WO2019037044A1 - 急性肾损伤的检测试剂盒 - Google Patents

急性肾损伤的检测试剂盒 Download PDF

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WO2019037044A1
WO2019037044A1 PCT/CN2017/098890 CN2017098890W WO2019037044A1 WO 2019037044 A1 WO2019037044 A1 WO 2019037044A1 CN 2017098890 W CN2017098890 W CN 2017098890W WO 2019037044 A1 WO2019037044 A1 WO 2019037044A1
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ngal
kit
monoclonal antibody
kidney injury
acute kidney
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PCT/CN2017/098890
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French (fr)
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李瑞净
刘俊鹏
胡金灵
阳馨滢
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菲鹏生物股份有限公司
广东菲鹏生物有限公司
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Publication of WO2019037044A1 publication Critical patent/WO2019037044A1/zh

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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
    • 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
    • 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
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the invention relates to the field of biological detection, in particular to a detection kit for acute kidney injury.
  • Acute kidney injury is a clinical syndrome caused by a sudden drop in renal function caused by various causes in a short period of time (hours to several days), and is a common disease that threatens the lives of critically ill patients.
  • people's understanding of the pathogenesis of AKI and the level of medical technology have been greatly improved, but the incidence and mortality of AKI are still high.
  • One of the main reasons for this situation is the lack of effective early diagnostic markers.
  • sCr Traditional diagnostic indicators (serum sCr, urine output, etc.) are affected by many factors, such as age, gender, muscle mass, muscle metabolism, drug use and hydration, etc., the level of sCr varies greatly. In addition, sCr levels tend to increase significantly after a few days of AKI. Therefore, sCr is a reliable renal function marker protein and plays an important role in the diagnosis of AKI.
  • sCr is not an ideal AKI early diagnostic marker protein.
  • the diagnosis of AKI according to the urine output index is often not well and effectively reflects the renal function damage due to factors such as oliguria, diuretic drug use and complicated operation process in some patients after surgery. Sensitivity and specificity do not meet the needs of early clinical diagnosis. At present, the search for specific, sensitive and stable early diagnostic markers to achieve early diagnosis and early prevention and treatment of AKI has become the key to reducing the mortality rate of critically ill patients.
  • a detection kit for acute kidney injury comprising a first detection solution containing a first anti-NGAL monoclonal antibody coated with magnetic particles, and a second detection solution containing the second detection solution A chemiluminescent label-labeled second anti-NGAL monoclonal antibody, wherein the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed against different NGAL epitopes, respectively.
  • the above detection kit for acute kidney injury comprises a first detection solution containing a first anti-NGAL monoclonal antibody coated with magnetic particles, and a second detection solution containing a chemiluminescent label The second anti-NGAL monoclonal antibody.
  • the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed to different NGAL epitopes, respectively.
  • the first detection liquid and the second detection liquid are added to the sample to be tested, and the NGAL in the sample to be tested can be sandwiched between the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody according to the chemiluminescent label.
  • the luminescence signal of the object can determine the content of NGAL in the sample to be tested.
  • the acute kidney injury detection kit is based on NGAL (neutrophil gelatinase-related apolipoprotein) as a marker for diagnostic detection, and can be rapidly detected and diagnosed in the early stage of acute kidney injury.
  • the first anti-NGAL monoclonal antibody is coated with magnetic particles
  • the second anti-NGAL monoclonal antibody is labeled with a chemiluminescent label
  • the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed to different NGAL epitopes, respectively.
  • the detection of the acute kidney injury detection kit has a good correlation, and the linear range is wide, the sensitivity is high, and the specificity is high.
  • Example 2 is a comparison diagram of correlation between a kit of Example 1 and a control kit for testing 18 NGAL clinical high value samples in Test Example 3;
  • Figure 3 is a graph showing the correlation between the kit of Example 1 in Test Example 3 and the control kit for detecting 22 NGAL-positive samples.
  • An assay kit for acute kidney injury includes a first detection solution and a second detection solution.
  • the first detection solution contains a first anti-NGAL monoclonal antibody coated with magnetic particles
  • the second detection solution contains a second anti-NGAL monoclonal antibody labeled with a chemiluminescent label.
  • the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed to different NGAL epitopes, respectively.
  • the test kit for acute kidney injury is based on NGAL (neutrophil gelatinase-associated apolipoprotein) as a marker for diagnostic detection, and can be rapidly detected and diagnosed in the early stage of acute kidney injury.
  • NGAL neurotrophil gelatinase-associated apolipoprotein
  • the first detection liquid and the second detection liquid are added to the sample to be tested, and the NGAL in the sample to be tested can be sandwiched between the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody according to the chemiluminescent label.
  • the luminescence signal of the object can determine the content of NGAL in the sample to be tested.
  • the detection is convenient and quick, and the operation is simple.
  • the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody respectively target two different hybridomas for different NGAL epitopes, for example, the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody, respectively.
  • the cells are secreted.
  • NGAL combines with the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody to form a sandwich structure through different epitopes, preventing the epitope on NGAL from being shielded, high detection sensitivity, good specificity, and improved detection. Determine the linear range.
  • the first anti-NGAL monoclonal antibody is secreted by the hybridoma cell line NGAL-4F6.
  • the second anti-NGAL monoclonal antibody is secreted by the hybridoma cell line NGAL-3B5.
  • the hybridoma cell line NGAL-3B5 which secretes the second anti-NGAL monoclonal antibody, was deposited at the China Center for Type Culture Collection (CCTCC) on December 14, 2016. Address: Wuhan University, Wuhan, China, with the accession number CCTCC No. :C2016214, classification and naming: hybridoma cell line NGAL-3B5.
  • the hybridoma cell line NGAL-4F6 and the hybridoma cell line NGAL-3B5 were screened, and the antibody titers secreted by the two hybridoma cell lines were high.
  • the first anti-NGAL monoclonal antibody secreted by the hybridoma cell line NGAL-4F6 was coated with magnetic particles to obtain a magnetic particle-coated first anti-NGAL monoclonal antibody.
  • the second anti-NGAL monoclonal antibody secreted by the hybridoma cell line NGAL-3B5 was labeled with a chemiluminescent label to obtain a chemiluminescent label-labeled second anti-NGAL monoclonal antibody.
  • the experimental results show that the above-mentioned magnetic particle-coated first anti-NGAL monoclonal antibody and the chemiluminescent label-labeled second anti-NGAL monoclonal antibody can detect NGAL in the sample to be tested with high sensitivity and high specificity.
  • the first anti-NGAL monoclonal antibody is secreted by the hybridoma cell line NGAL-3B5, and the second anti-NGAL monoclonal antibody is secreted by the hybridoma cell line NGAL-4F6.
  • the first detection solution contains a first anti-NGAL monoclonal antibody coated with magnetic particles, and the first anti-NGAL monoclonal antibody is cured by magnetic particle-coated first anti-NGAL monoclonal antibody to improve the first anti- The stability of binding of the NGAL monoclonal antibody to NGAL in the sample to be tested.
  • the second detection solution contains a chemiluminescent label-labeled second anti-NGAL monoclonal antibody, a chemiluminescent label is labeled on the second anti-NGAL monoclonal antibody, and a luminescent signal is generated by the chemiluminescent label to rapidly determine the sample to be tested.
  • the content of NGAL is a chemiluminescent label-labeled second anti-NGAL monoclonal antibody.
  • the first detection liquid and the second detection liquid are combined to form a magnetic particle-coated first anti-NGAL monoclonal antibody-in the sample to be tested.
  • NGAL-chemiluminescent label-labeled second anti-NGAL monoclonal antibody (antibody-antigen-antibody) stable sandwich structure and the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed to different NGAL epitopes, respectively , improve detection Sensitivity and specificity.
  • the concentration of the first anti-NGAL monoclonal antibody coated with the magnetic microparticles is from 0.1 mg/mL to 5 mg/mL.
