CN109738626B - NGAL latex immunoturbidimetry detection kit and preparation method thereof - Google Patents
NGAL latex immunoturbidimetry detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a NGAL latex immunoturbidimetry detection kit and a preparation method thereof, the kit consists of a reagent R1, a reagent R2, a calibrator and a quality control product, the reagent R1 comprises a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative, and the reagent R2 comprises a latex microsphere connected with an NGAL antibody, a buffer solution, a stabilizer and a preservative. Compared with the prior art, the anti-interference agent is used in the R1 reagent, so that the anti-interference capability of the reagent is obviously improved, the applicable population of the neutrophil gelatinase-associated lipocalin assay kit is expanded, meanwhile, the improved kit detection is high in sensitivity, strong in specificity and good in kit stability, the NGAL content in urine, plasma and serum can be efficiently detected, and the detection result is well related to the imported reagent.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a preparation method of a kit for detecting neutrophil gelatinase-associated lipocalin by a latex immunoturbidimetry, which has high sensitivity and strong specificity.
Background
Human neutrophil gelatinase-associated lipocalin (NGAL) has a molecular weight of about 25kDa and is covalently bound to neutrophil gelatinase. NGAL is a growth factor in physiological state, and mainly participates in the generation and growth of early renal epithelium. The expression level is low in normal tissues including epithelial tissues of kidney, lung, stomach and colon. The NGAL level is higher in the process of acute renal failure injury, and can be directly detected from urine and blood, and the prognosis will develop into acute renal failure. Acute Kidney Injury (AKI) is a disease with high morbidity and mortality, as the pathogenesis of the disease is unknown and there is a lack of accurate and reliable early diagnosis markers. Studies have shown that NGAL can be detected in the urine about 2 hours after induction of AKI, whereas a significant change in bleeding creatinine is detected over 3-4 days. In addition, the concentration of NGAL in blood and urine of a patient suffering from acute renal failure after heart surgery of children is obviously increased 2h after the surgery, and the concentration of blood creatinine is obviously increased 1-3d later, the measurement of the NGAL is more powerful for the early diagnosis of AKI, and can more sensitively reflect the renal function status than the traditional renal injury marker.
At present, NGAL detection methods in the market comprise an enzyme-linked immunosorbent assay (ELISA), a Radioimmunoassay (RIA), a latex-enhanced transmission immunoturbidimetry and the like, but the ELISA and RIA detection methods have the disadvantages of complicated operation and long detection time; the common transmission turbidimetry has insufficient sensitivity; the latex enhanced transmission immunoturbidimetry also has weak anti-interference capability, and special blood samples such as chylomicron blood samples and the like, and some kits can even measure negative values, which can not well reflect renal function conditions of patients with special physique.
Therefore, improvement needs to be made on the basis of the preparation of the conventional NGAL latex immunoturbidimetry detection kit, so as to improve the anti-interference capability of the NGAL latex immunoturbidimetry reagent and expand the applicable population.
Chinese patent CN102662064A discloses an immunoturbidimetric kit for detecting neutrophil gelatinase-associated lipocalin and a preparation method thereof, wherein an application liquid bottle, an antibody suspension liquid bottle and a standard substance bottle are placed in a box body, and the application liquid bottle is filled with an application liquid, which comprises the following components: 0.01-1% of surfactant, 0.01-0.5% of preservative, 1-20% of sodium chloride and 20-200 mmol/L of buffer solution; the antibody suspension liquid bottle is filled with latex suspension liquid of the monoclonal antibody of the lipid carrier protein related to the neutrophil gelatinase, and the components of the latex suspension liquid are as follows: 0.05-0.5% of latex for coating the neutrophil gelatinase-associated lipocalin monoclonal antibody, 0.01-1% of surfactant, 0.01-0.5% of preservative and 20-200 mmol/L of buffer solution; the standard bottle is filled with a neutrophil gelatinase-associated lipocalin standard. Realize the quantitative detection on a large scale on a biochemical instrument. However, the reagent produced by the method has insufficient anti-interference performance, and the health condition of patients with special physique cannot be accurately measured.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a neutrophil gelatinase-associated lipocalin latex immunoturbidimetry detection kit and a preparation method thereof.
The purpose of the invention can be realized by the following technical scheme:
the kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product,
the reagent R1 comprises buffer solution, an anti-interference agent, a sensitizing agent, electrolyte and a preservative,
the reagent R2 comprises latex microspheres connected with NGAL antibodies, buffer solution, stabilizing agent and preservative.
The anti-interference agent is prepared by compounding surfactant polyoxyethylene polyoxypropylene block polyether and triton in a mass ratio of 1:1-5:1 in a buffer solution in a reagent R1.
The polyoxyethylene polyoxypropylene block polyether is selected from one or more of polyoxyethylene polyoxypropylene block polyether L61, L64, F68 or F108, and the mass content of the polyoxyethylene polyoxypropylene block polyether in a buffer solution in a reagent R1 is 0.2-0.8%.
The triton comprises one or more of triton X-100 or triton X-45, and the mass content of the triton in a buffer solution in a reagent R1 is 0.15-0.6%.
The following are more preferred technical solutions:
the combination ratio of the polyoxyethylene polyoxypropylene block polyether L61 to the triton X-45 is 4: 3.
The combination ratio of the polyoxyethylene polyoxypropylene block polyether F68 to the triton X-45 is 3: 2.
