CN120559259B - Kit for diagnosing sepsis - Google Patents
Kit for diagnosing sepsisInfo
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- CN120559259B CN120559259B CN202511053167.9A CN202511053167A CN120559259B CN 120559259 B CN120559259 B CN 120559259B CN 202511053167 A CN202511053167 A CN 202511053167A CN 120559259 B CN120559259 B CN 120559259B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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Abstract
本申请属于生物医药技术领域，具体涉及一种诊断脓毒症的试剂盒。所述诊断脓毒症的试剂盒包括第一抗人CD14单克隆抗体和第二抗人CD14单克隆抗体；第一抗人CD14单克隆抗体为3B7抗体；第二抗人CD14单克隆抗体为13C2抗体。本申请的有益效果包括：本申请所述诊断脓毒症的试剂盒利用血浆诊断技术，在脓毒症诊断中具有操作简单、无介入、通量高、成本低的优势；本申请所述诊断脓毒症的试剂盒为脓毒症早期干预和诊治提供新的靶标。
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
The invention belongs to the technical field of biological medicines, and particularly relates to a kit for diagnosing sepsis.
Background
Sepsis is a syndrome of systemic inflammatory response caused by infection, commonly seen in severely wounded or infectious disease patients. The pathogenesis includes infection caused by bacteria, fungi, viruses, parasites and the like, and the inflammation reaction and the unbalance of the immunoregulation of the organism are caused. At present, suspected sepsis patients are diagnosed according to the sepsis diagnosis standard of Sepsis-3. The treatment principle of the sepsis patient is anti-infection, liquid resuscitation and multi-organ function support treatment. With the development of national economy and the improvement of medical level, the attention to the diagnosis and treatment of sepsis is continuously increased, but the cause and pathogenesis of sepsis in an intensive care unit are not completely clear. Sepsis is currently thought to be a complex disease based on infection, mediated by congenital and adaptive immune system disorders, and involving multiple factors such as coagulation dysfunction, hemoglobin reduction, etc.
CD14 is a glycoprotein with a molecular weight of 53-55kDa, consisting of 356 amino acids anchored to macrophages, monocytes, kupffer cells, neutrophils and part of the B cell membrane by Glycosyl Phosphatidylinositol (GPI). CD14 is a co-receptor for the Toll-like receptor TLR-4, playing a key role in the innate immune response.
CD14 can identify bacterial lipopolysaccharide, pathogen and injury related molecular patterns so as to promote inflammatory immune response, and recent researches show that the plasma soluble CD14 has a certain value for diagnosing and preventing neonatal sepsis, but CD14 serving as a diagnostic marker has not been reported in diagnosing sepsis.
Disclosure of Invention
The application provides a kit for diagnosing sepsis, which is simple to operate, has no intervention, high flux and low cost.
The application provides a kit for diagnosing sepsis, which comprises a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody;
The first anti-human CD14 monoclonal antibody is a 3B7 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 3B7 antibody is shown as 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 a 13C2 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 13C2 antibody is shown as 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.
according to some embodiments of the kit for diagnosing sepsis according to the present application, the kit detects elevated levels of sCD14 molecules in the plasma of a sepsis patient.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the test sample of the kit is venous blood.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the kit is an ELISA detection kit.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the kit further comprises a microporous enzyme-labeled plate, a sample diluent, a horseradish peroxidase-labeled antibody, a wash solution, a substrate, a stop solution, and a sealing plate membrane.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the sample diluent comprises PBS buffer.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the wash solution comprises PBS buffer.
According to some embodiments of the kit for diagnosing sepsis according to the present application, the stop solution comprises 2M H 2SO4.
The kit for diagnosing sepsis has the advantages of simplicity in operation, no intervention, high flux and low cost in sepsis diagnosis by utilizing a plasma diagnosis technology, and provides a new target for early intervention and diagnosis of sepsis.
Drawings
FIG. 1 is a schematic diagram of a standard test curve of a kit for diagnosing sepsis according to the present application;
FIG. 2 is a schematic diagram of the detection results of the reagent kit for diagnosing sepsis, which detects the level of soluble sCD14 in venous blood of different people;
FIG. 3 is a schematic diagram showing the change level of soluble sCD14 in venous blood on day 1, day 3 and day 7 of the administration of the kit for diagnosing sepsis according to the present application.