  • the mass ratio of the first anti-NGAL monoclonal antibody to the magnetic microparticles is 0.005 to 0.5:1.
  • the concentration of the first anti-NGAL monoclonal antibody coated by the magnetic particles is appropriate, and the content of NGAL in the sample to be tested is accurately determined.
  • the concentration of the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label is from 0.5 ⁇ g/mL to 10 ⁇ g/mL.
  • the concentration of the second anti-NGAL monoclonal antibody containing the chemiluminescent label is suitable for accurately determining the content of NGAL in the sample to be tested.
  • the first detection solution comprises magnetic particle-coated first anti-NGAL monoclonal antibody at a concentration of 0.1 mg/mL to 5 mg/mL, and HEPES at a concentration of 10 mmol/L to 100 mmol/L (4- Hydroxyethylpiperazineethanesulfonic acid), a stabilizer with a mass fraction of 0.1% to 30%, an inorganic salt having a mass fraction of 0.2% to 2%, a sucrose having a mass fraction of 0.2% to 20%, and a mass fraction of 0.01% ⁇ 1% of a surfactant and a bacteriostatic agent having a mass fraction of 0.01% to 1%.
  • the second detection solution comprises a chemiluminescent label-labeled second anti-NGAL monoclonal antibody at a concentration of 0.5 ⁇ g/mL to 10 ⁇ g/mL, and a concentration of 10 mmol/L to 100 mmol/L of Tris-HCl.
  • a stabilizer with a mass fraction of 0.1% to 30% an inorganic salt having a mass fraction of 0.2% to 2%, a glycerin having a mass fraction of 1% to 30%, a surfactant having a mass fraction of 0.01% to 1%, and a mass A bacteriostatic agent with a fraction of 0.01% to 1%.
  • the HEPES in the first detection liquid has a good buffering capacity, so that the pH of the first detection liquid is maintained at about 7.0, so that the magnetic particle-coated first anti-NGAL monoclonal antibody is not easily denatured.
  • the Tris-HCl in the second detection solution has a good buffering capacity, so that the pH of the second detection solution is maintained at about 8.0, so that the chemiluminescent label-labeled second anti-NGAL monoclonal antibody is not easily denatured.
  • the stabilizer is capable of promoting dispersion of the magnetic particle-coated first anti-NGAL monoclonal antibody in the first detection solution and the second anti-NGAL monoclonal antibody labeled by the chemiluminescent label in the second inspection Dispersion in the test liquid to avoid agglomeration and improve the accuracy of detection.
  • the stabilizer may be selected from at least one of bovine serum albumin (BSA), casein, and gelatin.
  • BSA bovine serum albumin
  • casein casein
  • gelatin gelatin
  • the inorganic salt is capable of adjusting the ion concentration such that the first anti-NGAL monoclonal antibody coated with the magnetic particles in the first detection liquid and the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label in the second detection liquid are not easily transsexual.
  • the inorganic salt may be selected from at least one of sodium chloride, potassium chloride, magnesium chloride, and potassium sulfate.
  • sucrose in the first detection liquid can increase the viscosity, and promote the stable binding of the magnetic particle-coated first anti-NGAL monoclonal antibody to the NGAL in the sample to be tested.
  • the glycerin in the second detecting solution can increase the viscosity, and the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label can form a stable binding with the NGAL in the sample to be tested, and the glycerol is stable in properties without affecting the chemiluminescent label.
  • the luminescence of matter can increase the viscosity, and the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label can form a stable binding with the NGAL in the sample to be tested, and the glycerol is stable in properties without affecting the chemiluminescent label. The luminescence of matter.
  • the surfactant is capable of lowering the surface tension of the liquid, promoting the dissolution of the magnetic particle-coated first anti-NGAL monoclonal antibody in the first detection solution, and the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label in the second Dissolved in the test solution.
  • the surfactant is selected from at least one of Tween-20 and Triton X-100.
  • the bacteriostatic agent is capable of inhibiting the growth of bacteria such that the first anti-NGAL monoclonal antibody coated with magnetic particles in the first detection solution and the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label in the second detection solution Has long-term stability.
  • the bacteriostatic agent is selected from at least one of sodium azide and PC-300.
  • the first detection solution comprises magnetic particle-coated first anti-NGAL monoclonal antibody at a concentration of 0.1 mg/mL to 5 mg/mL, and HEPES at a concentration of 40 mmol/L to 60 mmol/L (4- Hydroxyethylpiperazineethanesulfonic acid), BSA with a mass fraction of 0.5% to 2%, sodium chloride (NaCl) with a mass fraction of 0.5% to 2%, sucrose with a mass fraction of 1% to 10%, mass fraction It is 0.01% to 1% of Tween-20 and a mass fraction of 0.01% to 1% of PC-300.
  • the first test solution of the above formula is resistant when tested The interference ability is strong and the detection liquid is stable.
  • the second detection solution comprises a chemiluminescent label-labeled second anti-NGAL monoclonal antibody at a concentration of 0.5 ⁇ g/mL to 10 ⁇ g/mL, and a concentration of 40 mmol/L to 60 mmol/L of Tris-HCl. , BSA with a mass fraction of 0.5% to 2%, sodium chloride (NaCl) with a mass fraction of 0.5% to 2%, glycerin with a mass fraction of 1% to 10%, and Tween with a mass fraction of 0.01% to 1%. -20 and PC-300 with a mass fraction of 0.01% to 1%.
  • the second detection liquid of the above formula has strong anti-interference ability and stable detection liquid when tested.
  • the pH of the first detection liquid is 6.5 to 8.5.
  • the first anti-NGAL monoclonal antibody coated with magnetic particles was stable under the condition of pH 6.5-8.5, so the pH of the first detection solution was 6.5-8.5 to improve the detection accuracy.
  • the pH of the second detection liquid is 7.0 to 9.5.
  • the second anti-NGAL monoclonal antibody labeled with the chemiluminescent label was stable under the condition of pH 7.0-9.5, so the pH of the second detection solution was 7.0-9.5 to improve the detection accuracy.
  • the pH of the first detection liquid is smaller than the pH of the second detection liquid.
  • the magnetic particles are selected from the group consisting of carboxyl-modified magnetic particles, toluene-modified magnetic particles, amino-modified magnetic particles, and thiol-modified At least one of magnetic particles, epoxy-modified magnetic particles, and hydroxyl-modified magnetic particles.
  • the carboxyl group-modified magnetic particles include magnetic particles modified with a hydrophilic carboxyl group and magnetic particles modified with a hydrophobic carboxyl group.
  • the magnetic particles activated by the modification of a carboxyl group, a tosyl group, an amino group, a decyl group, an epoxy group or a hydroxyl group have the advantages of good hydrophilicity, low adsorption, and low background, and have little damage to antibody activity.
  • the magnetic fine particles are magnetic particles modified with a carboxyl group.
  • the magnetic particles are magnetic beads, and the magnetic beads have a particle diameter of 1.0 ⁇ m to 3.0 ⁇ m.
  • the chemiluminescent label-labeled second anti-NGAL monoclonal antibody after the second anti-NGAL monoclonal antibody binds to NGAL in the test solution, the chemiluminescent label can be under the action of the substrate liquid Generate a signal change, calculate the NGAL in the test solution according to the change of the signal content.
  • the chemiluminescent label is isoluminol, acridinium ester, acridinesulfonamide, terpyridine pyridinium, alkaline phosphatase or horseradish peroxidase.
  • the detection kit for acute kidney injury further comprises a luminescent substrate, the substrate being capable of reacting with the chemiluminescent label to produce a signal change.
  • the substrate liquid is, for example, a luminol sodium salt luminescent liquid.
  • the test kit for acute kidney injury further comprises an NGAL standard for preparing a standard curve to calculate the NGAL content in the sample to be tested.
