CN114047338B - Urine transferrin detection kit and detection method thereof - Google Patents

Urine transferrin detection kit and detection method thereof Download PDF

Info

Publication number
CN114047338B
CN114047338B CN202111323621.XA CN202111323621A CN114047338B CN 114047338 B CN114047338 B CN 114047338B CN 202111323621 A CN202111323621 A CN 202111323621A CN 114047338 B CN114047338 B CN 114047338B
Authority
CN
China
Prior art keywords
transferrin
detection
another preferred
reagent
detection reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111323621.XA
Other languages
Chinese (zh)
Other versions
CN114047338A (en
Inventor
陈怡滔
毛晓波
顾莉丽
王丽娇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Jiemen Biotechnology Co ltd
Original Assignee
Shanghai Jiemen Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Jiemen Biotechnology Co ltd filed Critical Shanghai Jiemen Biotechnology Co ltd
Priority to CN202111323621.XA priority Critical patent/CN114047338B/en
Publication of CN114047338A publication Critical patent/CN114047338A/en
Application granted granted Critical
Publication of CN114047338B publication Critical patent/CN114047338B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (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 relates to a urine transferrin detection kit and a detection method thereof. Specifically, the invention provides a transferrin detection kit, which comprises a detection reagent for detecting transferrin, wherein the detection reagent comprises a detection reagent R1 and a detection reagent R2. The transferrin detection kit can perform qualitative and quantitative determination on transferrin in a detection sample, and has the advantages of high detection repeatability, high accuracy, strong stability, coverage of most clinically ultrahigh-value samples, prevention of false negative results caused by antigen surplus and the like.

Description

Urine transferrin detection kit and detection method thereof
Technical Field
The invention relates to the technical field of medical biological detection, in particular to a urine transferrin detection kit and a detection method thereof.
Background
Transferrin, also known as Transferrin (TRF), is the major iron-containing protein in plasma, responsible for carrying iron absorbed by the digestive tract and released by erythrocyte degradation into the bone marrow for use in the production of mature erythrocytes. TRF is mainly synthesized by liver cells, the half life period is 7 days, the concentration of TRF in blood plasma is regulated by iron supply, and in the iron deficiency state, the concentration of TRF in blood plasma rises, and the TRF returns to normal level after effective treatment, so that the TRF level in blood plasma can be used for diagnosis and treatment monitoring of iron deficiency anemia. The TRF has a slightly larger molecular weight than albumin, but has fewer charges, and the less negative charges are more likely to penetrate through the negative charge barrier of the glomerulus, so that when the glomerulus filtration membrane of a patient with diabetes or hypertension is changed in charge, the TRF is more likely to filter from the glomerulus than trace albumin, so that the TRF can more sensitively reflect the damage condition of the glomerulus charge barrier, and because the damage of the renal injury usually occurs firstly, then the damage of the filtration membrane is caused, the detection of the urinary transferrin is favorable for finding early renal injury earlier than the urinary microalbumin, and the TRF is worthy of clinical popularization and use.
At present, urine transferrin is often measured by an immunoturbidimetry, wherein the immunoturbidimetry is based on the principle that a complex formed after antigen and antibody are combined can cause turbidity change of a liquid medium, the formed turbidity reduces the light transmission, and the light absorbed amount is positively correlated with the Immune Complex (IC) formation amount, so that the amount of an antigen to be measured can be calculated according to the measured absorbance value. However, the conventional detection reagent for detecting the urotransferrin by the immunoturbidimetry has a relatively narrow multi-linear range, such as the Baiottai, the Meikang organisms, the Bai Rong organisms and the like, the highest detection range is 30mg/L, the detection requirements of most patients cannot be covered, the HOOK effect range is not marked, or the HOOK effect range is smaller than 1000mg/L, and the detection requirements are difficult to meet.
Therefore, there is a need in the art to develop a method for detecting urinary transferrin that improves the measurement range, reproducibility, accuracy, etc. of transferrin.
Disclosure of Invention
The invention aims to provide a method for detecting urinary transferrin, which improves the measurement range, repeatability, accuracy and the like of transferrin.
In a first aspect of the present invention, there is provided a transferrin assay kit comprising an assay reagent for the detection of transferrin, the assay reagent comprising an assay reagent R1 and an assay reagent R2;
the detection reagent R1 comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polyethylene glycol, a surfactant and water;
the detection reagent R2 comprises a first transferrin latex antibody dispersion liquid and a second transferrin latex antibody dispersion liquid;
the first transferrin latex antibody dispersion comprises transferrin antibodies and first carboxyl polystyrene microspheres, wherein the transferrin antibodies are coupled to the first carboxyl polystyrene microspheres;
the second transferrin latex antibody dispersion comprises transferrin antibodies and second carboxyl polystyrene microspheres, and the transferrin antibodies are coupled to the second carboxyl polystyrene microspheres.
In another preferred embodiment, the transferrin comprises urinary transferrin.
In another preferred embodiment, the transferrin is human transferrin.
In another preferred embodiment, the particle size of the first carboxyl polystyrene microsphere is 40-100nm, preferably 50-90nm, more preferably 60-80nm, and even more preferably 65-85nm.
In another preferred embodiment, the particle size of the second carboxyl polystyrene microsphere is 150-250nm, preferably 170-230nm, more preferably 185-210nm, and even more preferably 190-200nm.
In another preferred embodiment, the weight ratio of the first carboxyl polystyrene microsphere to the second carboxyl polystyrene microsphere is 1:0.5-5, preferably 1:0.5-3, more preferably 1:1-2, more preferably 1:1.3-1.7, and most preferably 1:1.5.
In another preferred embodiment, the volume ratio of the first transferrin latex antibody dispersion to the second transferrin latex antibody dispersion is 1:0.5-5, preferably 1:0.5-3, more preferably 1:1-2, more preferably 1:1.3-1.7, most preferably 1:1.5.
In another preferred embodiment, the transferrin antibody comprises a goat anti-human transferrin antibody.
In another preferred embodiment, the dispersion medium in the first transferrin latex antibody dispersion comprises a latex protection buffer.
In another preferred embodiment, the dispersion medium in the second transferrin latex antibody dispersion comprises a latex protection buffer.
In another preferred embodiment, the latex protection buffer comprises an aqueous solution of phosphate buffer, serum albumin, sodium chloride, and sucrose.
