CN112946296A - Immunity turbidimetry kit - Google Patents
Immunity turbidimetry kit Download PDFInfo
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- CN112946296A CN112946296A CN202110119596.7A CN202110119596A CN112946296A CN 112946296 A CN112946296 A CN 112946296A CN 202110119596 A CN202110119596 A CN 202110119596A CN 112946296 A CN112946296 A CN 112946296A
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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
- G01N33/68—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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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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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
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
The invention discloses an immunoturbidimetric kit, which comprises a kit body, and a reagent R1, a reagent R2 and a reagent R3 which are arranged in the kit body; the reagent R1 comprises the following components: 0.01-1 wt% of surfactant, 0.05-0.5 wt% of preservative, 150-1000 mmol/L of sodium chloride and 5-200 mmol/L of buffer solution; the reagent R2 comprises the following components: 0.1-0.5 wt% of polystyrene latex microspheres, 0.01-1 wt% of surfactant, 0.05-0.1 wt% of preservative and 5-200 mmol/L of buffer solution; the surface of the polystyrene latex microsphere is crosslinked with a monoclonal antibody of lipocalin related to the encapsulated neutrophil gelatinase, and the monoclonal antibody and the polystyrene latex microsphere are connected in a covalent crosslinking or physical adsorption mode; reagent R3 is a buffer containing neutrophil gelatinase-associated lipocalin. The immunoturbidimetric kit realizes the quantitative mass detection of the human serum neutrophil gelatinase-associated lipocalin on a biochemical analyzer, has simple operation, high sensitivity, difficult interference by human factors and good detection stability and repeatability.
Description
Technical Field
The invention relates to the technical field of immunoturbidimetry, in particular to an immunoturbidimetry kit for detecting neutrophil gelatinase-associated lipocalin.
Background
The enzyme immunoassay kit for rapidly detecting the neutrophil gelatinase-associated lipocalin can be used for detecting human urine, blood plasma and blood serum. Acute Renal Failure (ARF) is a common complication of cardiac surgery, nephrotoxicity, and kidney transplantation.
Neutrophil gelatinase-associated Lipocalin (NGAL), also known as Lipocalin-2 (Lipocalin-2), is a new member of the Lipocalin family and is a biomarker in the early stages of renal function impairment. NGAL is not only present in neutrophils but also occurs in specific epithelial cells, for example during ischemic and toxic kidney injury, NGAL in renal tubular epithelial cells will increase significantly, within the first two hours, NGAL levels in urine and blood will increase significantly, and therefore NGAL is a sensitive marker of early acute kidney injury.
NGAL level is higher in the process of acute renal function injury, and prognosis will progress to acute renal failure. Higher NGAL levels are generally the following: cardiovascular surgery patients, critically ill persons, septic or hemorrhagic shock, renal transplantation, response to intravenous X-ray contrast agents, and nephrotoxic treatment response. Over 50% of patients develop a degree of acute renal failure, often with a large post-disease period leading to a significant increase in mortality rates.
Prognosis of acute renal failure has not been improved, and significant attention is paid to the prognosis in the next forty years. Common diagnostic methods such as the determination of serum creatinine or cystatin c are only detectable after a deterioration in renal function, that is within a few days or even a day after injury. The NGAL protein is used for rapidly detecting the enzyme immunoassay kit, so that a doctor can appropriately treat the acute renal dysfunction within hours of injury.
A particle-enhanced turbidimetric immunoassay (PETIA) is a relatively stable and accurate method for detecting humoral protein homogeneous phase immunoturbidimetric assay in recent years, and the basic principle is that surface cross-linked monoclonal or polyclonal antibodies of latex particles with certain particle sizes are rapidly aggregated together in a short time after microspheres with cross-linked antibodies are combined with antigens, so that the absorbance of a reaction system is changed. The change has a certain correlation with the concentration of the antigen to be detected, and can reflect the concentration of the antigen to be detected in a certain range. The technology amplifies antigen-antibody reaction in a particle enhancement mode, overcomes the defect of insufficient sensitivity of a common transmission turbidimetry, inherits the advantages of good stability, convenience and rapidness of the transmission turbidimetry, overcomes the defects of a zha ELISA method and an RIA method, and is increasingly and widely applied to quantitative detection of various trace proteins in clinic. At present, the latex enhanced immunity transmission turbidimetry area is widely used for detecting cystatin C, lipoprotein a, C reactive protein and antistreptolysin 0, and good social benefits are obtained, but no such neutrophilic granulocyte gelatinase related lipocalin detection kit is available at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing an immune turbidimetric kit for detecting neutrophil gelatinase-associated lipocalin, which realizes the large-scale quantitative detection of human serum neutrophil gelatinase-associated lipocalin on a biochemical analyzer, and has the advantages of simple operation, high sensitivity, difficult interference by human factors, and good detection stability and repeatability.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides an immunoturbidimetric kit, which comprises a kit body, and a reagent R1, a reagent R2 and a reagent R3 which are arranged in the kit body;
the reagent R1 comprises the following components: 0.01-1 wt% of surfactant, 0.05-0.5 wt% of preservative, 150-1000 mmol/L of sodium chloride and 5-200 mmol/L of buffer solution;
the reagent R2 comprises the following components: 0.1-0.5 wt% of polystyrene latex microspheres, 0.01-1 wt% of surfactant, 0.05-0.1 wt% of preservative and 5-200 mmol/L of buffer solution; the surface of the polystyrene latex microsphere is crosslinked with a monoclonal antibody of lipocalin related to neutrophil gelatinase, and the monoclonal antibody and the polystyrene latex microsphere are connected in a covalent crosslinking or physical adsorption mode;
the reagent R3 is a buffer containing neutrophil gelatinase-associated lipocalin.
