CN117607459B - Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof - Google Patents
Neutrophil gelatinase-associated lipocalin assay kit, and preparation method and application thereof Download PDFInfo
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- 102000013519 Lipocalin-2 Human genes 0.000 title claims abstract description 41
- 108010051335 Lipocalin-2 Proteins 0.000 title claims abstract description 41
- 238000003149 assay kit Methods 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 97
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000004816 latex Substances 0.000 claims abstract description 18
- 229920000126 latex Polymers 0.000 claims abstract description 18
- 239000004005 microsphere Substances 0.000 claims abstract description 15
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims abstract 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract 2
- 239000008118 PEG 6000 Substances 0.000 claims abstract 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims abstract 2
- 239000000872 buffer Substances 0.000 claims abstract 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 14
- 102000019298 Lipocalin Human genes 0.000 claims description 12
- 108050006654 Lipocalin Proteins 0.000 claims description 12
- 210000000440 neutrophil Anatomy 0.000 claims description 12
- 102000013382 Gelatinases Human genes 0.000 claims description 11
- 108010026132 Gelatinases Proteins 0.000 claims description 11
- SBKDIDITONHJHI-UHFFFAOYSA-N 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCCOCCOCCOCCO SBKDIDITONHJHI-UHFFFAOYSA-N 0.000 claims description 10
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 10
- -1 diethylene glycol fatty acid ester Chemical class 0.000 claims description 10
- 229930195729 fatty acid Natural products 0.000 claims description 10
- 239000000194 fatty acid Substances 0.000 claims description 10
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 5
- 239000004793 Polystyrene Substances 0.000 claims description 4
- 229920002223 polystyrene Polymers 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims 1
- 238000007710 freezing Methods 0.000 abstract description 10
- 108010004103 Chylomicrons Proteins 0.000 abstract description 8
- 230000008014 freezing Effects 0.000 abstract description 7
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- 239000004094 surface-active agent Substances 0.000 abstract description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 abstract description 2
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- 230000000052 comparative effect Effects 0.000 description 35
- 238000012360 testing method Methods 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
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- 208000007342 Diabetic Nephropathies Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 238000004737 colorimetric analysis Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 208000033679 diabetic kidney disease Diseases 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
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- 108010010234 HDL Lipoproteins Proteins 0.000 description 1
- 102000015779 HDL Lipoproteins Human genes 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 208000010159 IgA glomerulonephritis Diseases 0.000 description 1
- 206010021263 IgA nephropathy Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 239000007798 antifreeze agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
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- 201000001421 hyperglycemia Diseases 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 208000030761 polycystic kidney disease Diseases 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 description 1
- 229960005294 triamcinolone Drugs 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N33/531—Production of immunochemical test materials
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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
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a neutrophil gelatinase-associated lipocalin (NGAL) assay kit, which comprises a reagent R1 and a reagent R2; the reagent R1 contains the following components: tris-hydrochloric acid buffer, naCl, sodium bicarbonate, PEG6000, surfactant and preservative. Reagent R2:2- (N-morpholino) ethanesulfonic acid buffer, rabbit anti-human NGAL antibody coated latex microsphere, antifreeze and preservative. The invention also provides a preparation method and application of the kit, the kit can effectively avoid interference of chylomicron, and simultaneously adopts a novel antifreezing agent combination, so that the freezing resistance of the reagent can be effectively improved, and the kit is a liquid kit with strong stability, high sensitivity, good linearity and low cost.
Description
Technical Field
The invention relates to the technical field of biochemical reagent measurement, in particular to a neutrophil gelatinase related lipocalin measurement kit, and also relates to a preparation method and application of the neutrophil gelatinase related lipocalin measurement kit.
Background
Neutrophil gelatinase-associated lipocalin (NGAL) is one of lipocalins, initially a small molecular weight secreted protein found in activated neutrophils, and modern research has shown that NGAL is one of the most effective biological markers for diagnosing acute kidney injury, and also one of the effective markers for early diabetic nephropathy.