In another preferred embodiment, the latex protection buffer comprises an aqueous solution of phosphate buffer, serum albumin, sodium chloride, sucrose, and sodium azide.
In another preferred embodiment, the pH of the latex protection buffer is 7.0-7.4, preferably 7.2.
In another preferred embodiment, the serum albumin is bovine serum albumin.
In another preferred embodiment, the phosphate buffer is present in an amount of 0.01 to 0.05mol/L, preferably 0.01 to 0.03mol/L, most preferably 0.02mol/L.
In another preferred embodiment, the serum albumin is present in an amount of 0.08 to 0.5wt%, preferably 0.1 to 0.3wt%, more preferably 0.15 to 0.25wt%, most preferably 0.2wt%.
In another preferred embodiment, the sodium chloride is present in an amount of 1.5 to 2.6wt%, preferably 1.8 to 2.4wt%, more preferably 2.0 to 2.2wt%, most preferably 2.12wt%.
In another preferred embodiment, the sucrose is present in an amount of 1.5 to 2.5wt%, preferably 1.8 to 2.2wt%, more preferably 1.9 to 2.1wt%, most preferably 2.0wt%.
In another preferred embodiment, the sodium azide is present in an amount of 0.3 to 1.8 wt.%, preferably 0.5 to 1.5 wt.%, more preferably 0.8 to 1.2 wt.%, most preferably 1.0 wt.%.
In another preferred embodiment, the latex protection buffer comprises an aqueous solution of 0.01 to 0.03mol/L phosphate buffer, 0.15 to 0.25wt% serum albumin, 1.8 to 2.3wt% sodium chloride, and 1.8 to 2.2wt% sucrose.
In another preferred embodiment, the latex protection buffer comprises an aqueous solution of 0.01 to 0.03mol/L phosphate buffer, 0.15 to 0.25wt% serum albumin, 1.8 to 2.3wt% sodium chloride, 1.8 to 2.2wt% sucrose, and 0.8 to 1.2wt% sodium azide.
In another preferred embodiment, the first transferrin latex antibody dispersion is prepared by:
(1) And (3) coupling the transferrin antibody to the first carboxyl polystyrene microsphere, and dispersing the first carboxyl polystyrene microsphere in a latex protection buffer solution to obtain a first transferrin latex antibody dispersion.
In another preferred embodiment, the step (1) includes:
the first carboxyl polystyrene microsphere of (1-1) is activated by an activating agent to obtain an activated first carboxyl polystyrene microsphere;
(1-2) dispersing the activated first carboxyl polystyrene microsphere with MES buffer, adding transferrin antibody for coupling reaction, sealing the obtained precipitate by centrifugation with a sealing agent, and dispersing the obtained precipitate by centrifugation in latex protection buffer to obtain a first transferrin latex antibody dispersion.
In another preferred embodiment, in the step (1-1), the first carboxyl polystyrene microsphere is activated by an activator, and then centrifuged to remove the supernatant to obtain an activated first carboxyl polystyrene microsphere
In another preferred embodiment, the second transferrin latex antibody dispersion is prepared by:
(a) And (3) coupling the transferrin antibody to the second carboxyl polystyrene microsphere, and dispersing the second carboxyl polystyrene microsphere in a latex protection buffer solution to obtain a second transferrin latex antibody dispersion.
In another preferred embodiment, the step (a) includes:
the second carboxyl polystyrene microsphere of the (a-1) is activated by an activating agent to obtain an activated second carboxyl polystyrene microsphere;
and (c) dispersing the activated second carboxyl polystyrene microsphere by using MES buffer solution, adding transferrin antibody for coupling reaction, blocking the obtained precipitate by centrifugation by using a blocking agent, and dispersing the obtained precipitate by centrifugation in latex protection buffer solution to obtain a second transferrin latex antibody dispersion.
In another preferred embodiment, in the step (a-1), the second carboxyl polystyrene microsphere is activated by an activator, and then centrifuged to remove the supernatant, thereby obtaining the activated second carboxyl polystyrene microsphere.
In another preferred embodiment, the activator comprises EDC.
In another preferred embodiment, the activation is sufficient activation.
In another preferred embodiment, the activation temperature is 23-27 ℃.
In another preferred embodiment, the activation time is 8-12min.
In another preferred embodiment, the MES buffer is present at a concentration of 0.1 to 0.3mol/L, preferably 0.15 to 0.25mol/L.
In another preferred embodiment, the MES buffer has a pH of 5.5-6.5, preferably 6.0-6.5, more preferably 6.0-6.4.
In another preferred embodiment, the temperature of the coupling reaction is 18-25 ℃, preferably 18-22 ℃.
In another preferred embodiment, the coupling reaction is carried out for a period of time ranging from 1.5 to 2.5 hours, preferably from 1.8 to 2.2 hours.
In another preferred embodiment, the coupling reaction is a complete reaction.
In another preferred embodiment, the blocking agent comprises an aqueous serum albumin solution.
In another preferred embodiment, the serum albumin is bovine serum albumin.
In another preferred embodiment, the serum albumin is present in an amount of 0.1 to 0.3wt%, preferably 0.15 to 0.25
In another preferred embodiment, the temperature of the enclosure is 18-25 ℃, preferably 18-22 ℃.
In another preferred embodiment, the closing time is 1.5-2.5 hours, preferably 1.8-2.2 hours.
In another preferred embodiment, the closure is a substantially closed closure.