The combination ratio of the polyoxyethylene polyoxypropylene block polyether F108 to the triton X-100 is 15: 6.
The combination ratio of the polyoxyethylene polyoxypropylene block polyether L68 to the triton X-45 is 5: 4.
The invention is improved on the basis of the prior art, particularly in the aspect of using a surfactant, polyoxyethylene polyoxypropylene block polyether F68, F08 and the like with stronger anti-interference capability are selected, and the use ratio of triton and the polyoxyethylene polyoxypropylene block polyether is reasonably adjusted, so that the effects of ensuring the reaction sensitivity of a reagent and obviously improving the anti-interference performance of the reagent are achieved.
In the reagent R1, the reaction solution is,
the buffer solution includes but is not limited to phosphate buffer solution, HEPES buffer solution, MES buffer solution or MOPS buffer solution, the concentration is 25-150mM, the pH value is 6.5-9.0, and as a preferred technical scheme, the pH value of the buffer solution is 7.0-8.0.
The sensitizer is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 20000, and the mass content of the sensitizer in the buffer solution in the reagent R1 is 0.5-10%,
the electrolyte is sodium chloride, the mass content of the electrolyte in a buffer solution in a reagent R1 is 0.5-5%,
the preservative is one or two of sodium azide or Proclin300, and the mass content of the preservative in the buffer solution in the reagent R1 is 0.02-2%.
In the reagent R2, the reaction solution is,
the latex microsphere connected with the NGAL antibody is a NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microsphere, the diameter of the latex microsphere is 100-300nm, the mass content in the buffer solution in the reagent R1 is 0.1-5%,
the buffer solution is selected from one or more of Phosphate Buffer Solution (PBS), Tris (hydroxymethyl) aminomethane buffer solution (Tris), 3- (N-morpholinyl) propanesulfonic acid buffer solution (Mops), 2- (N-morpholine) ethanesulfonic acid buffer solution (MES), borate buffer solution or glycine buffer solution or hydroxyethyl piperazine ethanethiosulfonic acid buffer solution, the pH value is 6.5-9.0, and as a preferable technical scheme, the pH value of the buffer solution is 7.5-9.0.
The stabilizer is one or more of mannitol, bovine serum albumin and trehalose, the mass content of the stabilizer in a buffer solution in a reagent R1 is 0.5-5%,
the preservative is one or two of sodium azide or Proclin300, and the mass content of the preservative in the buffer solution in the reagent R1 is 0.02-2%.
The volume ratio of the reagent R1 to the reagent R2 is 3: 1.
The calibrator is recombinant or natural NGAL protein.
The quality control product is recombinant or natural NGAL protein.
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding buffer solution, anti-interference agent, sensitizer, electrolyte and preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 6.5-9.0, and filtering with a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, and mixing and activating for 30min at the temperature of 20-25 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 20-25 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 3-15 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
The anti-interference agent obtained by compounding the polyoxyethylene polyoxypropylene block copolymer and the triton is added into the reagent R1, and the dosage proportion is adjusted, so that the anti-interference capability of the reagent can be obviously improved, the applicable population is enlarged, and the good correlation with the existing reagent is kept. Triton can improve the sensitivity of the reagent and act on interferents with certain concentration, but when the concentration of the interferents is higher, particularly when the concentration of the ananas is high, the interference elimination capability is poorer. The polyoxyethylene polyoxypropylene block polyether F68, F08 and the like have extremely strong dispersion effects on extremely fine particles, have special anti-interference capability on lipids and the like in a viscous sample, but the sensitivity of a latex reagent is seriously reduced due to the strong dispersion capability, and the performance of the reagent is influenced. According to the invention, by reasonably adjusting the use ratio of the triton and the polyoxyethylene polyoxypropylene block polyether, the effects of ensuring the reaction sensitivity of the reagent and obviously improving the anti-interference performance of the reagent are achieved.
Compared with the prior art, the kit has the advantages of high sensitivity, strong specificity, good stability and better clinical application prospect. The kit is simple to prepare, is suitable for industrial production, can efficiently detect the NGAL content in urine, blood plasma and blood serum, has good correlation between the detection result and an imported reagent, and has great application value.
Drawings
FIG. 1 is a linear test chart of the kit;
FIG. 2 is a graph of a correlation test with an inlet reagent;
FIG. 3 is a serum sample anti-interference test chart;
FIG. 4 is a graph of anti-interference testing of a urine sample;
FIG. 5 is a reagent stability test chart.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1. Reagent R1 includes buffer solution, anti-interference agent, sensitizer, electrolyte, antiseptic, reagent R2 is including linking latex microsphere, buffer solution, stabilizer, antiseptic of NGAL antibody, calibrator and quality control article are recombination or natural NGAL protein.
In the case of the reagent R1, the reaction,
the anti-interference agent used is prepared by compounding surfactant polyoxyethylene polyoxypropylene block polyether and triton according to the mass ratio of 1:1-5:1 in a buffer solution in a reagent R1, wherein the polyoxyethylene polyoxypropylene block polyether is selected from one or more of polyoxyethylene polyoxypropylene block polyether L61, L64, F68 or F108, the mass content in the buffer solution in the reagent R1 is 0.2-0.8%, the triton comprises one or more of triton X-100 or triton X-45, the content is 0.15-0.6%, and the components can be selected according to actual needs, for example: the combination ratio of the polyoxyethylene polyoxypropylene block polyether L61 to the triton X-45 is 4:3, the combination ratio of the polyoxyethylene polyoxypropylene block polyether F68 to the triton X-45 is 3:2, the combination ratio of the polyoxyethylene polyoxypropylene block polyether F108 to the triton X-100 is 15:6, and the combination ratio of the polyoxyethylene polyoxypropylene block polyether L68 to the triton X-45 is 5: 4.
