acid sequence of the light chain variable region (mVL) is shown in SEQ ID NO.4: The sequence of SEQ ID NO.4 is: DIVLTQSQKFMSTSVGDRVSVTCKASQNVGTHVAWYQQQPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNGYPYTFGGGTKLEIK.

2. The kit for diagnosing sepsis according to claim 1, characterized in that: When detected by the kit, the level of sCD14 molecules in the plasma of sepsis patients is elevated.

3. The kit for diagnosing sepsis according to claim 1, characterized in that: The test sample of the kit is venous blood.

4. The kit for diagnosing sepsis according to claim 1, characterized in that The kit is an ELISA detection kit.

5. The kit for diagnosing sepsis according to claim 4, characterized in that: The kit also includes a microporous enzyme labeling plate, a sample diluent, an antibody labeled with horseradish peroxidase, a washing solution, a substrate, a stop solution and a plate sealing film.

6. The kit for diagnosing sepsis according to claim 5, characterized in that: The sample diluent includes PBS buffer.

7. The kit for diagnosing sepsis according to claim 5, characterized in that: The washing solution includes PBS buffer.

8. The kit for diagnosing sepsis according to claim 5, characterized in that: The stop solution includes 2M H2SO4.

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