1. Early marker for acute kidney disease
Experiments show that the NGAL can be detected in blood and urine in the very early stage (within 2 h) of ischemic kidney occurrence, and the injection of recombinant NGAL into an ischemia-reperfusion model mouse can reduce azotemia and reduce kidney injury. Human body researches also find that the blood and urine NGAL of patients after heart operation are obviously increased within 2 hours of the occurrence of the acute kidney disease, and accordingly, researchers speculate that the NGAL can be used as an early marker of the acute kidney disease after heart operation.
2. Early marker for chronic kidney disease
Along with the research on pathological types such as polycystic kidney disease, igA nephropathy, primary glomerulonephritis and the like, the urine NGAL is found to evaluate the severity and curative effect of the disease, can predict the occurrence of early chronic kidney disease and accurately predict the severity of the disease, and is possible to be used as an early marker of the chronic kidney disease.
3. Early marker for diabetic nephropathy
In human body detection, NGAL is positively correlated with obesity, hyperlipidemia, hyperglycemia, insulin resistance, and negatively correlated with high density lipoprotein levels. Urinary NGAL is presumed to be useful for renal disease in which renal tubular resorption is impaired, and the extent of injury and therapeutic effect can also be assessed.
The current methods for detecting the neutrophil gelatinase-associated lipocalin include latex immunoturbidimetry, double-antibody sandwich Immunochromatography (ILMA) and colloidal gold colorimetry. The double-antibody sandwich immunochemistry method (ILMA) has high accuracy and sensitivity, but the instrument and the equipment are expensive, the reagent is inconvenient to store and the price is high; the colloidal gold colorimetric method is simple to operate and quick in result, but only can be qualitatively researched and cannot be quantified. The latex immunoturbidimetry has the advantages of high specificity, simple and quick operation, accuracy and safety, automatic analysis and lower cost, but the Chinese production kit in the prior art has the defects of low analysis sensitivity, poor stability, incapability of resisting freezing, easiness in being influenced by Chylomicron (CM) and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a neutrophil gelatinase-associated lipocalin assay kit, a preparation method and application thereof, and the kit is a liquid kit with strong stability, high sensitivity, good linearity and low cost.
The invention is realized by the following technical scheme:
a neutrophil gelatinase-associated lipocalin assay kit comprising reagent R1 and reagent R2;
The reagent R1 contains the following components:
the reagent R2 contains the following components:
Wherein the percentages are by volume.
Preferably, the reagent R1 has a pH of 7.5-8.0.
Preferably, the reagent R2 has a pH of 6.5-7.5.
Preferably, the surfactant in the reagent R1 and the reagent R2 is selected from one or more of Tween-20, triton x-100, tetraethylene glycol monostearate and diethylene glycol fatty acid ester. More preferably, the surfactant is tween-20, tetraethylene glycol monostearate, diethylene glycol fatty acid ester.
Preferably, the antifreezing agent in the reagent R2 is one or more of glycerol, ethanol, tetraethylene glycol monostearate and triethanolamine. More preferably, the antifreeze agent consists of tetraethylene glycol monostearate and triethanolamine.
Preferably, the preservative in the reagents R1 and R2 is one or more of sodium azide, proclin300, MIT and sodium benzoate. More preferably, the preservative is proclin300.
Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5:1. More preferably, the volume ratio of reagent R1 to reagent R2 is 4:1.
The preparation method of the neutrophil gelatinase-associated lipocalin assay kit comprises the following steps: the preparation method of the rabbit anti-human neutrophil gelatinase related lipocalin antibody coated latex microsphere comprises the following steps: taking a proper amount of polystyrene latex microspheres with carboxylated surfaces, and adding the polystyrene latex microspheres into 10ml of buffer solution to make the final concentration of the latex microspheres be 1.0%; then adding a proper amount of rabbit anti-human neutrophil gelatinase related lipocalin antibody, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), mixing and stirring for about 3 hours at room temperature, adding 1ml of 10g/L BSA solution, centrifuging at 12000rpm for 40 minutes, and removing the supernatant to obtain a precipitate, namely the rabbit anti-human neutrophil gelatinase related lipocalin antibody coated latex microsphere. And adding other substances according to a proportion for dissolution to prepare the neutrophil gelatinase related lipocalin assay kit.