The buffer used may be phosphate buffer, HEPES buffer, MES buffer or MOPS buffer, with a concentration of 25-150mM, a pH of 6.5-9.0, preferably a pH of 7.0-8.0.
The sensitizer is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 20000, the content is 0.5-10%, the electrolyte is sodium chloride, the content is 0.5-5%, and the preservative is one or two of sodium azide or Proclin300, the content is 0.02-2%.
In the reagent R2, the reaction solution is,
the latex microsphere connected with the NGAL antibody is NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microsphere, the diameter of the latex microsphere is 100-300nm, the mass content of the buffer solution in the reagent R2 is 0.1-5%, the used buffer solution is selected from one or more of Phosphate Buffer Solution (PBS), Tris buffer solution (Tris), 3- (N-morpholinyl) propanesulfonic acid buffer solution (Mops), 2- (N-morpholinyl) ethanesulfonic acid buffer solution (MES), borate buffer solution or glycine buffer solution or hydroxyethylpiperazine ethanesulfonic acid buffer solution, the pH value is 6.5-9.0, and the pH value of the adopted buffer solution is preferably 7.5-9.0.
The stabilizer is one or more of mannitol, bovine serum albumin and trehalose, the buffer solution in the reagent R2 has a mass content of 0.5-5% (above), the preservative is one or two of sodium azide or Proclin300, and the buffer solution in the reagent R2 has a mass content of 0.02-2% (above).
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding buffer solution, anti-interference agent, sensitizer, electrolyte and preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 6.5-9.0, and filtering with a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, and mixing and activating for 30min at the temperature of 20-25 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 20-25 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 3-15 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
The following are more detailed embodiments, and the technical solutions and the technical effects obtained by the present invention will be further described by the following embodiments.
Example 1
Preparation of reagent R1: 995g of water is taken, then 4.8g of sodium dihydrogen phosphate, 22.8g of disodium hydrogen phosphate, 10.0g of polyethylene glycol 8000, 30.0g of NaCl, 2g of polyoxyethylene polyoxypropylene block copolymer L61, 1.5g of triton X-45(w/w) and 0.5g of Pr ocl in300 are added in sequence, each time the materials are added, stirring is required to be carried out until the materials are completely dissolved, and the pH is adjusted to 7.4: the mixture was filtered through a 0.22 μm filter to obtain lL of reagent R1.
Reagent R2 was prepared by the following steps:
(1) activation of polystyrene latex microspheres: adding 10mL of 500mM HEPES solution with the pH value of 7.4 and 4mL of freshly prepared EDC solution with the concentration of 20mg/mL into 30g of 200nm latex microspheres (100mg/mL), supplementing purified water to the final volume of 100mL, fully and uniformly mixing, and mixing at room temperature (20-25 ℃) for 30 min;
(2) coupling: according to the microsphere: adding the NGAL antibody into the reaction solution in the previous step according to the mass ratio of the NGAL antibody to the NGAL antibody of 12:1, and performing mixed coupling for 2 hours at room temperature;
(3) washing: selecting a suitable hollow fiber column (mPES/500KD/790cm2, SPECTRUM Co.), filtering the solution in step (2) by using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM Co.), exchanging the reaction buffer for the preservation buffer 100mM Tris 8.0, controlling the shear force value to 3000 and 4500, and filtering out 5 times the volume of the step (2) of 100mM Tris buffer solution to wash;
(4) and (3) sealing: adding 20mL of 20% bovine serum albumin solution into the system in the step (3), and mixing for 1h at room temperature;
(5) mixing and storing: diluting the latex microsphere primary reaction liquid obtained in the step (4) with an R2 diluent (containing 100mM glycine buffer solution, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide and pH value of 8.0) to a final volume of 1000mL, thereby obtaining a reagent R2.
III, preparing a NGAL calibrator: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred for complete dissolution every time, the pH value is adjusted to 8.0, 5g of commercial NGAL recombinant antigen is added after the solution is filtered by a 0.22 mu m filter membrane, and the NGAL calibrator solution with the final concentration of 5000ng/mL is prepared. The calibrator was assigned a value using a commercially available NGAL assay kit (latex immunoturbidimetry).
Preparing NGAL quality control products: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred to be completely dissolved each time, the pH value is adjusted to 8.0, 500mL of the solution is taken and added with 0.26g of commercial NGAL recombinant antigen after a 0.22-micron filter membrane is used for the solution, thus obtaining a NGAL quality control solution with the final concentration of 520ng/mL, and 500mL of the solution is taken and added with 0.085g of the commercial NGAL recombinant antigen, thus obtaining a NGAL quality control solution with the final concentration of 170ng/mL, and a calibration product is assigned by using a commercial NGAL determination kit (latex immunoturbidimetry).
The kit comprises the following components: the prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1 to R2 according to the ratio of 3:1 to form a kit, and the packaging specification of the kit is as follows: r1 (4X 45 ml/vial), R2 (4X 15 ml/vial).