Further, the surfactant is Triton-X100, Triton-X405, Tween-20, Tween-40 or Tween-80.
Further, the reagent R2 also comprises 0.1-2 wt% of PEG, and the molecular weight of the PEG is 5000-20000. Preferably, the PEG is at least one of PEG8000, PEG6000, PEG4000, PEG2000, PEG 1500.
Further, the reagent R2 also comprises 0.1-3 g/L of trehalose. The addition of trehalose is favorable for improving the stability of the latex suspension and prolonging the service life. Preferably, the concentration of the trehalose is 0.5-1 g/L.
Further, the buffer solution is PBS buffer solution, carbonate buffer solution, Tris-HCl buffer solution, 3- (N-morpholinyl) propanesulfonate buffer solution or glycine buffer solution.
Further, the preservative is sodium azide and/or Proclin-300.
In one embodiment, the concentration of neutrophil gelatinase-associated lipocalin in the reagent R1 is 3.0 mg/L.
Further, the reagent R2 is prepared by the following method:
uniformly mixing the polystyrene latex microspheres and the lipocalin monoclonal antibody related to the coated neutrophil gelatinase in a buffer solution, and stirring for reaction at normal temperature overnight; then, cleaning with a cleaning storage solution to obtain a reagent R2, and storing at the temperature of 2-8 ℃;
wherein, the cleaning storage liquid comprises the following components: 0.01-1 wt% of surfactant, 0.05-0.1 wt% of preservative and 5-200 mmol/L of buffer solution.
Further, the preparation method of the reagent R1 comprises the following steps: dissolving a surfactant, a preservative, sodium chloride and a buffer in water to obtain the reagent R1.
Further, the preparation method of the reagent R3 comprises the following steps: the neutrophil gelatinase-associated lipocalin was diluted with a buffer solution to a concentration of 3.0mg/L to obtain reagent R1.
Furthermore, the particle size of the polystyrene latex microspheres is 0.1-0.3 μm, and the functional groups on the surfaces of the polystyrene latex microspheres are amino groups or carboxyl groups.
Further, the immunoturbidimetric kit is used for detecting neutrophil gelatinase-associated lipocalin.
Compared with the prior art, the invention has the beneficial effects that:
1. the immunoturbidimetric kit amplifies antigen-antibody reaction in a latex particle enhancing mode, overcomes the defect of insufficient sensitivity of a common transmission turbidimetric method, inherits the advantages of good stability, convenience and rapidness of the transmission turbidimetric method, overcomes the defects of ELISA and RIA methods, and realizes the large-scale quantitative detection of the human serum neutrophil gelatinase-associated lipocalin on a biochemical analyzer.
2. The immunoturbidimetric kit is simple to operate, high in sensitivity, not prone to interference of human factors, good in detection stability and repeatability, capable of truly reflecting the content of a detected object, capable of detecting by using a biochemical analyzer, easy to realize automation and suitable for clinical popularization and application.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.
Example 1
The embodiment provides an immunoturbidimetric kit which comprises a kit body and a reagent R1, a reagent R2 and a reagent R3 which are independent of each other and are positioned in the kit body. Wherein, the composition of the reagent R1 is as follows: Triton-X1000.2wt%, sodium azide 0.1 wt%, sodium chloride 200mmol/L, and PBS buffer 50 mmol/L. The composition of reagent R2 was: 0.5 wt% of polystyrene latex microspheres, 0.5 wt% of Triton-X1000.2wt%, 0.1 wt% of sodium azide and 100mmol/L of PBS buffer solution. The composition of reagent R3 was: neutrophil gelatinase-associated lipocalin 3.0mg/L, PBS buffer 100 mmol/L.