The invention also discloses application of the neutrophil gelatinase-associated lipocalin assay kit for determining concentration of the neutrophil gelatinase-associated lipocalin in serum for non-disease diagnosis and treatment purposes.
The kit adopts a latex immunoturbidimetry, the reaction principle is that NGAL in a sample can be subjected to agglutination reaction with anti-NGAL antibodies adsorbed on latex microspheres to generate antigen antibodies, immune complexes are formed, and the content of NGAL in the sample can be calculated by measuring the change of absorbance of the immune complexes. The reaction has specificity, uses the full-automatic biochemical analyzer to detect, has simple operation and high automation degree, can reduce human errors, and is suitable for most clinical laboratories.
Advantageous effects
1) The stable neutrophil gelatinase related lipocalin assay kit is a liquid double reagent, does not need to be prepared by re-dissolution, and can be directly used after opening a bottle.
2) The triethanolamine and the tetraethylene glycol monostearate are added into the reagent R2 to form the composite antifreezing agent, the triethanolamine not only has the function of sealing the latex microspheres, but also can improve the dispersion capacity of the latex microspheres coated by the antibody with the tetraethylene glycol monostearate, effectively improve the freezing resistance of the reagent, facilitate the transportation of the reagent at low temperature, and facilitate the further popularization of the reagent in the market.
3) The sodium bicarbonate and the diethylene glycol fatty acid ester act together to form a polymer with chylomicrons, so that the chylomicrons can be effectively dispersed and masked, interference is avoided, the anti-interference capability of the reagent is greatly enhanced, and the stability of the reagent is improved to a certain extent.
4) The reagent has excellent performance indexes such as accuracy, repeatability, analysis sensitivity, linear range, stability and the like, is low in price and convenient to use, and is favorable for further popularization in the market.
Drawings
FIG. 1 is a correlation plot of the accuracy of the example 1 reagent versus the comparative example 1 reagent;
FIG. 2 is a linear correlation curve of the reagent of example 1;
FIG. 3 shows the concentration changes of the neutrophil gelatinase-associated lipocalin assay reagents provided in example 1 and comparative examples 1, 5, 6, 7 for stability testing.
Detailed Description
The invention is further illustrated by the following examples:
the kit of this embodiment, when in use, adopts a full-automatic biochemical analyzer of micrui 800 with dual reagent function, and uses an endpoint method to measure, and the main wavelength is 546nm, and the operation is as follows:
Adding 10 μl of physiological saline, sample or calibrator, adding 200 μl of R1 reagent, pre-incubating for 5min, adding 50 μl of R2 reagent, mixing, delaying for 1min, reading absorbance A1, reading absorbance A2 after 5min,
Calculate Δa= (A2-A1).
Neutrophil gelatinase-associated lipocalin content (ng/mL) = (Δa sample +.Δa calibrator) ×calibrator concentration.
Sample requirements:
1. Serum is not hemolyzed.
2. Sample stability: the specimen can be stored stably for 3 days at 2-8 ℃ and for 2 weeks at-20 ℃.
Example 1
A conventional neutrophil gelatinase-associated lipocalin assay kit comprising reagent R1 and reagent R2.
The reagent R1 contains the following components:
the pH of reagent R1 was 7.6.
The reagent R2 contains the following components:
the pH of reagent R2 was 7.0.
Wherein the percentages are by volume.
Comparative example 1
Commercial imported neutrophil gelatinase-associated lipocalin assay kits.
Comparative example 2
The difference from the neutrophil gelatinase-associated lipocalin assay kit of example 1 is only that the reagent R1 does not contain sodium bicarbonate, otherwise the same as in example 1.
Comparative example 3
The difference from the neutrophil gelatinase-associated lipocalin assay kit of example 1 is that the reagent R1 does not contain diethylene glycol fatty acid ester, and the other is the same as in example 1.