Example 2
Preparation of reagent R1: 995g of water is taken, then 4.8g of sodium dihydrogen phosphate, 22.8g of disodium hydrogen phosphate, 10.0g of polyethylene glycol 8000, 30.0g of NaCl, 8g of polyoxyethylene polyoxypropylene block copolymer L61, 6g of Triton X-45(w/w) and 0.5g of Pr ocl in300 are added in sequence, each time the materials are added, stirring is required to be carried out until complete dissolution is achieved, and the pH is adjusted to 7.4: the mixture was filtered through a 0.22 μm filter to obtain lL of reagent R1.
Reagent R2 was prepared by the following steps:
(1) activation of polystyrene latex microspheres: adding 10mL of 500mM HEPES solution with the pH value of 7.4 and 4mL of freshly prepared EDC solution with the concentration of 20mg/mL into 30g of 200nm latex microspheres (100mg/mL), supplementing purified water to the final volume of 100mL, fully and uniformly mixing, and mixing at room temperature (20-25 ℃) for 30 min;
(2) coupling: according to the microsphere: adding the NGAL antibody into the reaction solution in the previous step according to the mass ratio of the NGAL antibody to the NGAL antibody of 12:1, and performing mixed coupling for 2 hours at room temperature;
(3) washing: selecting a suitable hollow fiber column (mPES/500KD/790cm2, SPECTRUM Co.), filtering the solution in step (2) by using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM Co.), exchanging the reaction buffer for the preservation buffer 100mM Tris 8.0, controlling the shear force value to 3000 and 4500, and filtering out 5 times the volume of the step (2) of 100mM Tris buffer solution to wash;
(4) and (3) sealing: adding 20mL of 20% bovine serum albumin solution into the system in the step (3), and mixing for 1h at room temperature;
(5) mixing and storing: diluting the latex microsphere primary reaction liquid obtained in the step (4) with an R2 diluent (containing 100mM glycine buffer solution, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide and pH value of 8.0) to a final volume of 1000mL, thereby obtaining a reagent R2.
III, preparing a NGAL calibrator: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred for complete dissolution every time, the pH value is adjusted to 8.0, 5g of commercial NGAL recombinant antigen is added after the solution is filtered by a 0.22 mu m filter membrane, and the NGAL calibrator solution with the final concentration of 5000ng/mL is prepared. The calibrator was assigned a value using a commercially available NGAL assay kit (latex immunoturbidimetry).
Preparing NGAL quality control products: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred to be completely dissolved each time, the pH value is adjusted to 8.0, 500mL of the solution is taken and added with 0.26g of commercial NGAL recombinant antigen after a 0.22-micron filter membrane is used for the solution, thus obtaining a NGAL quality control solution with the final concentration of 520ng/mL, and 500mL of the solution is taken and added with 0.085g of the commercial NGAL recombinant antigen, thus obtaining a NGAL quality control solution with the final concentration of 170ng/mL, and a calibration product is assigned by using a commercial NGAL determination kit (latex immunoturbidimetry).
The kit comprises the following components: the prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1 to R2 according to the ratio of 3:1 to form a kit, and the packaging specification of the kit is as follows: r1 (4X 45 ml/vial), R2 (4X 15 ml/vial).
Example 3
Preparation of reagent R1: 995g of water is taken, then 4.8g of sodium dihydrogen phosphate, 22.8g of disodium hydrogen phosphate, 10.0g of polyethylene glycol 8000, 30.0g of NaCl, 7.5g of polyoxyethylene polyoxypropylene block copolymer F68, 5g of triton X-45(w/w) and 0.5g of Pr ocl in300 are added in sequence, each time the materials are added, stirring is required to be carried out until the materials are completely dissolved, and the pH is adjusted to 7.4: the mixture was filtered through a 0.22 μm filter to obtain l L reagent R1.
Reagent R2 was prepared by the following steps:
(1) activation of polystyrene latex microspheres: adding 10mL of 500mM HEPES solution with the pH value of 7.4 and 4mL of freshly prepared EDC solution with the concentration of 20mg/mL into 30g of 200nm latex microspheres (100mg/mL), supplementing purified water to the final volume of 100mL, fully and uniformly mixing, and mixing at room temperature (20-25 ℃) for 30 min;
(2) coupling: according to the microsphere: adding the NGAL antibody into the reaction solution in the previous step according to the mass ratio of the NGAL antibody to the NGAL antibody of 12:1, and performing mixed coupling for 2 hours at room temperature;
(3) washing: selecting a suitable hollow fiber column (mPES/500KD/790cm2, SPECTRUM Co.), filtering the solution in step (2) by using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM Co.), exchanging the reaction buffer for the preservation buffer 100mM Tris 8.0, controlling the shear force value to 3000 and 4500, and filtering out 5 times the volume of the step (2) of 100mM Tris buffer solution to wash;
(4) and (3) sealing: adding 20mL of 20% bovine serum albumin solution into the system in the step (3), and mixing for 1h at room temperature;
(5) mixing and storing: diluting the latex microsphere primary reaction liquid obtained in the step (4) with an R2 diluent (containing 100mM glycine buffer solution, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide and pH value of 8.0) to a final volume of 1000mL, thereby obtaining a reagent R2.
III, preparing a NGAL calibrator: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred for complete dissolution every time, the pH value is adjusted to 8.0, 5g of commercial NGAL recombinant antigen is added after the solution is filtered by a 0.22 mu m filter membrane, and the NGAL calibrator solution with the final concentration of 5000ng/mL is prepared. The calibrator was assigned a value using a commercially available NGAL assay kit (latex immunoturbidimetry).