(1) Preparation of reagent R1:
dissolving Triton-X100, sodium azide, sodium chloride and PBS buffer solution in water according to the proportion to obtain a reagent R1.
(2) Preparation of reagent R2:
according to the volume ratio of 1:1, uniformly mixing polystyrene latex microspheres with the average diameter of 200nm and the lipocalin monoclonal antibody related to the coated neutrophil gelatinase in PBS (phosphate buffer solution), carrying out rotary reaction at 30-40 ℃ overnight, washing with a washing storage solution to obtain a reagent R2, and storing at 2-8 ℃. The cleaning storage solution comprises the following components: Triton-X1000.2%, sodium azide 0.05%, PBS buffer 30 mmol/L.
(3) Preparation of reagent R3:
the neutrophil gelatinase-associated lipocalin was diluted to a concentration of 3.0mg/L with PBS buffer to obtain reagent R3.
Example 2
The embodiment provides an immunoturbidimetric kit which comprises a kit body and a reagent R1, a reagent R2 and a reagent R3 which are independent of each other and are positioned in the kit body. Wherein, the composition of the reagent R1 is as follows: Triton-X4050.2wt%, Proclin-3000.1 wt%, sodium chloride 200mmol/L, and glycine buffer 50 mmol/L. The composition of reagent R2 was: 0.5 wt% of polystyrene latex microspheres, 50.2 wt% of Triton-X4050, 78 wt% of Proclin-3000.1, 100mmol/L of glycine buffer solution and 0.5g/L of trehalose. The composition of reagent R3 was: 3.0mg/L of neutrophil gelatinase-associated lipocalin and 100mmol/L of glycine buffer.
(1) Preparation of reagent R1:
dissolving Triton-X100, sodium azide, sodium chloride and PBS buffer solution in water according to the proportion to obtain a reagent R1.
(2) Preparation of reagent R2:
uniformly mixing polystyrene latex microspheres with the average diameter of 300nm and the lipocalin monoclonal antibody related to the coated neutrophil gelatinase in glycine buffer solution according to the volume ratio of 1:1, carrying out rotary reaction at 30-40 ℃ overnight, washing with a washing storage solution to obtain a reagent R2, and storing at 2-8 ℃. The cleaning storage solution comprises the following components: Triton-X4050.2%, Proclin-3000.05%, glycine buffer solution 30mmol/L, and trehalose 0.5 g/L.
(3) Preparation of reagent R3:
the neutrophil gelatinase-associated lipocalin was diluted to a concentration of 3.0mg/L with PBS buffer to obtain reagent R3.
Example 3
The embodiment provides an immunoturbidimetric kit which comprises a kit body and a reagent R1, a reagent R2 and a reagent R3 which are independent of each other and are positioned in the kit body. Wherein, the composition of the reagent R1 is as follows: tween-200.2 wt%, sodium azide 0.1 wt%, sodium chloride 200mmol/L, and Tris-HCl buffer 50 mmol/L. The composition of reagent R2 was: 0.5 wt% of polystyrene latex microspheres, 0.2 wt% of Tween-200.2 wt%, 0.1 wt% of sodium azide, 100mmol/L of Tris-HCl buffer solution and PEG 60001 wt%. The composition of reagent R3 was: 3.0mg/L of neutrophil gelatinase-associated lipocalin and 100mmol/L of carbonate buffer.
(1) Preparation of reagent R1:
dissolving Tween-20, sodium azide, sodium chloride and Tris-HCl buffer solution in water according to a ratio to obtain a reagent R1.
(2) Preparation of reagent R2:
according to the volume ratio of 1:1, uniformly mixing polystyrene latex microspheres with the average diameter of 200nm and the lipocalin monoclonal antibody related to the coated neutrophil gelatinase in a Tris-HCl buffer solution, carrying out rotary reaction at 30-40 ℃ overnight, washing with a washing storage solution to obtain a reagent R2, and storing at 2-8 ℃. The cleaning storage solution comprises the following components: tween-200.2%, sodium azide 0.05% and Tris-HCl buffer solution 30 mmol/L.
(3) Preparation of reagent R3:
the neutrophil gelatinase-associated lipocalin was diluted to a concentration of 3.0mg/L with a carbonate buffer solution to obtain reagent R3.