  • the above-mentioned test kit for acute kidney injury may not include the substrate liquid and the NGAL standard, and the user may prepare it by himself.
  • the above-mentioned kit for detecting acute kidney injury is based on NGAL (neutrophil gelatinase-related apolipoprotein) as a marker for diagnostic detection, and can be rapidly detected and diagnosed in the early stage of acute kidney injury.
  • the first anti-NGAL monoclonal antibody is coated with magnetic particles
  • the second anti-NGAL monoclonal antibody is labeled with a chemiluminescent label
  • the first anti-NGAL monoclonal antibody and the second anti-NGAL monoclonal antibody are directed to different NGAL epitopes, respectively.
  • the detection of the acute kidney injury detection kit has a good correlation, and the linear range is wide, the sensitivity is high, and the specificity is high.
  • the test kit for acute kidney injury includes a first test solution, a second test solution, a substrate solution, and an NGAL standard.
  • the first detection solution contains a first anti-NGAL monoclonal antibody coated with magnetic particles at a concentration of 0.5 mg/mL (the mass ratio of the first anti-NGAL monoclonal antibody to the magnetic particles is 0.05:1), and the concentration is 50 mmol/L.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • BSA sodium chloride
  • NaCl sodium chloride
  • sucrose with a mass fraction of 5%
  • mass fraction of 0.1% Tween-20 and PC-300 with a mass fraction of 0.1%
  • the pH is 7.0.
  • the second detection solution contains a second anti-NGAL monoclonal antibody labeled with an HRP marker at a concentration of 1 ⁇ g/mL (a molar ratio of HRP to a second anti-NGAL monoclonal antibody is 1:1), and a concentration of 50 mmol/L of Tris.
  • the first anti-NGAL monoclonal antibody was secreted by the hybridoma cell line NGAL-4F6 (Accession No. CCTCC No: C2016216).
  • the second anti-NGAL monoclonal antibody was secreted by the hybridoma cell line NGAL-3B5 (Accession No. CCTCC No: C2016214).
  • the substrate solution is a luminol sodium salt illuminant, and the NGAL standard is a human NGAL protein.
  • the preparation method of the above test kit for acute kidney injury comprises the following steps:
  • mice 1. NGAL immunized mice
  • NGAL NGAL-Ag1, Philippine Bio
  • Freund's complete adjuvant Sigma-Aldrich, Cat. No. F5881
  • the emulsion was subcutaneously administered to BALB/c mice at a dose of 0.2 mL (Guangdong Medical Laboratory Animal Center: No. 119, Huangqiyang Road, Nanhai, Foshan City, Guangdong province, 6-week-old female, 5) back sites.
  • the abdominal cavity was boosted, that is, the same amount of antigen was mixed with the Freund's incomplete adjuvant (Sigma-Aldrich, F5506) in the same volume, and the immunization was boosted to four needles.
  • the tail blood was collected and the serum was separated for indirect ELISA.
  • the titer is determined by the method, and the titer is higher than 1:10000 for fusion.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • One day before the fusion BALB/c mice were sacrificed by neck-stretching, 75% alcohol was immersed in the whole body, and the abdominal skin was cut with scissors under sterile operation to expose the peritoneum.
  • mice Three days after the last immunization of the mice, the spleens were taken out under aseptic conditions, placed in a dish, rinsed once with RPMI 1640 base medium, and placed in a nylon beaker on a small beaker to be filtered to prepare a cell suspension. After centrifugation, the supernatant was discarded, and the RPMI 1640 base medium was resuspended, and this was repeated three times and counted.
  • Mouse myeloma cells Sp2/0 (preserved by Fei Peng Biotechnology Co., Ltd.) were screened by 8-azaguanine and cultured to logarithmic growth phase. Two large bottles were prepared to make cell suspension, centrifuged, and the supernatant was discarded. RPMI was used. The 1640 basal medium was resuspended and repeated three times, counting.
  • the myeloma cells and the immune spleen cells were mixed at a ratio of 1:10, and washed once with a RPMI 1640 base culture solution in a 50 mL plastic centrifuge tube, and centrifuged at 1200 rpm for 8 minutes. The supernatant was discarded, the cells were mixed, and 1 mL of 50% PEG 1500 fusion was slowly added. After 1 minute of fusion, 15 mL of RPMI 1640 basal medium was added to terminate the cell fusion. Centrifuge for 5 minutes at 1000 rpm.
  • the supernatant was discarded, and gently incubated with 50 mL of RPMI 1640 screening medium, aliquoted into 10 96-well plates plated with feeder cells, 50 ⁇ L/well, and cultured at 37 ° C, 5% CO 2 . The culture was continued until the sixth day, and the HT medium (HTMI-containing RPMI1640 complete medium) was changed twice.
  • the PK2-NGAL protein was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 2 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, it was blocked with 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder at 0.15 mL/well for 2 hours at 37 ° C for detection.
  • mice Six to eight weeks of robust BALB/c mice were selected, and each mouse was intraperitoneally injected with 0.5 mL of pristane; after 10 days, 1 x 10 6 hybridoma cells were intraperitoneally injected. After inoculation of cells for 7 to 10 days, ascites can be produced, and the animal's health and ascites signs should be closely observed. As much as possible of ascites, while the mice are killed before death, the mice are sacrificed and the ascites is inhaled into the test tube with a dropper. The mice can obtain 5-10 mL of ascites. The ascites was collected, centrifuged, and the supernatant was diluted with 3 volumes of PBS and filtered through a filter paper.
  • the resulting filtrate was applied to a protein G affinity chromatography column (GE) equilibrated with PBS at a flow rate of 1 mL/min.
  • the material not adsorbed by protein G was then washed with PBS at a flow rate of 1 mL/min until the adsorption value at OD280 nm reached the baseline.
  • the antibody was eluted and recovered with a 0.1 M glycine eluate (pH 2.5).
  • the recovered antibody was immediately neutralized with 0.1 M Tris (pH 8.8) and the concentration was measured by running a gel.
  • the above-mentioned secretory antibody against human NGAL hybridoma has an antibody titer of 2.77 ⁇ 10 6 or more.
  • the purified monoclonal antibody to be identified was diluted with 0.06 M pH 9.6 carbonate buffer solution to a final concentration of 1 ⁇ g/mL. 0.1 mL per well was added to a 96-well polystyrene plate and incubated at 37 ° C for 2 hours or 4 ° C overnight. The next day, 0.02 M pH 7.2 PBS containing 10% calf serum or 1% skim milk powder, 0.15 mL/well, blocked at 37 °C for 2 hours, and 2000-fold dilution of horseradish peroxidase-labeled NGAL epitope was added for identification.
  • the antigen was washed at 37 ° C for 30 minutes, PBST 5 times, patted dry, and 100 ⁇ L of 0.1% (M / V) o-phenylenediamine, 0.1% (V / V) hydrogen peroxide, pH 5.0 citrate phosphate buffer was added to each well.
  • dilute sulfuric acid solution was added, 50 ⁇ L per well, and the absorbance at 450 nm was measured, and the epitope was distinguished according to the reaction.
  • Two strains of different NGAL epitopes were screened, one of which was the hybridoma cell line NGAL-4F6, which secreted the first anti-NGAL monoclonal antibody, which was deposited on December 14, 2016 in China.
  • CTCC Type Culture Collection
  • the first anti-NGAL monoclonal antibody coated with the magnetic particles was washed twice with 1 mL of washing buffer, and the ultrasound was resuspended during the washing.
  • HRP horseradish peroxidase
  • Termination of oxidation 8 ⁇ l of ethylene glycol was dissolved in 40 ⁇ l of distilled water, and added to 2) at 4 ° C for 30 min to terminate the oxidation reaction.