Preparing NGAL quality control products: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred to be completely dissolved each time, the pH value is adjusted to 8.0, 500mL of the solution is taken and added with 0.26g of commercial NGAL recombinant antigen after a 0.22-micron filter membrane is used for the solution, thus obtaining a NGAL quality control solution with the final concentration of 520ng/mL, and 500mL of the solution is taken and added with 0.085g of the commercial NGAL recombinant antigen, thus obtaining a NGAL quality control solution with the final concentration of 170ng/mL, and a calibration product is assigned by using a commercial NGAL determination kit (latex immunoturbidimetry).
The kit comprises the following components: the prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1 to R2 according to the ratio of 3:1 to form a kit, and the packaging specification of the kit is as follows: r1 (4X 45 ml/vial), R2 (4X 15 ml/vial).
Example 4
Preparation of reagent R1: 995g of water is taken, then 4.8g of sodium dihydrogen phosphate, 22.8g of disodium hydrogen phosphate, 10.0g of polyethylene glycol 8000, 30.0g of NaCl, 7.5g of polyoxyethylene polyoxypropylene block copolymer F108, 3g of triton X-100(w/w) and 0.5g of Pr ocl in300 are added in sequence, each time the materials are added, stirring is required to be carried out until the materials are completely dissolved, and the pH is adjusted to 7.4: the mixture was filtered through a 0.22 μm filter to obtain l L reagent R1.
Reagent R2 was prepared by the following steps:
(1) activation of polystyrene latex microspheres: adding 10mL of 500mM HEPES solution with the pH value of 7.4 and 4mL of freshly prepared EDC solution with the concentration of 20mg/mL into 30g of 200nm latex microspheres (100mg/mL), supplementing purified water to the final volume of 100mL, fully and uniformly mixing, and mixing at room temperature (20-25 ℃) for 30 min;
(2) coupling: according to the microsphere: adding the NGAL antibody into the reaction solution in the previous step according to the mass ratio of the NGAL antibody to the NGAL antibody of 12:1, and performing mixed coupling for 2 hours at room temperature;
(3) washing: selecting a suitable hollow fiber column (mPES/500KD/790cm2, SPECTRUM Co.), filtering the solution in step (2) by using a tangential flow filtration system (KrosFlo research IIi TFF system, SPECTRUM Co.), exchanging the reaction buffer for the preservation buffer 100mM Tris 8.0, controlling the shear force value to 3000 and 4500, and filtering out 5 times the volume of the step (2) of 100mM Tris buffer solution to wash;
(4) and (3) sealing: adding 20mL of 20% bovine serum albumin solution into the system in the step (3), and mixing for 1h at room temperature;
(5) mixing and storing: diluting the latex microsphere primary reaction liquid obtained in the step (4) with an R2 diluent (containing 100mM glycine buffer solution, 4% bovine serum albumin, 0.5% trehalose, 0.05% sodium azide and pH value of 8.0) to a final volume of 1000mL, thereby obtaining a reagent R2.
III, preparing a NGAL calibrator: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred for complete dissolution every time, the pH value is adjusted to 8.0, 5g of commercial NGAL recombinant antigen is added after the solution is filtered by a 0.22 mu m filter membrane, and the NGAL calibrator solution with the final concentration of 5000ng/mL is prepared. The calibrator was assigned a value using a commercially available NGAL assay kit (latex immunoturbidimetry).
Preparing NGAL quality control products: 995mL of water is taken, 100mM Tris buffer, 1% sodium chloride, 0.2% disodium ethylene diamine tetraacetate, 3.0% bovine serum albumin and 0.1% sodium azide are sequentially added, materials are required to be stirred to be completely dissolved each time, the pH value is adjusted to 8.0, 500mL of the solution is taken and added with 0.26g of commercial NGAL recombinant antigen after a 0.22-micron filter membrane is used for the solution, thus obtaining a NGAL quality control solution with the final concentration of 520ng/mL, and 500mL of the solution is taken and added with 0.085g of the commercial NGAL recombinant antigen, thus obtaining a NGAL quality control solution with the final concentration of 170ng/mL, and a calibration product is assigned by using a commercial NGAL determination kit (latex immunoturbidimetry).
The kit comprises the following components: the prepared R1 reagent and R2 reagent are matched according to the volume ratio of R1 to R2 according to the ratio of 3:1 to form a kit, and the packaging specification of the kit is as follows: r1 (4X 45 ml/vial), R2 (4X 15 ml/vial).
Example 5
The kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1. Reagent R1 includes buffer solution, anti-interference agent, sensitizer, electrolyte, antiseptic, reagent R2 is including linking latex microsphere, buffer solution, stabilizer, antiseptic of NGAL antibody, calibrator and quality control article are recombinant NGAL protein.
In the case of the reagent R1, the reaction,
the used anti-interference agent is obtained by compounding surfactant polyoxyethylene polyoxypropylene block polyether L61 and triton X-100 according to the mass ratio of 1:1 in a buffer solution in a reagent R1, and the mass content of the polyoxyethylene polyoxypropylene block polyether L61 and the triton in the buffer solution in a reagent R1 is 0.2%.
The buffer solution used was phosphate buffer solution with a concentration of 25mM, a pH of 6.5, the sensitizer was polyethylene glycol 6000, the buffer solution contained 0.5% by mass in reagent R1, the electrolyte was sodium chloride, the buffer solution contained 0.5% by mass in reagent R1, the preservative was sodium azide, and the buffer solution contained 0.02% by mass in reagent R1.