Performance testing
The reagents prepared in the 3 examples are tested by a vertical 7080 biochemical analyzer, the testing wavelength is 570nm, a sample or a calibrator is taken for 3uL, then 200uL of reagent R1 is added, the temperature is kept at 37 ℃ for 5min, then 50uL of reagent R2 is added, the absorbance A1 is read after 20s, the absorbance A2 is read after 5min of incubation at 37 ℃, and then the reaction absorbance delta A is 2-A1; and (3) performing multi-point calibration by using a standard substance, automatically completing calibration by using an instrument, and detecting the conventional neutrophil gelatinase-associated lipocalin after the calibration is OK. The 3 examples were tested for accuracy, precision, stability, etc. The results obtained are shown in table 1:
TABLE 1 test results of immunoturbidimetric kits of examples 1-3
| Example 1 | Example 2 | Example 3 | |
| Rate of accuracy | 99.45％ | 99.68％ | 99.53％ |
| Precision degree | 1.91％ | 1.46％ | 1.76％ |
| Stability of | 18 months old | 18 months old | 18 months old |
In conclusion, the immunoturbidimetric kit amplifies antigen-antibody reaction in a latex particle enhancing mode, overcomes the defect of insufficient sensitivity of the common transmission turbidimetric method, inherits the advantages of good stability, convenience and rapidness of the transmission turbidimetric method, overcomes the defects of ELISA and RIA methods, and realizes the large-scale quantitative detection of the human serum neutrophil gelatinase-associated lipocalin on a biochemical analyzer. The method has the advantages of simple operation, high sensitivity, no easy interference of human factors, good detection stability and repeatability, capability of truly reflecting the content of the detected object, capability of detecting by using a biochemical analyzer, easy realization of automation and suitability for clinical popularization and application.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. An immunoturbidimetric kit, which is characterized by comprising a kit body, and a reagent R1, a reagent R2 and a reagent R3 which are arranged in the kit body;
the reagent R1 comprises the following components: 0.01-1 wt% of surfactant, 0.05-0.5 wt% of preservative, 150-1000 mmol/L of sodium chloride and 5-200 mmol/L of buffer solution;
the reagent R2 comprises the following components: 0.1-0.5 wt% of polystyrene latex microspheres, 0.01-1 wt% of surfactant, 0.05-0.1 wt% of preservative and 5-200 mmol/L of buffer solution; the surface of the polystyrene latex microsphere is crosslinked with a monoclonal antibody of lipocalin related to neutrophil gelatinase, and the monoclonal antibody and the polystyrene latex microsphere are connected in a covalent crosslinking or physical adsorption mode;
the reagent R3 is a buffer containing neutrophil gelatinase-associated lipocalin.
2. The immunoturbidimetric kit of claim 1, wherein the surfactant is Triton-X100, Triton-X405, Tween-20, Tween-40 or Tween-80.
3. The immunoturbidimetric kit of claim 1, wherein the buffer is PBS buffer, carbonate buffer, Tris-HCl buffer, 3- (N-morpholino) propanesulfonate buffer, or glycine buffer.
4. An immunoturbidimetric kit according to claim 1, wherein the preservative is sodium azide and/or Proclin-300.
5. The immunoturbidimetric kit of claim 1, wherein the reagent R2 further comprises 0.1-2 wt% PEG, wherein the PEG has a molecular weight of 5000-20000.
6. The immunoturbidimetric kit of claim 1, wherein the reagent R2 further comprises trehalose at a concentration of 0.1-3 g/L.
7. The immunoturbidimetric kit of claim 1, wherein the concentration of neutrophil gelatinase-associated lipocalin in the reagent R3 is 3.0 mg/L.
8. The immunoturbidimetric kit of claim 1, wherein the reagent R2 is prepared by the following method:
uniformly mixing the polystyrene latex microspheres and the lipocalin monoclonal antibody related to the coated neutrophil gelatinase in a buffer solution, and stirring for reaction at normal temperature overnight; then, cleaning with a cleaning storage solution to obtain a reagent R2, and storing at the temperature of 2-8 ℃;
wherein, the cleaning storage liquid comprises the following components: 0.01-1 wt% of surfactant, 0.05-0.1 wt% of preservative and 5-200 mmol/L of buffer solution.
9. The immunoturbidimetric kit of claim 8, wherein the polystyrene latex microspheres have a particle size of 0.1-0.3 μm, and the functional groups on the surface thereof are amino groups or carboxyl groups.
10. The immunoturbidimetric kit of any one of claims 1-9, for detecting neutrophil gelatinase-associated lipocalin.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607459A (en) * | 2023-11-28 | 2024-02-27 | 中拓生物有限公司 | Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof |
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| CN103995128A (en) * | 2014-05-08 | 2014-08-20 | 北京玖佳宜科技有限公司 | Neutrophil gelatinase-associated lipocalin detection kit and preparation |
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2021
- 2021-01-28 CN CN202110119596.7A patent/CN112946296A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102662064A (en) * | 2012-04-26 | 2012-09-12 | 苏州照康生物技术有限公司 | Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607459A (en) * | 2023-11-28 | 2024-02-27 | 中拓生物有限公司 | Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof |
| CN117607459B (en) * | 2023-11-28 | 2024-04-30 | 中拓生物有限公司 | Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof |
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