In another preferred embodiment, the weight ratio of transferrin to the first carboxyl polystyrene microsphere in the first transferrin latex antibody dispersion is 60-100:1, preferably 70-90:1, more preferably 75-85:1, preferably 78-82:1.
in another preferred embodiment, the volume to weight ratio (ml: mg) of the latex protection buffer to the first carboxypolystyrene microspheres in the first transferrin latex antibody dispersion is 60-100:1, preferably 70-90:1, more preferably 75-85:1, preferably 78-82:1.
in another preferred embodiment, the weight ratio of transferrin to the second carboxyl polystyrene microsphere in the second transferrin latex antibody dispersion is 60-100:1, preferably 70-90:1, more preferably 75-85:1, preferably 78-82:1.
in another preferred embodiment, the volume to weight ratio (ml: mg) of the latex protection buffer to the second carboxypolystyrene microspheres in the second transferrin latex antibody dispersion is 60-100:1, preferably 70-90:1, more preferably 75-85:1, preferably 78-82:1.
in another preferred embodiment, the first transferrin latex antibody dispersion is prepared by:
adding 0.8-1.2mL of first carboxyl polystyrene microsphere latex containing 4.8-5.2wt% into a reaction vessel, adding 0.2-0.4mL of 3.1-3.5wt% EDC solution into the reaction vessel, vibrating, fully activating the first carboxyl polystyrene microsphere at 23-27 ℃ for 8-12min, dispersing the precipitate obtained by centrifugation with 1.8-2.2mL of 0.15-0.25mol/L MES buffer solution (pH 6.0-6.4), adding 3.8-4.2mg of transferrin antibody, fully reacting for 1.8-2.2h at 18-22 ℃, adding 1.8-2.2mL of 0.18-0.22wt% bovine serum albumin aqueous solution, fully blocking and reacting for 1.8-2.2h at 18-22 ℃, and fully dispersing the precipitate obtained by centrifugation with 3.8-4.2mL of latex protection buffer solution to obtain the first transferrin latex antibody dispersion.
In another preferred embodiment, the second transferrin latex antibody dispersion is prepared by:
adding 0.8-1.2mL of polystyrene microsphere latex containing 4.8-5.2wt% of second carboxyl into a reaction vessel, adding 0.2-0.4mL of 3.1-3.5wt% EDC solution into the reaction vessel, vibrating, fully activating the polystyrene microsphere with the second carboxyl at the temperature of 23-27 ℃ for 8-12min, dispersing the precipitate obtained by centrifugation with 1.8-2.2mL of 0.15-0.25mol/L MES buffer solution (pH 6.0-6.4), adding 3.8-4.2mg of transferrin antibody, fully reacting for 1.8-2.2h at the temperature of 18-22 ℃, adding 1.8-2.2mL of 0.18-0.22wt% bovine serum albumin aqueous solution, fully blocking and reacting for 1.8-2.2h at the temperature of 18-22 ℃, and fully dispersing the precipitate obtained by centrifugation with latex protection buffer solution 3.8-4.2mL to obtain the second transferrin latex antibody dispersion.
In another preferred embodiment, the detection reagent R1 is a liquid.
In another preferred embodiment, the disodium hydrogen phosphate comprises disodium hydrogen phosphate dodecahydrate.
In another preferred embodiment, the sodium dihydrogen phosphate comprises sodium dihydrogen phosphate dihydrate.
In another preferred embodiment, the polyethylene glycol has a molecular weight of 200-10000.
In another preferred embodiment, the polyethylene glycol comprises polyethylene glycol-6000 (PEG-6000).
In another preferred embodiment, the surfactant comprises tween.
In another preferred embodiment, the tween is selected from the group consisting of: tween-20, tween-40, tween-60, tween-80, or a combination thereof.
In another preferred embodiment, the detection reagent R1 further comprises a preservative.
In another preferred embodiment, the preservative comprises sodium azide.
In another preferred embodiment, the disodium hydrogen phosphate is present in an amount of 0.4 to 0.5wt%, preferably 0.45 to 0.48wt%, based on the weight of the detection reagent R1.
In another preferred embodiment, the sodium dihydrogen phosphate is present in an amount of 0.04 to 0.08wt%, preferably 0.05 to 0.06wt%, based on the weight of the detection reagent R1.
In another preferred embodiment, the sodium chloride is present in an amount of 2.5 to 3.5wt%, preferably 2.7 to 3.1wt%, based on the weight of the detection reagent R1.
In another preferred embodiment, the polyethylene glycol is present in an amount of 1.5 to 2.1wt%, preferably 1.6 to 2.0wt% based on the weight of the detection reagent R1.
In another preferred embodiment, the surfactant is present in an amount of 0.15 to 0.25wt%, preferably 0.18 to 0.22wt%, based on the weight of the detection reagent R1.
In another preferred embodiment, the Tween is present in an amount of 0.15-0.25v/v%, preferably 0.18-0.22v/v%, based on the volume of the detection reagent R1.
In another preferred embodiment, the tween is present in an amount of 0.05 to 0.15 wt.%, preferably 0.08 to 0.12 wt.%, based on the weight of the detection reagent R1.
In another preferred embodiment, the reagent R1 to be detected is prepared by the following method:
disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polyethylene glycol, surfactant, water and optionally antiseptic are mixed to obtain detection reagent R1.
In another preferred embodiment, the reagent R1 to be detected is prepared by the following method:
dissolving 4.4-4.8g of disodium hydrogen phosphate, 0.5-0.65g of sodium dihydrogen phosphate and 28-31g of sodium chloride in 780-820mL of purified water, adding PEG-6000 17-19g, 1.8-2.2mL of Tween 20 and optionally 4.8-5.2mL of 18-22wt% sodium azide aqueous solution, mixing and dissolving, and then fixing the volume to 980-1020mL by using the purified water to obtain a detection reagent R1.
In a second aspect, the present invention provides the use of a transferrin assay kit according to the first aspect of the present invention for the preparation of a assay kit for the detection of transferrin in a sample.
In another preferred embodiment, the sample comprises a sample to be tested.
In another preferred embodiment, the sample contains transferrin to be tested.
In another preferred embodiment, the sample is urine or blood.
In another preferred embodiment, the sample is of human origin.
In another preferred embodiment, the transferrin comprises urinary transferrin.
In another preferred embodiment, the transferrin is human transferrin.
In another preferred embodiment, the assay is an in vitro assay.
In another preferred embodiment, the detection is an auxiliary detection.
In another preferred embodiment, the assays are non-diagnostic and non-therapeutic assays.
In another preferred embodiment, the detection is qualitative and/or quantitative.
In another preferred embodiment, the assay is an immunoturbidimetry assay.
In another preferred embodiment, the detection comprises biochemical analyzer detection.
In another preferred embodiment, the biochemical analyzer comprises a fully automated biochemical analyzer.
In another preferred embodiment, the parameters of the biochemical analyzer include one or more parameters selected from the group consisting of:
sample amount: 1-3 mu L;
amount of detection reagent R1: 200-220 mu L;
amount of detection reagent R2: 60-80 mu L;
reaction temperature: 35-37 ℃;
reaction time (T1/T2) 290-310 seconds/290-310 seconds;
detection wavelength: 560-580nm;
the reaction direction is as follows: an increment (upward); and/or
Standard curve fitting mode: 6-point scaling, nonlinear computation modes, such as log/Spline.