In the reagent R2, the reaction solution is,
the latex microspheres connected with NGAL antibody are NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microspheres, the diameter of the latex microspheres is 100nm, the mass content of the latex microspheres in a buffer solution in a reagent R2 is 0.1%, the used buffer solution is Phosphate Buffer Solution (PBS), the pH value is 6.5, a stabilizing agent is mannitol, the mass content of the buffer solution in a reagent R2 is 0.5-5%, a preservative is sodium azide, and the mass content of the buffer solution in a reagent R2 is 0.02%.
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 6.5, and filtering the materials by using a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, mixing and activating for 30min at 20 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 20 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 3 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
Example 6
The kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1. Reagent R1 includes buffer solution, anti-interference agent, sensitizer, electrolyte, antiseptic, reagent R2 is including linking latex microsphere, buffer solution, stabilizer, antiseptic of NGAL antibody, calibrator and quality control article are recombination or natural NGAL protein.
In the case of the reagent R1, the reaction,
the used anti-interference agent is obtained by compounding surfactant polyoxyethylene polyoxypropylene block polyether L61 and triton X-45 according to the mass ratio of 4:3 in a buffer solution in a reagent R1, the polyoxyethylene polyoxypropylene block polyether L61 accounts for 0.8% in the buffer solution in the reagent R1, and the triton X-45 accounts for 0.6%.
The buffer solution used is HEPES buffer solution, the pH value is 7.0, the sensitizer is a mixture of polyethylene glycol 6000 and polyethylene glycol 8000, the buffer solution in the reagent R1 contains 2% by mass of electrolyte, sodium chloride and preservative, and the content of the preservative is 0.5%.
In the reagent R2, the reaction solution is,
the latex microsphere connected with the NGAL antibody is formed by connecting NGAL monoclonal antibody or polyclonal antibody to polystyrene carboxyl microsphere, the diameter of the latex microsphere is 200nm, the mass content of the buffer solution in the reagent R2 is 2%, the used buffer solution is Tris buffer solution (Tris), and the pH value is 7.5.
The stabilizer is bovine serum albumin, the buffer solution in the reagent R2 has the mass content of 1%, the preservative is sodium azide, and the buffer solution in the reagent R2 has the mass content of 0.5%.
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 7.0, and filtering the materials by using a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, mixing and activating for 30min at 20 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 20 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 5 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
Example 7
The kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1. Reagent R1 includes buffer solution, anti-interference agent, sensitizer, electrolyte, antiseptic, reagent R2 is including linking latex microsphere, buffer solution, stabilizer, antiseptic of NGAL antibody, and calibrator and quality control article are natural NGAL protein.
In the case of the reagent R1, the reaction,
the anti-interference agent is obtained by compounding surfactant polyoxyethylene polyoxypropylene block polyether F68 and triton X-45 according to the mass ratio of 5:4 in a buffer solution in a reagent R1, wherein the polyoxyethylene polyoxypropylene block polyether F68 accounts for 0.5% of the buffer solution in the reagent R1, and the content of the triton X-45 accounts for 0.4%.
The buffer solution used was MES buffer solution with a concentration of 100mM, a pH of 8.0, a sensitizer of polyethylene glycol 20000 with a content of 6%, an electrolyte of sodium chloride with a content of 3%, and a preservative of Proclin300 with a content of 1%.
In the reagent R2, the reaction solution is,
the latex microsphere connected with the NGAL antibody is a NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microsphere, the diameter of the latex microsphere is 300nm, the mass content of the buffer solution in the reagent R2 is 4%, the used buffer solution is 2- (N-morpholine) ethanesulfonic acid buffer solution (MES), and the pH value is 8.0.
The stabilizing agent is trehalose, the buffer solution in the reagent R2 contains 4% by mass of the stabilizing agent, the preservative is Proclin300, and the buffer solution in the reagent R2 contains 1% by mass of the preservative. .
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 8.0, and filtering the materials by using a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, and mixing and activating for 30min at 25 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 25 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 3-15 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
Example 8
The kit for measuring the immunoturbidimetry of the neutrophil gelatinase-associated lipocalin latex consists of a reagent R1, a reagent R2, a calibrator and a quality control product, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1. Reagent R1 includes buffer solution, anti-interference agent, sensitizer, electrolyte, antiseptic, reagent R2 is including linking latex microsphere, buffer solution, stabilizer, antiseptic of NGAL antibody, and calibrator and quality control article are natural NGAL protein.
In the case of the reagent R1, the reaction,
the used anti-interference agent is obtained by compounding surfactant polyoxyethylene polyoxypropylene block polyether F108 and triton X-100 according to the mass ratio of 5:1 in a buffer solution in a reagent R1, wherein the polyoxyethylene polyoxypropylene block polyether F108 accounts for 0.8% in the buffer solution in a reagent R1, and the triton X-100 accounts for 0.16%.
The buffer solution used was MOPS buffer solution, the concentration was 150mM, the pH was 9.0, the sensitizer was polyethylene glycol 8000, the content was 10%, the electrolyte was sodium chloride, the content was 5%, the preservative was Proclin300, the content was 2%.