Comparative example 4
The difference from the kit for measuring neutrophil gelatinase-associated lipocalin in example 1 was that the reagent R1 did not contain sodium hydrogencarbonate or diethylene glycol fatty acid ester, and was replaced with the same mass of triamcinolone X-100, and the other was the same as in example 1.
Comparative example 5
The difference from the neutrophil gelatinase-associated lipocalin assay kit of example 1 is only that the reagent R2 does not contain triethanolamine, otherwise the same as in example 1.
Comparative example 6
The difference from the neutrophil gelatinase-associated lipocalin assay kit of example 1 is that the reagent R2 does not contain tetraethylene glycol monostearate, and the other is the same as in example 1.
Comparative example 7
The difference from the kit for measuring neutrophil gelatinase-associated lipocalin in example 1 was that the reagent R1 did not contain sodium hydrogencarbonate or diethylene glycol fatty acid ester, but was replaced with glycerol of the same mass, and the other was the same as in example 1.
Performance verification
Test one
Accuracy comparison test: correlation experiments: the experimental scheme is as follows: the reagent of the example 1 and the reagent of the comparative example 1 detect 20 clinical serum samples simultaneously, perform correlation analysis on two groups of detection results, and calculate a correlation coefficient r; the relative deviation (r) of 20 pairs of data was calculated using the results of the test of the reagent of comparative example 1 as a control value. It is required that r is not less than 0.99 and the relative deviation is not more than + -10%. The results of the detection are shown in Table 1, and correlation curves (shown in FIG. 1) of the example 1 reagent and the comparative example 1 reagent were obtained.
TABLE 1 correlation vs. experimental results
TABLE 2 correlation coefficients of the comparative example 1 reagents and the example 1 reagents, respectively
As can be seen from Table 1 and FIG. 1, the maximum value of the serum test deviation of the kit of the example 1 reagent and the comparative example 1 reagent is 2.68%, the correlation coefficient of the two reagents is more than 0.99, and the detection results of the example 1 reagent and the comparative example 1 reagent are very close, so that the reagent of the example 1 provided by the invention has good correlation with the imported reagent, can completely replace the imported reagent, and meets clinical requirements.
Test II
Linear experiments: taking a high-value sample of the neutrophil gelatinase-associated lipocalin at 5000ng/mL, diluting, preparing 6 samples with different concentrations, and sequentially taking samples with the concentrations of 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL and 0ng/mL, detecting by using the reagent of the embodiment 1, measuring each sample with each concentration level three times, and taking the average value of each sample. The detection results are shown in the table:
table 3 test data sheet for linear correlation verification experiments
| Theoretical concentration (ng/mL) | EXAMPLE 1ng/mL |
| 0 | 0.0 |
| 312.5 | 313.4 |
| 625 | 628.1 |
| 1250 | 1256.4 |
| 2500 | 2508.3 |
| 5000 | 5009.2 |
| Correlation coefficient R 2 | 1 |
As can be seen from Table 3 and FIG. 2, the linear correlation coefficient of example 1 of the present invention varies linearly with the dilution concentration, and reaches 1, more than 0.990, which meets the standard requirements. The linear range of example 1 is illustrated to be preferred. Example 1 the linear range is entirely alternative to the inlet reagent. The better linear variation of the example 1 reagent is more able to meet the requirements of clinical case specimens.
Test three
Interference experiment: taking a quality control substance with traceability, and diluting a sample with the median quality control substance (target value of 600.0+/-40.0 ng/mL) and simultaneously releasing 100.0ng/mL, and respectively adding the sample into the chylomicron content in the table. Then, the reagent of example 1, the reagent of comparative example 2, the reagent of comparative example 3 and the reagent of comparative example 4 are used for simultaneously detecting the content of the neutrophil gelatinase-associated lipocalin in the sample, and the measurement results of each group are shown in the table 3-1 and the table 3-2 after the average value is obtained for 5 times.