In another preferred embodiment, the detection wavelength is 570nm.
In another preferred embodiment, the method of detecting includes:
after the sample and the detection reagent R1 are subjected to incubation reaction, the detection reagent R2 is added for continuous incubation reaction, the absorbance of the first light measuring point is read first, then the absorbance of the second light measuring point is read, the difference value of the absorbance of the two light measuring points is the reactivity, and qualitative and/or quantitative detection is carried out on transferrin according to the reactivity.
In another preferred embodiment, the absorbance of the first photometric points are all absorbance at the same detection wavelength.
In another preferred embodiment, the time interval between the absorbance of the first photosite and the absorbance of the second photosite is 3.8-4.2min.
In another preferred embodiment, the incubation is carried out at a temperature of 35-37 ℃.
In another preferred embodiment, the incubation with the detection reagent R1 takes about 4.5 to 5.5 minutes.
In another preferred embodiment, the incubation time with the addition of the detection reagent R2 is 0.8-1.2min.
In another preferred embodiment, the method of detecting includes:
mixing and incubating the sample and the detection reagent R1 at 35-37 ℃ for 4.5-5.5min, adding the detection reagent R2, continuing the incubation, reading the absorbance of the first light measuring point after 0.8-1.2min, reading the absorbance of the second light measuring point after 4.5-5.5min, and carrying out qualitative and/or quantitative detection on transferrin according to the reactivity, wherein the difference value of the absorbance of the two light measuring points is the reactivity.
In another preferred embodiment, the method of detecting includes:
the sample adding amount of the sample, the sample adding amount of the detection reagent R1 and the sample adding amount of the detection reagent R2 are respectively 1-3 mu L, 200-220 mu L and 60-80 mu L in sequence, the sample and the sample are mixed and incubated at the temperature of 35-37 ℃ for 4.5-5.5min, then the sample and the sample are added with the detection reagent R2 for continuous incubation, the absorbance of the first light measuring point is read after 0.8-1.2min, the absorbance of the second light measuring point is read after 4.5-5.5min, the difference value of the absorbance of the two light measuring points is the reactivity, and the qualitative and/or quantitative detection of transferrin is carried out according to the reactivity.
In another preferred embodiment, the absorbance of the first photometric point is the absorbance after 0.8-1.2min of the reaction with the addition of the detection reagent R2.
In another preferred example, the absorbance of the second photometric point is the absorbance after 4.5-5.5min of the reaction with the addition of the detection reagent R2.
In another preferred embodiment, the quantitative detection comprises a standard curve method detection.
In another preferred embodiment, the standard curve assay comprises plotting a standard curve based on the concentration and reactivity of different transferrin standard solutions.
In another preferred embodiment, the abscissa of the standard curve is the concentration of different transferrin standard solutions and the ordinate is the reactivity.
In another preferred embodiment, the different transferrin standard solutions are prepared by the following method:
different amounts of human transferrin standard are added to the standard dilutions, thereby preparing different transferrin standard solutions.
In another preferred embodiment, the standard diluent comprises:
disodium hydrogen phosphate 4.4-4.8g/L, sodium dihydrogen phosphate 0.5-0.7g/L, sodium chloride 2.4-3.0wt%, bovine serum albumin 0.6-1.0wt%, sucrose 1.8-2.2wt%, trehalose 1.8-2.2wt% and optionally 4.8-3.2wt% sodium azide (18-22 wt%) with water as the solvent.
In another preferred embodiment, the reactivity of transferrin in the sample is substituted into the standard curve, and the transferrin content in the sample is calculated.
In a third aspect of the invention there is provided a method of detecting transferrin in a sample, the method comprising detecting transferrin in a sample with a transferrin detection kit according to the first aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a standard curve of human transferrin concentration versus reactivity in standard solutions.
Detailed Description
The invention develops a transferrin detection kit, which comprises a detection reagent for detecting transferrin, wherein the detection reagent comprises a detection reagent R1 and a detection reagent R2. The transferrin detection kit can perform qualitative and quantitative determination on transferrin in a detection sample (such as urine), and has the advantages of high detection repeatability, high accuracy, strong stability, coverage of most clinical ultrahigh-value samples, prevention of false negative results caused by excessive antigens and the like.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only open-ended definitions, but also semi-closed, and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, the term "PEG" refers to polyethylene glycol.
As used herein, the term "tween" is a polyoxyethylene sorbitan fatty acid ester.
As used herein, the term "MES" refers to 2- (N-morpholino) ethanesulfonic acid.
As used herein, the term "EDC" refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
As used herein, the term "wt%" refers to weight percent.
As used herein, the term "v/v%" refers to volume percent.
In the present invention, 20wt% sodium azide aqueous solution means that the weight percentage of sodium azide in the sodium azide aqueous solution is 20wt%.
As used herein, the terms "first" and "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated.
Transferrin detection kit
The invention provides a transferrin detection kit, which comprises a detection reagent for detecting transferrin, wherein the detection reagent comprises a detection reagent R1 and a detection reagent R2;
the detection reagent R1 comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polyethylene glycol, a surfactant and water;
the detection reagent R2 comprises a first transferrin latex antibody dispersion liquid and a second transferrin latex antibody dispersion liquid;
the first transferrin latex antibody dispersion comprises transferrin antibodies and first carboxyl polystyrene microspheres, wherein the transferrin antibodies are coupled to the first carboxyl polystyrene microspheres;
the second transferrin latex antibody dispersion comprises transferrin antibodies and second carboxyl polystyrene microspheres, and the transferrin antibodies are coupled to the second carboxyl polystyrene microspheres.
In a preferred embodiment of the present invention, the particle size of the first carboxyl polystyrene microsphere is 40 to 100nm, preferably 50 to 90nm, more preferably 60 to 80nm, still more preferably 65 to 85nm.
In a preferred embodiment of the present invention, the particle size of the second carboxyl polystyrene microsphere is 150 to 250nm, preferably 170 to 230nm, more preferably 185 to 210nm, and even more preferably 190 to 200nm.