In the reagent R2, the reaction solution is,
the latex microspheres connected with NGAL antibody are NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microspheres, the diameter of the latex microspheres is 300nm, the mass content of the latex microspheres in a buffer solution in a reagent R2 is 5%, the used buffer solution is a mixture of glycine buffer solution and hydroxyethyl piperazine ethanethiosulfonic acid buffer solution, the pH value is 9.0, the stabilizer is bovine serum albumin, the mass content of the buffer solution in a reagent R2 is 5%, the preservative is Proclin300, and the mass content of the buffer solution in a reagent R2 is 2%.
The preparation method of the neutrophil gelatinase-associated lipocalin latex immunoturbidimetry kit comprises the following steps:
I. preparation of reagent R1:
adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 9.0, and filtering the materials by using a 0.22 mu m filter membrane;
II. Preparation of reagent R2:
(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, and mixing and activating for 30min at 25 ℃;
(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 25 ℃ for 2h to carry out covalent coupling;
(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;
(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;
(5) mixing and storing: diluting the solution obtained in the step (4) by 3-15 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;
III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;
VI, forming a kit:
the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (3) packaging the R2-3: 1 into bottles, and combining the bottles with a calibrator and a quality control material to form a kit.
Example 9
Performance test of NGAL assay kit of the present invention
A detection tool: a full-automatic biochemical analyzer of Dirui AU680 model.
The analysis method comprises the following steps: two-point endpoint method. Dominant wavelength: 570 nm; secondary wavelength: none. Mu.l of the reagent R1150 prepared in example 2 was added to 3. mu.l of the serum sample, and after incubation at 37 ℃ for 5min, 250. mu.l of the reagent R was added, and the absorbance A1 was read, and after 5min of the reaction, the absorbance A2 was read, and the final absorbance of the reaction was calculated as the difference between A2 and A1.
A calibration mode: multipoint non-linearity. The reaction direction is as follows: and (4) rising.
The calculation method comprises the following steps: and (5) making a calibration curve according to the absorbance and different concentrations of the calibrator. And testing the serum sample to obtain absorbance, and calculating according to the calibration curve to obtain the sample content.
Reference ranges: 0-5000 ng/mL.
I. Linear testing of the kits of the invention
The NGAL calibrator prepared in example 4 was diluted with water to 0ng/mL, 312.5ng/mL, 625ng/mL, 1250ng/mL, 2500ng/mL, 5000ng/mL, respectively, and calibration curves were tested using reagents R1, R2 prepared in example 2. As shown in FIG. 1, the X-axis represents theoretical value (ng/mL), the Y-axis represents measured value (ng/mL), and R20.9992, Y1.0431X-35.071. The kit has good linear relation in the concentration range of 0-5000 ng/mL.
II. Sensitivity and correlation test of the kit and imported control kit
The example 4 kit and the imported control kit (latex immunoturbidimetry) were tested for different concentrations of calibrators, respectively, and the absorbance vs. ratio is shown in table 1. The kit has higher sensitivity.
TABLE 1
The calibration curves were tested separately for the example 4 kit and the imported control kit (latex immunoturbidimetry). The results of testing 60 clinical serum samples with different concentrations are shown in fig. 2, and the linear correlation coefficient R2 of the two samples is 0.992, which is good in correlation.
III inter-batch Difference test of the kit of the invention
Three batches of NGAL kits were prepared in duplicate according to the experimental procedure described in example 4, and each was tested for a different concentration of calibrator, the absorbance vs. ratio being shown in table 2. The result shows that the NGAL latex immunoturbidimetry kit used in the invention has small batch difference and good repeatability.
TABLE 2
| Calibrator concentration (ng/mL) | Absorbance of the first batch | Absorbance of the second batch | Absorbance of |
| 0 | 0.00023 | 0.00019 | 0.00024 |
| 312.5 | 0.014 | 0.015 | 0.018 |
| 625 | 0.028 | 0.027 | 0.024 |
| 1250 | 0.165 | 0.154 | 0.162 |
| 2500 | 0.251 | 0.255 | 0.249 |
IV, anti-interference test of the kit
A blood sample and a urine sample (the samples are from Shanghai Hospital affiliated to Shanghai, Fudan university) of a healthy person who is detected to be normal are taken, recombinant NGAL antigen is added to prepare samples with the concentration of 600ng/mL, high-concentration interferent is precisely measured and mixed with serum and urine respectively to prepare mixed samples with different concentrations of the interferent (shown in a table 3), each sample is repeatedly measured for 3 times, and the average value of the samples is taken. The method for calculating the interference degree comprises the following steps:
the interference degree is the mean value of the samples with the interferents/the mean value of the samples without the interferents multiplied by 100 percent
The interference degree is between 90% and 110% and is an acceptable interference range.
TABLE 3
The results show that: as shown in FIG. 3 and FIG. 4, compared with the sample without the interferent, the measured value after the addition can be controlled within + -5%, and simultaneously, compared with the previous invention, the kit has higher acceptable interferent and stronger anti-interference capability.
V, Effect-Limited stability test of the kit of the invention
Under the storage condition of 2-8 ℃, the kit prepared in example 2 is used for detecting the same high-value quality control product (500ng/mL) and low-value blood quality control product (170ng/mL) in 0 month, 3 months, 6 months, 9 months, 12 months and 14 months respectively, and each sample is measured for 5 times to obtain an average value (the result is shown in figure 5). The results show that the kit can be stably stored for one year at the temperature of 2-8 ℃.