Detailed Description
The following detailed description of embodiments of the invention, examples of which are illustrative and intended to be used to illustrate the invention and not to be construed as limiting the invention.
For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
The embodiment of the application provides a kit for diagnosing sepsis, which comprises a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody;
The first anti-human CD14 monoclonal antibody is a 3B7 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 3B7 antibody is shown as 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 a 13C2 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 13C2 antibody is shown as 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.
the technical scheme of the application is further described below with reference to specific embodiments.
Example 1
8 Healthy people, 7 sepsis patients, 8 pancreatitis patients and 8 cervical cancer patients are respectively collected, whole blood samples are placed in an anticoagulation tube, and the sample is required to be venous blood. After blood sample collection, centrifugation was performed for 30min at 1000 Xg, and under this condition, centrifugation was performed for 10min to obtain a plasma sample and immediately detection was performed.
The level of CD14 in the above plasma samples was measured with the kit for diagnosing sepsis according to the present application and the average value of each group was determined.
The kit for diagnosing sepsis comprises a microporous enzyme-labeled plate, a standard substance corresponding to sCD14 molecules, sample diluent, an antibody marked by horseradish peroxidase, a washing solution, a substrate 3,3', 5' -tetramethyl benzidine, a stop solution and a sealing plate membrane.
The specific detection method comprises the following steps:
(1) Dilution and use of sCD14 standard the standard was prepared within 2 hours prior to use.
Preparing 5000pg/ml sCD14 standard substance, namely adding 1000 mu l PBS diluent into a standard substance tube containing sCD14, repeatedly reversing/oscillating to aid dissolution after covering, and standing for more than 10 min.
8 Concentration gradients of sCD14 standard were prepared by preparing 8 centrifuge tubes (Eppendorf tubes), adding 500. Mu.l of diluent to each tube, labeling 2500pg/ml,1250pg/ml,625pg/ml,312.5pg/ml,156.25pg/ml,78.125pg/ml,39.0625pg/ml,0pg/ml, adding 500. Mu.l of 5000pg/ml standard to the tube labeled 2500pg/ml, mixing well, and adding 500. Mu.l to the next tube.
(2) Sample addition, namely adding 100 mu lsCD of a standard substance (each concentration is added in sequence) and a plasma sample into each hole, after incubating for 2 hours at 37 ℃, throwing away liquid in the holes, adding 300 mu l of cleaning liquid into each hole, washing the plate for 3 times, discarding the liquid in the holes, and sucking water by using filter paper/absorbent paper.
(3) Adding detection antibody, namely adding 100 μl/hole of diluted detection antibody, covering a sealing plate film, incubating at 37 ℃ for 1h, then throwing away liquid in the hole, adding 300 μl of cleaning liquid in each hole, washing the plate for 3 times, discarding the liquid in the hole, and sucking water by using filter paper/absorbent paper.
(4) Adding enzyme-labeled antibody, namely adding 100 μl/hole of horseradish peroxidase labeled antibody, covering a sealing plate film, incubating at 37 ℃ for 1h, removing liquid in the hole, adding 300 μl of cleaning liquid in each hole, washing the plate for 6 times, discarding the liquid in the hole, and absorbing water by using filter paper/absorbent paper.
(5) Color development, namely adding chromogenic substrate 3,3', 5' -tetramethyl benzidine of enzyme-labeled antibody, 100 μl/well, and adding 2M H 2SO4 stop solution 50 μl/well after incubation at 37deg.C for 15min in dark place.
(6) Reading the plate Thermo MultiskanTM Fc enzyme-labeled detector to detect OD value at 450 nm.
And drawing a standard curve, namely, the abscissa is named as standard concentration, the ordinate is named as OD value, drawing a standard linear regression curve as shown in figure 1, and calculating the concentration value of each sample according to a curve equation.
The linear regression correlation coefficient R2 of the standard curve is more than or equal to 0.99, the accuracy is 95 percent, and the repeatability is that the variation coefficient is less than 5 percent.