  • the first detection solution comprises 0.5 mg/mL of magnetic particle coated first anti-NGAL monoclonal antibody, 50 mmol /L of HEPES (4-hydroxyethylpiperazineethanesulfonic acid), BSA with a mass fraction of 1%, sodium chloride (NaCl) with a mass fraction of 1%, sucrose with a mass fraction of 5%, mass fraction of 0.1 % Tween-20 and PC-300 with a mass fraction of 0.1%, the balance of ultrapure water.
  • the pH of the first test solution was 7.0.
  • a chemiluminescent label-labeled second anti-NGAL monoclonal antibody Dissolving a chemiluminescent label-labeled second anti-NGAL monoclonal antibody in a buffer to obtain a second detection solution, wherein the second detection solution comprises 1 ⁇ g/mL of a chemiluminescent label-labeled second anti-NGAL monoclonal antibody, 50mmol/L Tris-HCl, 1% BSA, 1% sodium chloride (NaCl), 5% glycerol, 0.1% Tween-20 and mass fraction For 0.1% PC-300, the balance of ultrapure water. The pH of the second test solution was 8.0.
  • NGAL (NGAL-Ag1, Philippine Bio) was formulated to a concentration of 1500 ng/mL, 300 ng/mL, 60 ng, respectively, using a calibration buffer (40 mmol/L Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0). /mL, 12ng/mL, 2.4ng/mL, 0ng/mL, 0.5mL per bottle is lyophilized, and stored at 4 degrees for use.
  • a calibration buffer 40 mmol/L Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0.
  • /mL, 12ng/mL, 2.4ng/mL, 0ng/mL, 0.5mL per bottle is lyophilized, and stored at 4 degrees for use.
  • NGAL calibrator Concentration of 1500 ng/mL, 300 ng/mL, 60 ng/mL, 12 ng/mL, 2.4 ng/mL, 0 ng/mL
  • the second test solution was reacted for 15 min, and washed 5 times with 50 mmol/L Tris + 0.05% Tween-20, pH 7.5.
  • 100 ⁇ L of luminescent substrate Luminol sodium luminescent solution
  • FIG. 1 A corresponding relationship between the NGAL content and the luminescence value is established, and the result is shown in Fig. 1.
  • Each point on the curve in Fig. 1 represents a standard product, wherein the x-axis represents the content of NGAL and the y-axis represents the luminescence value.
  • the NGAL was detected by the kit of Example 1, and the established NGAL content had a good linear relationship with the standard curve established by the luminescence value, and the linear range was wide.
  • the kit of Example 1 has high detection sensitivity.
  • 102 clinical urine and 22 positive specimens were tested according to the method of Test 1, and the values were read by the Zhongsheng Baike BK-L96C chemiluminescence immunoassay analyzer and brought into the sample.
  • the standard curve calculates the NGAL content.
  • the results showed that 102 clinical random urine samples were basically in the range of 2 ng / mL ⁇ 30 ng / mL. 18 of the concentrations detected ranged from 40 ng/mL to 130 ng/mL.
  • the potential interference/cross-substance and different pH values in the urine samples were evaluated using the kit of Example 1.
  • the pH value of the urine sample was adjusted or the following concentration substances were added to the negative sample, and the negative urine sample which was not treated was used as a control, and the NGAL content was measured according to the method of Test Example 1. The results are shown in Table 2.
  • the results show that the potential interference/crossings are almost non-intersecting.
  • the kit of Example 1 has strong anti-interference ability and can adapt to different test samples.
  • the first detection solution and the second detection solution in the test kit for acute kidney injury of Example 1 were subjected to a 37-degree, 3-day, and 7-day thermal break test, and four concentration points of the calibration product were selected for detection. The results are shown in Table 3.

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Abstract

一种急性肾损伤的检测试剂盒，包括第一检测液和第二检测液，第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体。第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位。

Description

急性肾损伤的检测试剂盒 技术领域
本发明涉及生物检测领域，特别是涉及一种急性肾损伤的检测试剂盒。