From the above embodiments, it can be seen that the NGAL assay kit researched and prepared by the invention has the advantages of strong anti-interference capability, good linearity, good stability, small batch-to-batch difference and the like, can be used for large-scale industrial production, and has good clinical application prospects.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (7)
- The NGAL latex immunoturbidimetry detection kit is characterized by consisting of a reagent R1, a reagent R2, a calibrator and a quality control product,the reagent R1 comprises a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative, wherein the anti-interference agent is prepared by compounding surfactant polyoxyethylene polyoxypropylene block polyether and triton according to the mass ratio of 1:1-5: 1;the reagent R2 comprises latex microspheres connected with NGAL antibody, buffer solution, stabilizer and preservative;the polyoxyethylene polyoxypropylene block polyether is selected from one or more of polyoxyethylene polyoxypropylene block polyether L61, F68 or F108, and the mass content of the polyoxyethylene polyoxypropylene block polyether in a buffer solution in a reagent R1 is 0.2-0.8%;the triton comprises one or more of triton X-100 or triton X-45, and the mass content of the triton in a buffer solution in a reagent R1 is 0.15-0.6%.
- 2. The NGAL latex immunoturbidimetry assay kit of claim 1, wherein in the reagent R1,the buffer solution includes but is not limited to phosphate buffer solution, HEPES buffer solution, MES buffer solution or MOPS buffer solution, the concentration is 25-150mM, the pH value is 6.5-9.0,the sensitizer is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 20000, the mass content of the sensitizer in the buffer solution in the reagent R1 is 0.5-10%,the electrolyte is sodium chloride, the mass content of the electrolyte in the buffer solution in the reagent R1 is 0.5-5%,the preservative is one or two of sodium azide or Proclin300, and the mass content of the preservative in the buffer solution in the reagent R1 is 0.02-2%.
- 3. The NGAL latex immunoturbidimetry assay kit of claim 1, wherein in the reagent R2,the latex microsphere connected with the NGAL antibody is a NGAL monoclonal antibody or polyclonal antibody connected with polystyrene carboxyl microsphere, the diameter of the latex microsphere is 100-300nm, the mass content in the buffer solution in the reagent R2 is 0.1-5%,the buffer solution is selected from one or more of Phosphate Buffer Solution (PBS), Tris buffer solution (Tris), 3- (N-morpholinyl) propanesulfonic acid buffer solution (Mops), 2- (N-morpholine) ethanesulfonic acid buffer solution (MES), borate buffer solution or glycine buffer solution or hydroxyethylpiperazine ethanethiosulfonic acid buffer solution, the pH value is 6.5-9.0,the stabilizer is one or more of mannitol, bovine serum albumin and trehalose, the mass content of the stabilizer in a buffer solution in a reagent R2 is 0.5-5%,the preservative is one or two of sodium azide or Proclin300, and the mass content of the preservative in the buffer solution in the reagent R2 is 0.02-2%.
- 4. The NGAL latex immunoturbidimetry assay kit of claim 1, wherein the volume ratio of the reagent R1 to the reagent R2 is 3: 1.
- 5. The NGAL latex immunoturbidimetry detection kit of claim 1, wherein the calibrator is recombinant or native NGAL protein.
- 6. The NGAL latex immunoturbidimetry assay kit of claim 1, wherein the quality control is recombinant or native NGAL protein.
- 7. The method of making a NGAL latex immunoturbidimetry test kit according to any one of claims 1 to 6, using the steps of:I. preparation of reagent R1:adding buffer solution, anti-interference agent, sensitizer, electrolyte and preservative into purified water in sequence, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 6.5-9.0, and filtering with a 0.22 mu m filter membrane;II. Preparation of reagent R2:(1) activated polystyrene latex microspheres: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the latex microspheres, and mixing and activating for 30min at the temperature of 20-25 ℃;(2) coupling antibody: adding the neutrophil gelatinase-associated lipocalin antibody into the activated polystyrene latex microspheres, and reacting at 20-25 ℃ for 2h to carry out covalent coupling;(3) washing to remove the unconjugated successful antibody: centrifuging the obtained latex microsphere reaction solution to remove supernatant and remove free antibodies and micromolecular impurities;(4) blocking carboxyl sites of unconjugated antibody on the microspheres: adding bovine serum albumin into the obtained latex microspheres, and sealing for 1 h;(5) mixing and storing: diluting the solution obtained in the step (4) by 3-15 times by using R2 diluent containing a stabilizer, a preservative and a buffer solution to finally obtain a reagent R2, and storing at 4 ℃;III, preparing a calibration material and a quality control material: diluting natural or recombinant NGAL protein to target concentration with lyophilized diluent, lyophilizing, and storing at 4 deg.C;VI, forming a kit:the reagent R1 and the reagent R2 prepared above were mixed in a volume ratio of R1: and (4) packaging the R2=3:1 into bottles, and forming a kit with the calibrator and the quality control material.
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| CN101467044A (en) * | 2006-07-13 | 2009-06-24 | 奥林巴斯株式会社 | Method of confirming the specimen-detecting activity of microparticles |
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| CN107015004A (en) * | 2017-03-21 | 2017-08-04 | 深圳市汇松科技发展有限公司 | A kind of serum amyloid A protein determines kit and preparation method thereof |
| CN107942069A (en) * | 2017-11-15 | 2018-04-20 | 捷和泰（北京）生物科技有限公司 | A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof |
| CN108089008A (en) * | 2017-12-01 | 2018-05-29 | 苏州康和顺医疗技术有限公司 | The immunoturbidimetry detection reagent and method of a kind of NGAL |
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