In a preferred embodiment of the present invention, the weight ratio of the first carboxyl polystyrene microsphere to the second carboxyl polystyrene microsphere is 1:0.5-5, preferably 1:0.5-3, more preferably 1:1-2, more preferably 1:1.3-1.7, and most preferably 1:1.5.
In a preferred embodiment of the invention, the volume ratio of the first transferrin latex antibody dispersion to the second transferrin latex antibody dispersion is 1:0.5-5, preferably 1:0.5-3, more preferably 1:1-2, more preferably 1:1.3-1.7, most preferably 1:1.5.
In a preferred embodiment of the present invention, the dispersion medium in the first transferrin latex antibody dispersion comprises a latex protection buffer.
In a preferred embodiment of the present invention, the dispersion medium in the second transferrin latex antibody dispersion comprises a latex protection buffer.
In a preferred embodiment of the present invention, the latex protection buffer comprises an aqueous solution of phosphate buffer, serum albumin, sodium chloride and sucrose.
In another preferred embodiment, the latex protection buffer comprises an aqueous solution of phosphate buffer, serum albumin, sodium chloride, sucrose, and sodium azide.
In a preferred embodiment of the invention, the pH of the latex protection buffer is 7.0-7.4, preferably 7.2.
More specifically, the transferrin detection kit according to the present invention is as described above in the first aspect of the present invention.
Use and method
The invention also provides application of the transferrin detection kit, which is used for preparing the detection kit for detecting transferrin in a sample.
The invention also provides a method of detecting transferrin in a sample, the method comprising detecting transferrin in a sample with a transferrin detection kit according to the invention.
In a preferred embodiment of the invention, the sample is urine or blood.
In another preferred embodiment, the assay is an in vitro assay.
In another preferred embodiment, the detection is an auxiliary detection.
In another preferred embodiment, the assays are non-diagnostic and non-therapeutic assays.
In another preferred embodiment, the detection is qualitative and/or quantitative.
In another preferred embodiment, the assay is an immunoturbidimetry assay.
In another preferred embodiment, the detection comprises biochemical analyzer detection.
In another preferred embodiment, the biochemical analyzer comprises a fully automated biochemical analyzer.
In a preferred embodiment of the present invention, the parameters of the biochemical analyzer include one or more parameters selected from the group consisting of:
sample amount: 1-3 mu L;
amount of detection reagent R1: 200-220 mu L;
amount of detection reagent R2: 60-80 mu L;
reaction temperature: 35-37 ℃;
reaction time (T1/T2) 290-310 seconds/290-310 seconds;
detection wavelength: 560-580nm;
the reaction direction is as follows: an increment (upward); and/or
Standard curve fitting mode: 6-point scaling, nonlinear computation modes, such as log/Spline.
In another preferred embodiment, the detection wavelength is 570nm.
In a preferred embodiment of the present invention, the method of detection includes:
after the sample and the detection reagent R1 are subjected to incubation reaction, the detection reagent R2 is added for continuous incubation reaction, the absorbance of the first light measuring point is read first, then the absorbance of the second light measuring point is read, the difference value of the absorbance of the two light measuring points is the reactivity, and qualitative and/or quantitative detection is carried out on transferrin according to the reactivity.
In another preferred embodiment, the absorbance of the first photometric points are all absorbance at the same detection wavelength.
In another preferred embodiment, the time interval between the absorbance of the first photosite and the absorbance of the second photosite is 3.8-4.2min.
In another preferred embodiment, the incubation is carried out at a temperature of 35-37 ℃.
In another preferred embodiment, the incubation with the detection reagent R1 takes about 4.5 to 5.5 minutes.
In another preferred embodiment, the incubation time with the addition of the detection reagent R2 is 0.8-1.2min.
In another preferred embodiment, the method of detecting includes:
mixing and incubating the sample and the detection reagent R1 at 35-37 ℃ for 4.5-5.5min, adding the detection reagent R2, continuing the incubation, reading the absorbance of the first light measuring point after 0.8-1.2min, reading the absorbance of the second light measuring point after 4.5-5.5min, and carrying out qualitative and/or quantitative detection on transferrin according to the reactivity, wherein the difference value of the absorbance of the two light measuring points is the reactivity.
In another preferred embodiment, the method of detecting includes:
the sample adding amount of the sample, the sample adding amount of the detection reagent R1 and the sample adding amount of the detection reagent R2 are respectively 1-3 mu L, 200-220 mu L and 60-80 mu L in sequence, the sample and the sample are mixed and incubated at the temperature of 35-37 ℃ for 4.5-5.5min, then the sample and the sample are added with the detection reagent R2 for continuous incubation, the absorbance of the first light measuring point is read after 0.8-1.2min, the absorbance of the second light measuring point is read after 4.5-5.5min, the difference value of the absorbance of the two light measuring points is the reactivity, and the qualitative and/or quantitative detection of transferrin is carried out according to the reactivity.
In another preferred embodiment, the absorbance of the first photometric point is the absorbance after 0.8-1.2min of the reaction with the addition of the detection reagent R2.
In another preferred example, the absorbance of the second photometric point is the absorbance after 4.5-5.5min of the reaction with the addition of the detection reagent R2.
In a preferred embodiment of the invention, the quantitative detection comprises standard curve detection.
In another preferred embodiment, the standard curve assay comprises plotting a standard curve based on the concentration and reactivity of different transferrin standard solutions.
In another preferred embodiment, the abscissa of the standard curve is the concentration of different transferrin standard solutions and the ordinate is the reactivity.
In another preferred embodiment, the different transferrin standard solutions are prepared by the following method:
different amounts of human transferrin standard are added to the standard dilutions, thereby preparing different transferrin standard solutions.
In another preferred embodiment, the standard diluent comprises:
disodium hydrogen phosphate 4.4-4.8g/L, sodium dihydrogen phosphate 0.5-0.7g/L, sodium chloride 2.4-3.0wt%, bovine serum albumin 0.6-1.0wt%, sucrose 1.8-2.2wt%, trehalose 1.8-2.2wt% and optionally 4.8-3.2wt% sodium azide (18-22 wt%) with water as the solvent.
In another preferred embodiment, the reactivity of transferrin in the sample is substituted into the standard curve, and the transferrin content in the sample is calculated.