背景技术
急性肾损伤(acute kidney injury，AKI)是由各种原因引起的肾功能在短时间(几小时至几天)内突然下降而出现的临床综合征，是威胁重症患者生命的常见疾病。通常50％以上的重症监护的病人都有不同程度的肾损伤，急性肾功能衰竭死亡率处于危重病死亡率前列。在过去的几十年里人们对AKI发病机理的认识和医疗技术水平都有了很大的提高，但AKI的发病率和病死率仍然居高不下。导致这一局面的一个主要原因是缺乏有效的早期诊断标志物。传统的诊断指标(血肌酐sCr、排尿量等)受诸多因素影响，如由于受年龄、性别、肌肉的质量、肌肉新陈代谢、药物使用及水合作用等因素的影响，sCr水平变化很大。另外sCr水平往往在AKI发生几天后才有显著升高。因此，sCr虽然是可靠的肾功能标志蛋白并在AKI的诊断过程中起了重要的作用。但由于以上缺点，sCr不是理想的AKI早期诊断标志蛋白。另外根据排尿量指标进行AKI诊断常常由于手术后一些病人的少尿症、利尿药物的使用及复杂操作过程等因素的影响而不能很好的、有效的反映肾功能损伤情况。敏感性和特异性均无法满足临床早期诊断的需求。目前，寻找特异、敏感、稳定的早期诊断标志物，从而达到早诊断、早防治AKI，成为降低重症患者病死率的关键。
然而，传统的急性肾损伤的检测试剂盒灵敏度较低、特异性较差。
发明内容
基于此，有必要提供一种检测灵敏度较高、特异性较高的急性肾损伤的检测试剂盒。
一种急性肾损伤的检测试剂盒，包括第一检测液和第二检测液，所述第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，所述第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体，其中，所述第一抗NGAL单克隆抗体和所述第二抗NGAL单克隆抗体分别针对不同的NGAL表位。
上述急性肾损伤的检测试剂盒，包括第一检测液和第二检测液，第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体。其中，第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位。检测时，向待测样品中加入第一检测液和第二检测液，待测样品中的NGAL能够夹心于第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体之间，根据化学发光标记物的发光信号即可测定待测样品中NGAL的含量。该急性肾损伤的检测试剂盒基于NGAL(中性粒细胞明胶酶相关载脂蛋白)作为诊断检测的标志物进行检测，能够在急性肾损伤发生的早期快速检测诊断。第一抗NGAL单克隆抗体上包被磁微粒，第二抗NGAL单克隆抗体上标记化学发光标记物，且第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位，避免NGAL上的表位被屏蔽。该急性肾损伤的检测试剂盒检测的相关性好可测定线性范围宽且敏度较高、特异性较高。
附图说明
图1为测试例一中得到NGAL的含量与发光值的对应关系曲线；
图2为测试例三中实施例1的试剂盒与对照试剂盒检测18份NGAL临床高值样本相关性对比图；
图3为测试例三中实施例1的试剂盒与对照试剂盒检测22份NGAL阳性样本相关性对比图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似改进，因此本发明不受下面公开的具体实施的限制。
一实施方式的急性肾损伤的检测试剂盒，包括第一检测液和第二检测液。第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体。其中，第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位。
该急性肾损伤的检测试剂盒基于NGAL(中性粒细胞明胶酶相关载脂蛋白)作为诊断检测的标志物，能够在急性肾损伤发生的早期快速检测诊断。检测时，向待测样品中加入第一检测液和第二检测液，待测样品中的NGAL能够夹心于第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体之间，根据化学发光标记物的发光信号即可测定待测样品中NGAL的含量。检测方便快捷、操作简单。
具体地，第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位，例如第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别通过两株不同的杂交瘤细胞分泌得到。NGAL通过不同的表位分别与第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体结合从而形成夹心结构，避免NGAL上的表位被屏蔽，检测灵敏度高，特异性好，提高检测的可测定线性范围。
在一个实施方式中，第一抗NGAL单克隆抗体由杂交瘤细胞株NGAL-4F6分泌得到。可分泌第一抗NGAL单克隆抗体的杂交瘤细胞株NGAL-4F6于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016216，分类命名：杂 交瘤细胞株NGAL-4F6。
在一个实施方式中，第二抗NGAL单克隆抗体由杂交瘤细胞株NGAL-3B5分泌得到。可分泌第二抗NGAL单克隆抗体的杂交瘤细胞株NGAL-3B5于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016214，分类命名：杂交瘤细胞株NGAL-3B5。
经人的NGAL免疫小鼠后，筛选获得的杂交瘤细胞株NGAL-4F6和杂交瘤细胞株NGAL-3B5，两株杂交瘤细胞株分泌的抗体效价高。用磁微粒包被杂交瘤细胞株NGAL-4F6分泌的第一抗NGAL单克隆抗体，得到磁微粒包被的第一抗NGAL单克隆抗体。用化学发光标记物标记杂交瘤细胞株NGAL-3B5分泌的第二抗NGAL单克隆抗体，得到化学发光标记物标记的第二抗NGAL单克隆抗体。实验结果表明，上述磁微粒包被的第一抗NGAL单克隆抗体与化学发光标记物标记的第二抗NGAL单克隆抗体配合能够高灵敏度、高特异性的检测待测样品中的NGAL。
在其他实施方式中，还可以是第一抗NGAL单克隆抗体由杂交瘤细胞株NGAL-3B5分泌得到，而第二抗NGAL单克隆抗体由杂交瘤细胞株NGAL-4F6分泌得到。
具体地，第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，通过磁微粒包被的第一抗NGAL单克隆抗体，将第一抗NGAL单克隆抗体固化，提高第一抗NGAL单克隆抗体与待测样品中的NGAL结合的稳定性。第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体，在第二抗NGAL单克隆抗体上标记化学发光标记物，通过化学发光标记物产生发光信号，快速测定待测样品中NGAL的含量。检测时，向待测样品中加入第一检测液和第二检测液后，第一检测液和第二检测液配合，形成磁微粒包被的第一抗NGAL单克隆抗体-待测样品中的NGAL-化学发光标记物标记的第二抗NGAL单克隆抗体(抗体-抗原-抗体)稳定的夹心结构，且第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位，提高检测 的灵敏度和特异性。
具体地，第一检测液中，磁微粒包被的第一抗NGAL单克隆抗体的浓度为0.1mg/mL～5mg/mL。
具体地，磁微粒包被的第一抗NGAL单克隆抗体中，第一抗NGAL单克隆抗体与磁微粒的质量比为0.005～0.5∶1。磁微粒包被的第一抗NGAL单克隆抗体浓度适宜，准确测定待测样品中的NGAL的含量。
具体地，第二检测液中，化学发光标记物标记的第二抗NGAL单克隆抗体的浓度为0.5μg/mL～10μg/mL。含有化学发光标记物标记的第二抗NGAL单克隆抗体浓度适宜，准确测定待测样品中的NGAL的含量。
在一个实施方式中，第一检测液中包括浓度为0.1mg/mL～5mg/mL的磁微粒包被的第一抗NGAL单克隆抗体、浓度为10mmol/L～100mmol/L的HEPES(4-羟乙基哌嗪乙磺酸)、质量分数为0.1％～30％的稳定剂、质量分数为0.2％～2％的无机盐、质量分数为0.2％～20％的蔗糖、质量分数为0.01％～1％的表面活性剂和质量分数为0.01％～1％的抑菌剂。
在一个实施方式中，第二检测液中包括浓度为0.5μg/mL～10μg/mL的化学发光标记物标记的第二抗NGAL单克隆抗体、浓度为10mmol/L～100mmol/L的Tris-HCl、质量分数为0.1％～30％的稳定剂、质量分数为0.2％～2％的无机盐、质量分数为1％～30％的甘油、质量分数为0.01％～1％的表面活性剂和质量分数为0.01％～1％的抑菌剂。
具体地，第一检测液中的HEPES具有较好的缓冲能力，使得第一检测液的pH维持在7.0左右，使得磁微粒包被的第一抗NGAL单克隆抗体不易变性。
具体地，第二检测液中的Tris-HCl具有较好的缓冲能力，使得第二检测液的pH维持在8.0左右，使得化学发光标记物标记的第二抗NGAL单克隆抗体不易变性。
具体地，稳定剂能够促进磁微粒包被的第一抗NGAL单克隆抗体在第一检测液中分散以及化学发光标记物标记的第二抗NGAL单克隆抗体在第二检 测液中分散，避免产生团聚，提高检测的准确度。