TABLE 3-1 target 600ng/mL results of anti-interference verification of reagents
TABLE 3-2 target 100ng/mL results of reagent anti-interference verification
As can be seen from tables 3-1,3-2, the reagent of example 1 was not significantly disturbed, and the reagent of comparative example 1, the reagent of comparative example 2, the reagent of comparative example 3, and the reagent of comparative example 4 were significantly disturbed, both at low and high values of detection, when chylomicrons were 2ml/L and 4 ml/L. Example 1 the example 1 reagent comprising the two components has a greater interference resistance than the comparative example 2 reagent, the comparative example 3 reagent. The results show that the anti-interference performance of the reagent of the example 1 is obviously improved by the synergistic effect of the sodium bicarbonate and the diethylene glycol fatty acid ester after the sodium bicarbonate and the diethylene glycol fatty acid ester are added, the reagent is superior to the reagent of the comparative example 1, and the clinical requirements are met.
Test four
Stability experiment: the stability test was performed on the neutrophil gelatinase-associated lipocalin assay reagents provided in example 1 and comparative example 1, comparative example 5, comparative example 6, and comparative example 7, with the following test protocol: the reagents of example 1 and comparative example 1, comparative example 5, comparative example 6 and comparative example 7 were stored in a refrigerator at-20℃and frozen for 24 hours each time, and the quality control product having a target value of 600.+ -. 60ng/mL was detected seven times, and each group was tested 5 times, averaged, and the change in the measured value of the quality control product was monitored, and the results are shown in Table 4.
TABLE 4 data for verifying the freeze stability of reagents
As can be seen from table 4 and fig. 3, the reagent in example 1 provided by the invention has no change in 5 times of repeated freezing, and has better anti-freezing effect; the reagents of comparative examples 1, 5, 6 and 7 all changed significantly after freezing. The freezing resistance of the kit of the example 1 is better than that of the kits of the comparative examples 1, 5, 6 and 7, which shows that the buffer solution of the invention and the addition of triethanolamine and tetraethylene glycol monostearate cooperate to obviously improve the freezing resistance of the kit for measuring the neutrophil gelatinase-associated lipocalin and obviously improve the performance of the kit.
In conclusion, the kit is added with sodium bicarbonate and diethylene glycol fatty acid ester, so that interference of chylomicron is reduced better, and triethanolamine and tetraethylene glycol monostearate effectively improve the anti-freezing performance of the reagent, so that the kit is a liquid kit with strong anti-freezing performance, strong stability, high sensitivity, good linearity and low cost. Provides a good development space for the kit and enhances the competitive power of the kit in the market.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the invention pertains or may be substituted in a similar manner without departing from the spirit of the invention or beyond the scope of the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (3)
1. A neutrophil gelatinase-associated lipocalin assay kit, characterized in that the kit comprises a reagent R1 and a reagent R2;
The reagent R1 contains the following components:
50mmol/L of tris-hydrochloric acid buffer;
NaCl 9g/L；
sodium bicarbonate 0.2mmol/L;
PEG6000 15g/L；
0.2% of diethylene glycol fatty acid ester;
proclin300 1ml/L；
The pH value of the reagent R1 is 7.6;
the reagent R2 contains the following components:
50mmol/L of 2- (N-morpholino) ethanesulfonic acid buffer;
the rabbit anti-human NGAL antibody coats 30mg/L of the latex microsphere;
10ml/L triethanolamine;
1.5% of tetraethylene glycol monostearate;
tween-20.1%;
proclin300 1m/L；
The pH value of the reagent R2 is 7.0;
Wherein the percentages are by volume.
2. The neutrophil gelatinase-associated lipocalin assay kit of claim 1, wherein the volume ratio of reagent R1 to reagent R2 is 4:1.