The main effects of the invention include:
the invention develops a transferrin detection kit which can carry out qualitative and quantitative determination on transferrin in a detection sample (such as urine), has the advantages of high detection repeatability, high accuracy and strong stability, covers most clinical ultrahigh-value samples, avoids false negative results caused by excessive antigens and the like, and is simple and convenient to operate and easy to detect in a large scale.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1 determination of urinary transferrin
Sheep anti-human transferrin antibodies were purchased from Shanghai cymbal Leshi biotechnology limited.
1. Reagents and apparatus
1.1 reagents
1.1.1 reagent R1:
dissolving 4.65g of disodium hydrogen phosphate dodecahydrate, 0.56g of sodium dihydrogen phosphate dihydrate and 29.25g of sodium chloride in 800mL of purified water, adding PEG-6000 18g, 2mL of Tween 20 and 5mL of 20wt% sodium azide aqueous solution, mixing and dissolving, and then fixing the volume to 1000mL by using the purified water to obtain a reagent R1.
1.1.2 reagent R2:
latex protection buffer: the pH was determined to be 7.2 in an aqueous solution of 0.02mol/L phosphate buffer, 0.2wt% bovine serum albumin, 2.12wt% sodium chloride, 2wt% sucrose, 1wt% sodium azide.
The preparation method of the reagent R2 comprises the following steps:
adding 1mL of polystyrene microsphere latex containing 5wt% of carboxyl groups (JSR groups in Japan) into a reaction container, adding 0.3mL of 3.3wt% EDC solution into the reaction container, vibrating, fully activating the carboxyl polystyrene microspheres at 25 ℃ for 10min, dispersing the obtained precipitate by centrifugation with 2mL of 0.2mol/L MES buffer (pH 6.2), adding 4mg of goat anti-human transferrin antibody to fully react for 2h at 20 ℃, adding 2mL of 0.2wt% bovine serum albumin water solution into the obtained precipitate by centrifugation to fully seal and react for 2h at 20 ℃, and fully dispersing the obtained precipitate with 4mL of latex protection buffer to obtain transferrin latex antibody dispersion.
Wherein, the small particle size transferrin latex antibody dispersion liquid is prepared by adopting 70nm carboxyl polystyrene microsphere; the large-particle-size transferrin latex antibody dispersion liquid is prepared by adopting 195nm carboxyl polystyrene microspheres; the small particle size transferrin latex antibody dispersion and the large particle size transferrin latex antibody dispersion are mixed according to the volume ratio of 1:1.5 to obtain the reagent R2.
1.2. Instrument for measuring and controlling the intensity of light
Full-automatic biochemical analyzer, model: hitachi 7180.
The Hitachi 7180 biochemical analyzer belongs to a discrete multichannel analyzer, and can realize the detection of multiple samples and multiple items by automatically controlling the actions of sample and reagent feeding, damage incubation, photometry cleaning and the like, and the Hitachi 7180 biochemical analyzer is a multi-wavelength spectrophotometer capable of continuously monitoring absorbance of reaction liquid. The measurement principle is that the absorbance obtained according to the lambert beer law, namely after monochromatic light vertically penetrates through a solution with a certain thickness, is in direct proportion to the concentration of the solution.
2. Preparation of a Standard Curve
Standard curves were measured on a fully automated biochemical analyzer:
principle of measurement
The measurement principle is an immunoturbidimetry, which is an antigen-antibody binding dynamic measurement method. The basic principle is as follows: when an antigen and an antibody react in a special dilution system and the ratio is proper (the excessive amount of the antibody is generally regulated), the formed soluble immune complex is separated out from a liquid phase under the action of a polymerization promoter (polyethylene glycol and the like) in the dilution system to form particles, so that turbidity of a reaction solution occurs. When the concentration of the antibody is fixed, the amount of the immunocomplex formed increases with an increase in the amount of the antigen in the sample, and the turbidity of the reaction solution increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction liquid and comparing with a series of standard substances.
Setting parameters: sample size: 2. Mu.L; amount of reagent (R1/R2): 210. Mu.L/70. Mu.L, reaction temperature: 37 ℃; reaction time (T1/T2) 300 seconds/300 seconds; wavelength: 570nm; units: mg/L; the reaction direction is as follows: an increment (upward); standard curve fitting mode: 6-point scaling, nonlinear computation modes, such as log/Spline.
The standard dilutions were prescribed as follows:
disodium hydrogen phosphate 4.65g/L, sodium dihydrogen phosphate 0.56g/L, sodium chloride 2.7wt%, bovine serum albumin 0.8wt%, sucrose 2wt%, trehalose 2wt% and 5wt% sodium azide (20 wt%) and the solvent is water.
Preparation of standard solutions containing different concentrations of human transferrin: different amounts of human transferrin standard are added to the standard diluent to prepare standard solutions containing different concentrations of human transferrin.
The measuring method comprises the following steps:
the sample adding amounts of the standard substance solution containing the human transferrin with different concentrations, the reagent R1 and the reagent R2 are respectively 2 mu L, 210 mu L and 70 mu L in sequence, the standard substance solution containing the human transferrin with different concentrations and the reagent R1 are mixed and incubated at 37 ℃ for 5min, then the reagent R2 is added for continuous incubation, the absorbance of a first light measuring point is read after 1min, the absorbance of a second light measuring point is read after 5min, the difference value of the absorbance of the two light measuring points is the reactivity, a standard curve is drawn according to the concentration and the corresponding reactivity of the standard substance, and the relation between the concentration and the reactivity of the human transferrin in the standard substance solution is shown in table 1 and figure 1.
The absorbance of the first light measuring point is the reaction absorbance after 1min of reaction after adding the reagent R2, the absorbance of the second light measuring point is the reaction absorbance after 5min of reaction after adding the reagent R2, the absorbance value is enhanced along with the increase of time (antigen-antibody reaction), and the difference value of the two points is the reactivity under the standard concentration.
TABLE 1 relationship between human transferrin concentration and reactivity in standard solutions
As can be seen from Table 1 and FIG. 1, the concentration of human transferrin in the standard solution has a good linear relationship in the range of 0-60mg/L, and can be accurately used for quantitative determination of transferrin.
3. Repeatability assay
1mg/L or 5mg/L of human transferrin standard solution was taken, respectively, and 10 times of measurement was performed for each sample according to the method in "preparation of standard curve" and the reproducibility of the reagent at two concentrations was calculated, and the results are shown in Table 2 below.