The levels of sCD14 in plasma of 7 cases of sepsis patients, 8 cases of healthy persons, 8 cases of pancreatitis patients, 8 cases of cervical cancer patients (average value of each group was calculated) were analyzed by enzyme-linked immunosorbent assay technique, and the results are shown in fig. 2.
As can be seen from FIG. 2, the expression level of sCD14 in venous blood of healthy people is lower, while the expression level of sCD14 in sepsis patients is obviously higher than that of healthy people, and the expression level of sCD14 in venous blood of pancreatitis patients is slightly higher than that of healthy people, and the expression level of sCD14 in venous blood of cervical cancer patients is not different from that of healthy people.
Example 2
Collecting whole blood of 15 sepsis patients on 1 day, 3 days and 7 days, placing the sample in an anticoagulant tube, centrifuging the collected blood sample in 30min at a centrifugation speed of 1000 Xg, centrifuging under the condition of 10min to obtain a plasma sample, and immediately detecting.
The levels of sCD14 in plasma on day 1, day 3 and day 7 of admission (averages of each group were calculated) were analyzed by enzyme-linked immunosorbent assay technique for 15 sepsis patients and the results are shown in fig. 3.
As can be seen from FIG. 3, comparing sCD14 expression on day 1, day 3, and day 7 of hospitalization of sepsis patients, no difference in soluble sCD14 levels was found on day 1 and day 3, and sCD14 expression was higher overall than on days 1 and 3 as disease progressed to day 7. The method speculates that excessive inflammatory reaction and immune metabolism change exist in the sepsis pathogenesis, causes release of a large number of metabolites and immune related biomarkers, explores expression and dynamic change of different stages of plasma sCD14 of patients with sepsis, and can provide new targets for early intervention and diagnosis of sepsis.
While the above embodiments have been shown and described, it should be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations of the above embodiments may be made by those of ordinary skill in the art without departing from the scope of the invention.
Claims (8)
1. A kit for diagnosing sepsis, comprising a first anti-human CD14 monoclonal antibody and a second anti-human CD14 monoclonal antibody;
The first anti-human CD14 monoclonal antibody is a 3B7 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 3B7 antibody is shown as 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 a 13C2 antibody, and the amino acid sequence of a heavy chain variable region (mVH) of the 13C2 antibody is shown as 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 the level of sCD14 molecules in the plasma of a sepsis patient is increased when the kit is tested.
3. The kit for diagnosing sepsis according to claim 1, wherein 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, further comprising a microporous enzyme-labeled plate, a sample diluent, a horseradish peroxidase-labeled antibody, a wash solution, a substrate, a stop solution, and a sealing plate membrane.
6. The kit for diagnosing sepsis according to claim 5, wherein the sample diluent comprises PBS buffer.
7. The kit for diagnosing sepsis according to claim 5, wherein the wash solution includes PBS buffer.
8. The kit for diagnosing sepsis according to claim 5, wherein the stop solution includes 2M H 2SO4.
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| CN103497250A (en) * | 2013-10-11 | 2014-01-08 | 中国海洋大学 | WSSA (white spot syndrome virus disease) double-antibody sandwiched ELISA (enzyme linked immunosorbent assay) detection kit |
| CN110922484A (en) * | 2020-02-18 | 2020-03-27 | 南京诺艾新生物技术有限公司 | anti-EGFRvIII antibody and application thereof in disease diagnosis or treatment |
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| US10537637B2 (en) * | 2017-01-05 | 2020-01-21 | Gensun Biopharma Inc. | Checkpoint regulator antagonists |
| CN114702574B (en) * | 2022-05-07 | 2024-03-15 | 中国海洋大学 | Recombinant antibody of single-chain variable region of white spot virus, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103497250A (en) * | 2013-10-11 | 2014-01-08 | 中国海洋大学 | WSSA (white spot syndrome virus disease) double-antibody sandwiched ELISA (enzyme linked immunosorbent assay) detection kit |
| CN110922484A (en) * | 2020-02-18 | 2020-03-27 | 南京诺艾新生物技术有限公司 | anti-EGFRvIII antibody and application thereof in disease diagnosis or treatment |
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