在一个实施方式中，稳定剂可以选自牛血清蛋白(BSA)、酪蛋白和明胶中的至少一种。
具体地，无机盐能够调节离子浓度，使得第一检测液中的磁微粒包被的第一抗NGAL单克隆抗体以及第二检测液中的化学发光标记物标记的第二抗NGAL单克隆抗体不易变性。
在一个实施方式中，无机盐可以选自氯化钠、氯化钾、氯化镁和硫酸钾中的至少一种。
具体地，第一检测液中的蔗糖能够增加的黏度，促进磁微粒包被的第一抗NGAL单克隆抗体与待测样品中的NGAL形成稳定的结合。
具体地，第二检测液中的甘油能够增加黏度，促进化学发光标记物标记的第二抗NGAL单克隆抗体与待测样品中的NGAL形成稳定的结合，且甘油性质稳定，不影响化学发光标记物的发光。
具体地，表面活性剂能够降低液体的表面张力，促进磁微粒包被的第一抗NGAL单克隆抗体在第一检测液中溶解以及化学发光标记物标记的第二抗NGAL单克隆抗体在第二检测液中溶解。
在一个实施方式中，表面活性剂选自吐温-20和TritonX-100中的至少一种。
具体地，抑菌剂能够抑制细菌的生长，使得第一检测液中的磁微粒包被的第一抗NGAL单克隆抗体以及第二检测液中化学发光标记物标记的第二抗NGAL单克隆抗体具有长期稳定性。
在一个实施方式中，抑菌剂选自叠氮钠和PC-300中的至少一种。
在一个实施方式中，第一检测液中包括浓度为0.1mg/mL～5mg/mL的磁微粒包被的第一抗NGAL单克隆抗体、浓度为40mmol/L～60mmol/L的HEPES(4-羟乙基哌嗪乙磺酸)、质量分数为0.5％～2％的BSA、质量分数为0.5％～2％的氯化钠(NaCl)、质量分数为1％～10％的蔗糖、质量分数为0.01％～1％的吐温-20和质量分数为0.01％～1％的PC-300。上述配方的第一检测液在检测时，抗 干扰性能力强，检测液稳定。
在一个实施方式中，第二检测液中包括浓度为0.5μg/mL～10μg/mL的化学发光标记物标记的第二抗NGAL单克隆抗体、浓度为40mmol/L～60mmol/L的Tris-HCl、质量分数为0.5％～2％的BSA、质量分数为0.5％～2％的氯化钠(NaCl)、质量分数为1％～10％的甘油、质量分数为0.01％～1％的吐温-20和质量分数为0.01％～1％的PC-300。上述配方的第二检测液在检测时，抗干扰性能力强，检测液稳定。
具体地，第一检测液的pH值为6.5～8.5。研究过程中发现，磁微粒包被的第一抗NGAL单克隆抗体在pH值为6.5～8.5条件下性质稳定，因而采用第一检测液的pH值为6.5～8.5提高检测的准确性。
具体地，第二检测液的pH值为7.0～9.5。研究过程中发现，化学发光标记物标记的第二抗NGAL单克隆抗体在pH值为7.0～9.5条件下性质稳定，因而采用第二检测液的pH值为7.0～9.5提高检测的准确性。
进一步地，第一检测液的pH值小于第二检测液的pH值。
在一个实施方式中，磁微粒包被的第一抗NGAL单克隆抗体中，磁微粒选自经羧基修饰的磁微粒、经甲苯磺酰基修饰的磁微粒、经氨基修饰的磁微粒、经巯基修饰的磁微粒、经环氧基修饰的磁微粒和经羟基修饰的磁微粒中的至少一种。
具体地，经羧基修饰的磁微粒包括经亲水性羧基修饰的磁微粒和经疏水性羧基修饰的磁微粒。
经羧基、甲苯磺酰基、氨基、巯基、环氧基或羟基修饰活化后的磁微粒具有亲水性好、低吸附、本底低的优点，对抗体活性损伤小。
本实施方式中，磁微粒为经羧基修饰的磁微粒。
在一个实施方式中，磁微粒为磁珠，磁珠的粒径为1.0μm～3.0μm。
在一个实施方式中，化学发光标记物标记的第二抗NGAL单克隆抗体中，第二抗NGAL单克隆抗体与待测液中的NGAL结合后，化学发光标记物能够在底物液的作用下产生信号变化，根据信号的变化计算待测液中的NGAL的 含量。具体地，化学发光标记物为异鲁米诺、吖啶酯、吖啶磺酰胺、三联吡啶钌、碱性磷酸酶或辣根过氧化物酶。
在一个实施方式中，该急性肾损伤的检测试剂盒还包括发光底物，底物能够与化学发光标记物反应产生信号变化。底物液例如为鲁米诺钠盐发光液。
在一个实施方式中，该急性肾损伤的检测试剂盒还包括NGAL标准品，NGAL标准品用于制备标准曲线，从而计算待测样品中的NGAL含量。
在其他的实施例中，上述急性肾损伤的检测试剂盒也可以不包括底物液和NGAL标准品，使用者自行配制即可。
上述急性肾损伤的检测试剂盒基于NGAL(中性粒细胞明胶酶相关载脂蛋白)作为诊断检测的标志物进行检测，能够在急性肾损伤发生的早期快速检测诊断。第一抗NGAL单克隆抗体上包被磁微粒，第二抗NGAL单克隆抗体上标记化学发光标记物，且第一抗NGAL单克隆抗体和第二抗NGAL单克隆抗体分别针对不同的NGAL表位，避免NGAL上的表位被屏蔽。该急性肾损伤的检测试剂盒检测的相关性好可测定线性范围宽且敏度较高、特异性较高。
以下为具体实施例。
实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，通常按照常规条件，例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。
实施例1
该急性肾损伤的检测试剂盒中包括第一检测液、第二检测液、底物液以及NGAL标准品。其中第一检测液中含有浓度为0.5mg/mL磁微粒包被的第一抗NGAL单克隆抗体(第一抗NGAL单克隆抗体与磁微粒的质量比为0.05∶1)、浓度为50mmol/L的HEPES(4-羟乙基哌嗪乙磺酸)、质量分数为1％的BSA、质量分数为1％的氯化钠(NaCl)、质量分数为5％的蔗糖、质量分数为0.1％的吐温-20和质量分数为0.1％的PC-300，余量的超纯水，第一检测液 的pH值为7.0。第二检测液中含有浓度为1μg/mL的HRP标记物标记的第二抗NGAL单克隆抗体(HRP与第二抗NGAL单克隆抗体的摩尔比为1∶1)、浓度为50mmol/L的Tris-HCl、质量分数为1％的BSA、质量分数为1％的氯化钠(NaCl)、质量分数为5％的甘油、质量分数为0.1％的吐温-20和质量分数为0.1％的PC-300，余量的超纯水，第二检测液的pH值为8.0。第一抗NGAL单克隆抗体由杂交瘤细胞株NGAL-4F6(保藏号为CCTCC No：C2016216)分泌得到。第二抗NGAL单克隆抗体由杂交瘤细胞株NGAL-3B5(保藏号为CCTCC No：C2016214)分泌得到。底物液为鲁米诺钠盐发光液，NGAL标准品为人源的NGAL蛋白。
上述急性肾损伤的检测试剂盒的制备方法包括以下步骤：
一、杂交瘤细胞株的建立
1、NGAL免疫小鼠
将NGAL(NGAL-Ag1，菲鹏生物)稀释到1.0mg/mL，与弗氏完全佐剂(Sigma-Aldrich公司，货号：F5881)等体积混合，并充分乳化，得到油状乳液。将该乳液以0.2mL的剂量皮下施给BALB/c小鼠(广东省医学实验动物中心：广东省佛山市南海黄岐鄱阳路119号，6周龄雌性，5只)背部位点。第一次免疫14天后腹腔增强免疫，即等量抗原与弗氏不完全佐剂(Sigma-Aldrich公司，F5506)等体积混合，增强免疫到四针后，采尾血，分离血清，用间接ELISA法测定效价，效价高于1∶10000即可用于融合。
融合前3天，用相同剂量抗原与等体积0.9％氯化钠注射液混合腹腔注射追加免疫，免疫方法同上。
2、杂交瘤细胞系的制备
(1)饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜。用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT 的RPMI 1640完全培养液中)重悬，调整细胞浓度1×105个/mL，加入96孔板，150μL/孔，37℃，5％CO2培养过夜。
(2)免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
(3)骨髓瘤细胞的制备
小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)经8-氮鸟嘌呤筛选后，培养至对数生长期，取两大瓶制成细胞悬液，离心，弃上清，用RPMI 1640基础培养液重悬，如些重复三次，计数。
(4)细胞融合及HAT选择杂交瘤
将骨髓瘤细胞与免疫脾细胞按1∶10比例混合，在50mL塑胶离心管内用RPMI 1640基础培养液洗1次，1200rpm，离心8分钟。弃上清，将细胞混匀，缓慢加入1mL 50％的PEG1500融合，融合1分钟后加入15mL的RPMI 1640基础培养液终止细胞融合。1000rpm，离心5分钟。弃上清，用50mL的RPMI 1640筛选培养液轻轻混悬，平分于10块铺有饲养细胞的96孔板，50μL/孔，37℃，5％CO2培养。培养至第六天，换HT培养液(含HT的RPMI1640完全培养液)两次。
(5)抗体的检测
用0.06M pH9.6碳酸缓冲溶液稀释PK2-NGAL蛋白使其终浓度为2μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2 PBS，0.15mL/孔，37℃封闭2小时，用于检测。上述杂交瘤细胞重组融合后第七天，取细胞上清稀释不同倍数后取0.1mL于上述96孔检测板中，37℃30分钟，PBST洗五次后加入2000倍稀释的辣根过氧化酶标记的羊抗鼠IgG(菲鹏生物股份有限公司生产，货号GRCGAMS001)，37℃30分钟同上洗后，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃ 15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值。RPMI 1640完全培养液作为阴性对照，共检测有杂交瘤细胞的384孔，最终获得32株稳定分泌抗人NGAL的细胞株。细胞培养上清效价2.28×103以上。