TABLE 2 repeatability assay (reactivity)
As can be seen from Table 2, the reproducibility of both high and low values is less than 4%, and the low value is 3.3% at 1mg/L, which is superior to the 10% requirement of the general reagents on the market, so that the reagents R1 and R2 have excellent reproducibility for measuring transferrin by using an immunoturbidimetry, thereby improving the accuracy of the measurement.
The medical decision level of urine transferrin is 2mg/L, and the reagent has excellent repeatability at 1mg/L, and can improve the accuracy of low-value sample measurement in clinical measurement of samples, thereby improving the accuracy of measurement distributed around the medical decision level.
HOOK Effect
The high value of human transferrin is mixed in a low value urine sample according to a certain proportion, the composition concentration is 100mg/L,500mg/L,1000mg/L,2000mg/L,4000mg/L,6000mg/L and 8000mg/L urine sample, the measurement value is measured according to the method in the preparation of the standard curve of 2, and the result is shown in the following table 3.
TABLE 3 measurement of human transferrin at various concentrations
As can be seen from the test results in Table 3, the test values from 100mg/L to 8000mg/L are all greater than the linear 60mg/L of the reagent, and the reagent covers 99% of clinically ultrahigh-value samples, so that the reagent can greatly avoid false negative results caused by excessive antigens.
5. Measurement accuracy
Accuracy measurement: and (3) taking a verified reference substance, repeatedly detecting 3 times on a full-automatic biochemical analyzer in the preparation of a standard curve by using a reagent R1 and a reagent R2, taking a mean value (M) of test results, and calculating a relative deviation (B) according to a formula (1), wherein the relative deviation is less than or equal to 10%.
In the formula: m is the average value of the test result;
t-value of witness mark
The witness reference substances are: ERM-DA470K/IFCC human serum standard, source: european standard office. Wherein the Transferrin (TRF) target is 2.36.+ -. 0.08g/L.
The measuring method comprises the following steps: the ERM-DA470K/IFCC was diluted 100-fold and 1000-fold with physiological saline, respectively, and the reagent R1 and the reagent R2 were taken, and were measured three times on a full-automatic biochemical analyzer according to the "preparation of standard curve" in the following manner, and the mean value (M) of the test results was taken, and the relative deviation (B) was calculated according to the formula (1), and the data are shown in Table 4 below:
table 4 measurement accuracy
Dilution factor 1000 times 100 times of
Measurement value 1 2.26 23.22
Measurement value 2 2.23 23.21
Measurement value 3 2.28 23.16
Average value of 2.26 23.20
Target value 2.36mg/L 23.6mg/L
Deviation of -4.38％ -1.71％
As can be seen from the results in Table 4, the measurement of the reagent R1 and the reagent R2 on the full-automatic biochemical analyzer showed that the international standard substance deviation was within 5%, indicating that the measurement accuracy of the reagent R1 and the reagent R2 on the full-automatic biochemical analyzer was high.
6. Stability of opening bottle
And (3) respectively placing the reagent R1 and the reagent R2 in reagent bottles, opening the reagent bottles, placing the reagent bottles in an instrument reagent tray, calibrating, reserving samples, observing for 14 days, and measuring and controlling the quality on a full-automatic biochemical analyzer in the preparation of a standard curve according to '2' every day. The results are shown in Table 5 below:
TABLE 5 stability to opening bottles
As can be seen from Table 5, the loss rate of reagent R1 and reagent R2 in 14 days after bottle opening is within 5%, and the bottle opening stability is good.
Comparative example 1
Comparative example 1 is the same as example 1, except that,
in the reagent R2, the small-particle-size transferrin latex antibody dispersion liquid and the large-particle-size transferrin latex antibody dispersion liquid are mixed according to the volume ratio of 1:1, 1:2 or 1:3 to obtain the reagent R2
Repeatability assay
Repeatability was measured according to the method of "3. Repeatability measurement" in example 1.
The results of reproducibility of the reagent R2 measurements obtained after mixing the small particle size transferrin latex antibody dispersion and the large particle size transferrin latex antibody dispersion in a volume ratio of 1:1, 1:2 or 1:3 are respectively shown in Table 6, table 7 and Table 8.
TABLE 6 mixing small particle size transferrin latex antibody dispersion and large particle size transferrin latex antibody dispersion at a volume ratio of 1:1 to obtain reproducible results of reagent R2 assay
TABLE 7 mixing small particle size transferrin latex antibody dispersion and large particle size transferrin latex antibody dispersion at a volume ratio of 1:2 to obtain reproducible results of reagent R2 assay
TABLE 8 mixing of small particle size transferrin latex antibody dispersions and large particle size transferrin latex antibody dispersions at a volume ratio of 1:3 gave reproducible results for reagent R2 assay
As can be seen from tables 6 to 8, the small particle size transferrin latex antibody dispersion and the large particle size transferrin latex antibody dispersion were mixed in a volume ratio of 1:1, 1:2 or 1:3 to give a poor reproducibility of the reagent R2 assay, with CV greater than 2.0%.
HOOK effect
The HOOK effect was measured according to the method of "4.hook effect" in example 1.
The HOOK effects measured by the small particle size transferrin latex antibody dispersion and the large particle size transferrin latex antibody dispersion mixed in a volume ratio of 1:1, 1:2 or 1:3 to obtain the reagent R2 are shown in tables 9, 10 and 11, respectively.