3. A method of preparing a neutrophil gelatinase-associated lipocalin assay kit according to claim 1, characterized in that: the method comprises the following steps: firstly, preparing rabbit anti-human neutrophil gelatinase related lipocalin antibody coated latex microspheres, wherein the preparation method comprises the following steps: taking a proper amount of polystyrene latex microspheres with carboxylated surfaces, and adding the polystyrene latex microspheres into 10ml of buffer solution to make the final concentration of the latex microspheres be 1.0%; then adding a proper amount of rabbit anti-human neutrophil gelatinase related lipocalin antibody, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, mixing and stirring for 3 hours at room temperature, adding 1ml of 10g/L BSA solution, centrifuging for 40 minutes with 12000 rpm, and removing the supernatant to obtain a precipitate, namely the rabbit anti-human neutrophil gelatinase related lipocalin antibody coated latex microsphere; and adding other substances according to a proportion for dissolution to prepare the neutrophil gelatinase related lipocalin assay kit.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680698A (en) * | 2011-07-29 | 2012-09-19 | 南京诺尔曼生物技术有限公司 | Neutrophil gelatinase-associated lipocalin (NGAL) assay kit (latex-enhanced immunoturbidimetry) |
| CN106093422A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring neutrophil gelatinase-associated lipocalin NGAL |
| CN107918021A (en) * | 2017-11-17 | 2018-04-17 | 南通伊仕生物技术股份有限公司 | Neutrophil gelatinase-associated lipocalin detection kit |
| CN109061182A (en) * | 2018-08-03 | 2018-12-21 | 广州市伊川生物科技有限公司 | A kind of cysteine protease inhibitor C test kit and its detection method |
| CN111337691A (en) * | 2020-03-20 | 2020-06-26 | 中拓生物有限公司 | Sensitive and stable serum procalcitonin determination kit and preparation method and application thereof |
| CN111912990A (en) * | 2020-08-12 | 2020-11-10 | 山东博科生物产业有限公司 | Neutrophil gelatinase-associated lipocalin detection kit |
| CN112946296A (en) * | 2021-01-28 | 2021-06-11 | 苏州为度生物技术有限公司 | Immunity turbidimetry kit |
| CN113406323A (en) * | 2021-06-18 | 2021-09-17 | 上海奥普生物医药股份有限公司 | Latex immunoturbidimetry second reagent, composition containing same and preparation method thereof |
| CN116577513A (en) * | 2023-06-05 | 2023-08-11 | 中元汇吉生物技术股份有限公司 | Long-term stable immune turbidimetry reagent and preparation method thereof |
-
2023
- 2023-11-28 CN CN202311601142.9A patent/CN117607459B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680698A (en) * | 2011-07-29 | 2012-09-19 | 南京诺尔曼生物技术有限公司 | Neutrophil gelatinase-associated lipocalin (NGAL) assay kit (latex-enhanced immunoturbidimetry) |
| CN106093422A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring neutrophil gelatinase-associated lipocalin NGAL |
| CN107918021A (en) * | 2017-11-17 | 2018-04-17 | 南通伊仕生物技术股份有限公司 | Neutrophil gelatinase-associated lipocalin detection kit |
| CN109061182A (en) * | 2018-08-03 | 2018-12-21 | 广州市伊川生物科技有限公司 | A kind of cysteine protease inhibitor C test kit and its detection method |
| CN111337691A (en) * | 2020-03-20 | 2020-06-26 | 中拓生物有限公司 | Sensitive and stable serum procalcitonin determination kit and preparation method and application thereof |
| CN111912990A (en) * | 2020-08-12 | 2020-11-10 | 山东博科生物产业有限公司 | Neutrophil gelatinase-associated lipocalin detection kit |
| CN112946296A (en) * | 2021-01-28 | 2021-06-11 | 苏州为度生物技术有限公司 | Immunity turbidimetry kit |
| CN113406323A (en) * | 2021-06-18 | 2021-09-17 | 上海奥普生物医药股份有限公司 | Latex immunoturbidimetry second reagent, composition containing same and preparation method thereof |
| CN116577513A (en) * | 2023-06-05 | 2023-08-11 | 中元汇吉生物技术股份有限公司 | Long-term stable immune turbidimetry reagent and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 中性粒细胞明胶酶相关脂质运载蛋白检测试剂盒（胶乳免疫比浊法）检测性能验证;蔡爱华;李启平;陈丽娟;李静沛;李伟华;;中国食品药品监管;20191215(第12期);第54-57页 * |
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