3.单克隆抗体的制备
选6-8周健壮的BALB/c小鼠，每只小鼠腹腔注射0.5mL的降植烷；10天后腹腔注射1×106个杂交瘤细胞。接种细胞7～10天后可产生腹水，密切观察动物的健康状况与腹水征象，待腹水尽可能多，而小鼠频于死亡之前，处死小鼠，用滴管将腹水吸入试管中，一般一只小鼠可获5～10mL腹水。收集腹水，离心取上清，用3倍体积的PBS稀释后滤纸过滤。将所得的滤液在1mL/min的流速下加到一个已用PBS平衡的蛋白G亲和层析柱(GE公司)。然后用PBS以1mL/min的流速洗涤未被蛋白G吸附的物质直至在OD280nm下的吸附值达到基线为止。再用0.1M的甘氨酸洗脱液(pH2.5)洗脱并回收该抗体。所回收的抗体立即用0.1M Tris(pH8.8)中和，跑胶测浓度。上述可分泌抗人NGAL杂交瘤腹水抗体效价2.77×106以上。
4.单克隆抗体表位鉴定
用0.06M pH9.6碳酸缓冲溶液稀释纯化好的待鉴定单抗使其终浓度为1μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2 PBS，0.15mL/孔，37℃封闭2小时，加入2000倍稀释的辣根过氧化酶标记的NGAL表位鉴定抗原，37℃30分钟，PBST洗5次，拍干，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值，根据反应区分表位。筛选获得两株分别针对不同的NGAL表位的细胞株，其中一株可分泌第一抗NGAL单克隆抗体的杂交瘤细胞株NGAL-4F6，于2016年12月14日保藏在中国典型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016216，分类命名：杂交瘤细胞株NGAL-4F6。另一株可分泌第二抗NGAL单克隆抗体的杂交瘤细胞株NGAL-3B5，于2016年12月14日保藏在中国典 型培养物保藏中心(CCTCC)，地址：中国.武汉.武汉大学，保藏号为CCTCC No：C2016214，分类命名：杂交瘤细胞株NGAL-3B5。
二、制备磁微粒包被的第一抗NGAL单克隆抗体
1)将0.2mL的1mg/mL第一抗NGAL单克隆抗体(由杂交瘤细胞株NGAL-4F6分泌)装入透析袋，在活化缓冲液(15mmol/L的MES：2-(N-吗啡啉)乙磺酸)中透析至pH 6.0。
2)取10mg粒径为3.0μm的羧基化磁微粒(JSR MagnosphereTM MS300/Carboxyl)悬浮液，磁分离去上清，用清洗缓冲液(15mmol/LMES+0.1％Tween20，pH 6.0)洗涤4次，每次1mL，最后加800μL活化缓冲液(15mmol/L的MES)，超声分散。
3)分别称取1mg EDC，1mgNHS用0.1mL活化缓冲液(预冷)溶解至10mg/mL。将溶好的NHS和EDC先后加入清洗好并定容的磁珠中，混合比例为：800μL磁珠+100μLNHS+100μLEDC混合均匀后旋转孵育器上25度，20rmp反应30min。
4)活化好的磁珠用1mL清洗缓冲液清洗一次，加入1mL清洗缓冲液重悬。
5)将透析后的第一抗NGAL单克隆抗体加入清洗好并重悬的磁珠，混合均匀后旋转孵育器上4度，20rmp反应16h～24h，得到磁微粒包被的第一抗NGAL单克隆抗体。
6)将磁微粒包被的第一抗NGAL单克隆抗体用1mL清洗缓冲洗两次，洗的过程中超声重悬。
7)将洗好的磁微粒包被的第一抗NGAL单克隆抗体用1mL封闭液(清洗缓冲液+1％BSA)重悬，并超声，旋转孵育器上25度，20rmp反应2h。
8)将封闭完成的磁珠用1mL存储液(50mM Tris+1％BSA)重悬，终浓度10mg/mL固体含量，+2～+8℃保存备用。
三、制备化学发光标记物标记的第二抗NGAL单克隆抗体(采用采用NaIO4氧化法)
1)将0.5mL的2.0mg/mL的第二抗NGAL单克隆抗体(由杂交瘤细胞株NGAL-3B5分泌)装入透析袋，用20mmol/L CB缓冲液，pH＝9.51在4℃透析过夜。
2)氧化：8mg辣根过氧化物酶(HRP)溶解于0.4mL蒸馏水中，1∶1加入新鲜配置的20mg/mL NaIO4 4℃下避光搅拌20min。
3)终止氧化：取8微升乙二醇溶于40微升蒸馏水中，4℃下加入2)中搅拌30min，终止氧化反应。
4)抗体交联：将3)溶液加入1)中，装入透析袋，置0.025mol/L，pH9.51 CB缓冲液中，4℃透析28h。
5)还原：取出透析袋中液体，加入80微升5mg/mLNaBH4，4℃1h。
6)提纯：加40％饱和(NH4)2SO4(PH7.6)溶液，小牛血清20％沉淀结合物，4℃沉淀15min，12000r/min离心10min，弃去上清。
7)将沉淀物溶于1mL 0.02M PB(PH7.4)+20％小牛血清1mL复溶。
8)等体积加入甘油，-20℃保存备用。
四、制备第一检测液
将磁微粒包被的第一抗NGAL单克隆抗体溶解在缓冲液中得到第一检测液，其中第一检测液中包括0.5mg/mL的磁微粒包被的第一抗NGAL单克隆抗体、50mmol/L的HEPES(4-羟乙基哌嗪乙磺酸)、质量分数为1％的BSA、质量分数为1％的氯化钠(NaCl)、质量分数为5％的蔗糖、质量分数为0.1％的吐温-20和质量分数为0.1％的PC-300，余量的超纯水。第一检测液的pH值为7.0。
五、制备第二检测液
将化学发光标记物标记的第二抗NGAL单克隆抗体溶解在缓冲液中得到第二检测液，其中第二检测液中包括1μg/mL的化学发光标记物标记的第二抗NGAL单克隆抗体、50mmol/L的Tris-HCl、质量分数为1％的BSA、质量分数为1％的氯化钠(NaCl)、质量分数为5％的甘油、质量分数为0.1％的吐温-20和质量分数为0.1％的PC-300，余量的超纯水。第二检测液的pH值为8.0。
六、制备NGAL校准品
用定标缓冲液(40mmol/L Tris-HCl，0.5％BSA，1％Nacl，pH8.0)将NGAL(NGAL-Ag1，菲鹏生物)配置成浓度分别为1500ng/mL、300ng/mL、60ng/mL、12ng/mL、2.4ng/mL、0ng/mL，每瓶0.5mL分装冻干，4度保存备用。
测试例一
制定标准工作曲线
在反应容器中分别加入20μL NGAL校准品(浓度分别为1500ng/mL、300ng/mL、60ng/mL、12ng/mL、2.4ng/mL、0ng/mL)，然后加入50μL的第一检测液和50μL第二检测液，反应15min，用50mmol/L Tris+0.05％吐温-20，pH 7.5，洗5次。加入100μL发光底物(鲁米诺钠盐发光液)反应1min，用百克BK-L96C化学发光免疫分析仪读值。建立NGAL含量与发光值的对应关系曲线，结果如图1所)，图1中曲线上每个点代表一个含量的标准品，其中x轴表示NGAL的含量，y轴表示发光值。从图中可以看出，用实施例1的试剂盒检测NGAL，建立的NGAL的含量与发光值建立的标准曲线线性关系好、线性范围宽。
测试例二
分析灵敏度测定
用实施例1的试剂盒检测24次定标缓冲液(40mmol/L Tris-HCl，0.5％BSA，1％Nacl，pH8.0)，定标缓冲液中不含NGAL，以定标缓冲液的数据作为检测的基准线。按测试一的方法检测记录发光值，结果见表1。
表1：分析灵敏度实验数据
Figure PCTCN2017098890-appb-000001
Figure PCTCN2017098890-appb-000002
然后计算平均值MEAN和标准偏差SD，然后计算MEAN+2SD，利用校准曲线换算过来的浓度即试剂盒分析灵敏度为0.59ng/mL。说明实施例1的试剂盒检测灵敏度高。
测试例三
临床相关性检测
使用实施例1的试剂盒按测试一的方法检测102份临床尿液及22份阳性标本(急性肾损伤患者样本)，通过中生百克BK-L96C化学发光免疫分析仪读值，并带入标准曲线计算NGAL含量。结果显示102份临床随机尿样浓度基本都在2ng/mL～30ng/mL。检出的其中18份浓度在40ng/mL～130ng/mL。对18份样本用雅培尿中性粒细胞明胶酶相关脂质运载蛋白测定试剂盒(对照试剂盒，化学发光微粒子免疫检测法)再次检测，并与实施例1的试剂盒的检测结果进行相关性分析，结果如图2所示。同时对22份阳性标本也做了相关性分析，结果见图3。结果显示实施例1的试剂盒的检测结果与对照试剂盒的检测结果相关性很高。说明实施例1的试剂盒检测灵敏度高、特异性高。
测试例四
干扰性分析
采用实施例1的试剂盒对尿液标本中潜在干扰/交叉物质及不同pH值进行评估。调节尿液标本pH值或将以下浓度物质加入阴性标本中，以不处理的阴性尿液标本为对照，按测试例一的方法进行NGAL含量测定，结果如表2。
表2：实施例1的试剂盒对潜在干扰/交叉物质分析
潜在干扰/交叉物 浓度及pH 结果差异
丙酮 2mg/mL ＜5％
碳酸氢盐 20mg/mL ＜3％
胆红素 0.04mg/mL ＜3％
乙醇 4mg/mL ＜3％
葡萄糖 20mg/mL ＜3％
血红蛋白 0.2mg/mL ＜3％
微球蛋白 0.2mg/mL ＜3％
硫酸卡那霉素 0.06mg/mL ＜7％
低PH值 4.5 5％
高PH值 10 -8％
结果显示潜在干扰/交叉物几乎无交叉。实施例1的试剂盒抗干扰能力强，能够适应不同检测样本。
测试例五
将实施例1的急性肾损伤的检测试剂盒中的第一检测液和第二检测液进行37度3天、7天热破实验，选取校准品4个浓度点进行检测，结果如表3。
表3：实施例1试剂盒稳定性分析
校准品浓度(ng/mL) 6.25 25 100 400
4度保存 100％ 100％ 100％ 100％
37℃3天实测浓度下降百分比 2.10％ 1.65％ 3.56％ 5.42％
37℃7天实测浓度下降百分比 4.50％ 2.84％ 3.98％ 6.75％
从表3可以看出，热破后波动均在10％以内，稳定性合格。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (20)

  1. 一种急性肾损伤的检测试剂盒，其特征在于，包括第一检测液和第二检测液，所述第一检测液中含有磁微粒包被的第一抗NGAL单克隆抗体，所述第二检测液中含有化学发光标记物标记的第二抗NGAL单克隆抗体，其中，所述第一抗NGAL单克隆抗体和所述第二抗NGAL单克隆抗体分别针对不同的NGAL表位。
  2. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第一抗NGAL单克隆抗体由杂交瘤细胞株NGAL-4F6分泌得到，所述杂交瘤细胞株NGAL-4F6的保藏号为CCTCC NO：C2016216。
  3. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第二抗NGAL单克隆抗体由杂交瘤细胞株NGAL-3B5分泌得到，所述杂交瘤细胞株NGAL-3B5的保藏号为CCTCC NO：C2016214。
  4. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第一检测液中，所述磁微粒包被的第一抗NGAL单克隆抗体的浓度为0.1mg/mL～5mg/mL。
  5. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述磁微粒包被的第一抗NGAL单克隆抗体中，所述第一抗NGAL单克隆抗体与所述磁微粒的质量比为0.005～0.5∶1。
  6. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第二检测液中，所述化学发光标记物标记的第二抗NGAL单克隆抗体的浓度为0.05μg/mL～10μg/mL。
  7. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第一检测液中还包括浓度为10mmol/L～100mmol/L的HEPES、质量分数为0.1％～30％的稳定剂、质量分数为0.2％～2％的无机盐、质量分数为0.2％～20％的蔗糖、质量分数为0.01％～1％的表面活性剂和质量分数为0.01％～1％的抑菌剂。
  8. 根据权利要求7所述的急性肾损伤的检测试剂盒，其特征在于，所述 稳定剂选自牛血清蛋白、酪蛋白和明胶中的至少一种。
  9. 根据权利要求7所述的急性肾损伤的检测试剂盒，其特征在于，所述无机盐选自氯化钠、氯化钾、氯化镁和硫酸钾中的至少一种。
  10. 根据权利要求7所述的急性肾损伤的检测试剂盒，其特征在于，所述表面活性剂选自吐温-20和TritonX-100中的至少一种。
  11. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第一检测液的pH值为6.5～8.5。
  12. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第二检测液中还包括浓度为10mmol/L～100mmol/L的Tris-HCl、质量分数为0.1％～30％的稳定剂、质量分数为0.2％～2％的无机盐、质量分数为1％～30％的甘油、质量分数为0.01％～1％的表面活性剂和质量分数为0.01％～1％的抑菌剂。
  13. 根据权利要求12所述的急性肾损伤的检测试剂盒，其特征在于，所述稳定剂选自牛血清蛋白、酪蛋白和明胶中的至少一种。
  14. 根据权利要求12所述的急性肾损伤的检测试剂盒，其特征在于，所述无机盐选自氯化钠、氯化钾、氯化镁和硫酸钾中的至少一种。
  15. 根据权利要求12所述的急性肾损伤的检测试剂盒，其特征在于，所述表面活性剂选自吐温-20和TritonX-100中的至少一种。
  16. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述第二检测液的pH值为7.0～9.5。
  17. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述磁微粒包被的第一抗NGAL单克隆抗体中，所述磁微粒选自经羧基修饰的磁微粒、经甲苯磺酰基修饰的磁微粒、经氨基修饰的磁微粒、经巯基修饰的磁微粒、经环氧基修饰的磁微粒和经羟基修饰的磁微粒中的至少一种。
  18. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，所述化学发光标记物标记的第二抗NGAL单克隆抗体中，所述化学发光标记物为异鲁米诺、吖啶酯、吖啶磺酰胺、三联吡啶钌、碱性磷酸酶或辣根过氧化 物酶。
  19. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，还包括底物液，所述底物液能够与所述化学发光标记物反应产生信号变化。
  20. 根据权利要求1所述的急性肾损伤的检测试剂盒，其特征在于，还包括NGAL标准品。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102967714A (zh) * 2012-12-10 2013-03-13 天津市协和医药科技集团有限公司 中性粒细胞明胶酶相关脂质运载蛋白化学发光检测试剂盒
CN106645762A (zh) * 2016-12-27 2017-05-10 菲鹏生物股份有限公司 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒
CN107525938A (zh) * 2017-08-24 2017-12-29 菲鹏生物股份有限公司 急性肾损伤的检测试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102967714A (zh) * 2012-12-10 2013-03-13 天津市协和医药科技集团有限公司 中性粒细胞明胶酶相关脂质运载蛋白化学发光检测试剂盒
CN106645762A (zh) * 2016-12-27 2017-05-10 菲鹏生物股份有限公司 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒
CN107525938A (zh) * 2017-08-24 2017-12-29 菲鹏生物股份有限公司 急性肾损伤的检测试剂盒

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIPPI, GET ET AL., NEUTROPHIL GELATINASE ASSOCIATED LIPOCALIN (NGAL): ANALYTICAL ISSUES. LIGANDASSAY, vol. 18, no. 4, 31 January 2013 (2013-01-31) *
QI, JIALONG ET AL.: "Preparation of Monoclonal Antibodies Against Neutrophil Gelatinase-Associated lipocalin (NGAL) and Development of an Antibody-Based Chemiluminescence Immune Quantification Assay", CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS, vol. 35, no. 4, 31 December 2015 (2015-12-31) *
RUAN, Q. Q. ET AL.: "Supporting Immunoassay Design with Biophysical Tools", ANALYTICAL BIOCHEMISTRY, vol. 437, no. 1, 27 February 2013 (2013-02-27), XP028545919, DOI: 10.1016/j.ab.2013.02.012 *
RUAN, Q. Q.: "Supporting Immunoassay Design with Biophysical Tools", ANALYTICAL BIOCHEMISTRY, vol. 437, no. 1, 27 February 2013 (2013-02-27), XP028545919, DOI: 10.1016/j.ab.2013.02.012 *

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