TABLE 9 HOOK Effect measured with reagent R2 after mixing small particle size transferrin latex antibody dispersion and large particle size transferrin latex antibody dispersion in a volume ratio of 1:1
Concentration (mg/L) Measuring value (mg/L)
100 101.22
500 95.43
1000 96.15
2000 84.37
4000 77.66
6000 72.14
8000 70.18
TABLE 10 HOOK Effect measured with reagent R2 after mixing small particle size transferrin latex antibody dispersion and large particle size transferrin latex antibody dispersion in a volume ratio of 1:2
TABLE 10 HOOK Effect measured with reagent R2 after mixing small particle size transferrin latex antibody dispersion and large particle size transferrin latex antibody dispersion in a volume ratio of 1:3
Concentration (mg/L) Measuring value (mg/L)
100 80.13
500 71.25
1000 68.87
2000 61.24
4000 58.85
6000 51.24
8000 47.33
From the results, it can be seen that the HOOK effect measured by the reagent R2 obtained by mixing the small particle size transferrin latex antibody dispersion and the large particle size transferrin latex antibody dispersion at a volume ratio of 1:2 or 1:3 was poor.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (5)

1. A transferrin detection kit, which is characterized in that the kit is used for detecting urine, the kit comprises a detection reagent for detecting transferrin, the detection reagent comprises a detection reagent R1 and a detection reagent R2, and a standard substance diluent;
the detection reagent R1 is prepared by the following method:
dissolving 4.4-4.8g of disodium hydrogen phosphate, 0.5-0.65g of sodium dihydrogen phosphate and 28-31g of sodium chloride in 780-820mL of purified water, adding PEG-6000 17-19g, 1.8-2.2mL of Tween 20 and 4.8-5.2mL of 18-22wt% sodium azide aqueous solution, mixing and dissolving, and then fixing the volume to 980-1020mL by using purified water;
the detection reagent R2 is prepared by the following method:
adding first carboxyl polystyrene microspheres and second carboxyl polystyrene microspheres into a reaction container, adding 3.3wt% EDC solution into the reaction container, fully activating the first carboxyl polystyrene microspheres and the second carboxyl polystyrene microspheres for 10min at 25 ℃, dispersing the obtained precipitate by centrifugation with 0.2mol/L MES buffer solution, adding goat anti-human transferrin antibody for fully reacting for 2h at 20 ℃, adding 0.2wt% bovine serum albumin aqueous solution into the obtained precipitate by centrifugation for fully sealing reaction for 2h at 20 ℃, and fully dispersing the obtained precipitate with latex protection buffer solution to obtain transferrin latex antibody dispersion;
the particle size of the first carboxyl polystyrene microsphere is 65-85nm;
the particle size of the second carboxyl polystyrene microsphere is 190-200nm;
the weight ratio of the first carboxyl polystyrene microsphere to the second carboxyl polystyrene microsphere is 1:1.3-1.7;
the components of the standard diluent are as follows:
disodium hydrogen phosphate 4.65g/L, sodium dihydrogen phosphate 0.56g/L, sodium chloride 2.7wt%, bovine serum albumin 0.8wt%, sucrose 2wt%, trehalose 2wt% and 5wt% sodium azide, the solvent is water.
2. The transferrin assay kit of claim 1, wherein the weight ratio of the first carboxypolystyrene microsphere to the second carboxypolystyrene microsphere is 1:1.5.
3. Use of a transferrin assay kit according to claim 1 for preparing a assay kit for detecting transferrin in a sample.
4. The use according to claim 3, wherein the assay is an immunoturbidimetry assay.
5. A method of detecting transferrin in a sample, the method comprising detecting transferrin in a sample with the transferrin detection kit of claim 1, the kit for detecting urine.
CN202111323621.XA 2021-11-10 2021-11-10 Urine transferrin detection kit and detection method thereof Active CN114047338B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111323621.XA CN114047338B (en) 2021-11-10 2021-11-10 Urine transferrin detection kit and detection method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111323621.XA CN114047338B (en) 2021-11-10 2021-11-10 Urine transferrin detection kit and detection method thereof

Publications (2)

Publication Number Publication Date
CN114047338A CN114047338A (en) 2022-02-15
CN114047338B true CN114047338B (en) 2024-03-12

Family

ID=80207915

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111323621.XA Active CN114047338B (en) 2021-11-10 2021-11-10 Urine transferrin detection kit and detection method thereof

Country Status (1)

Country Link
CN (1) CN114047338B (en)

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5522192A (en) * 1978-08-02 1980-02-16 Hoffmann La Roche Immunine reagent
US4342739A (en) * 1979-01-09 1982-08-03 Fuji Photo Film Co., Ltd. Novel material for immunological assay of biochemical components and a process for the determination of said components
WO2010011315A2 (en) * 2008-07-25 2010-01-28 Viral Genetics, Inc. Proteins for use in diagnosing and treating infection and disease
CN102128924A (en) * 2010-01-12 2011-07-20 上海景源医疗器械有限公司 Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof
CN102628867A (en) * 2011-12-30 2012-08-08 北京九强生物技术股份有限公司 Double antibody latex enhanced retinol binding protein detection kit
CN102628864A (en) * 2011-12-30 2012-08-08 北京九强生物技术股份有限公司 Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay
WO2012123419A1 (en) * 2011-03-11 2012-09-20 Vib Vzw Molecules and methods for inhibition and detection of proteins
CN103954766A (en) * 2014-01-24 2014-07-30 武汉生之源生物科技有限公司 Transferring receptor detection kit and preparation method thereof
CN104215770A (en) * 2014-08-14 2014-12-17 上海睿康生物科技有限公司 Two-particle-based retinol binding protein detection kit
CN105044352A (en) * 2015-07-01 2015-11-11 南京普朗医疗设备有限公司 Urinary transferrin immuno-turbidimetry reagent and preparation method thereof
CN105675887A (en) * 2016-04-13 2016-06-15 柏荣诊断产品（上海）有限公司 High-performance human urine microalbumin detection kit
CN106053838A (en) * 2016-07-12 2016-10-26 安徽伊普诺康生物技术股份有限公司 Kit for determining transferrin and preparation method thereof
CN106324239A (en) * 2016-07-28 2017-01-11 武汉景川诊断技术股份有限公司 preparation method and application of C-reactive protein immuno latex microsphere
CN106918708A (en) * 2015-12-28 2017-07-04 北京九强生物技术股份有限公司 A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin
CN106932588A (en) * 2015-12-30 2017-07-07 上海复星长征医学科学有限公司 Detection α1Kit of-microglobulin and preparation method thereof
CN107643282A (en) * 2016-07-20 2018-01-30 长春市诺克因生物科技有限公司 A kind of kit of Chemiluminescence quantitative immunoassay detection urinary metabolites and preparation method thereof
CN107727867A (en) * 2017-09-30 2018-02-23 安徽伊普诺康生物技术股份有限公司 A kind of preparation method of Complement C_3 detection kit
CN108593940A (en) * 2018-07-20 2018-09-28 迪瑞医疗科技股份有限公司 A kind of urine transferrins detection kit
CN108761070A (en) * 2018-05-03 2018-11-06 柏荣诊断产品（上海）有限公司 A kind of urine transferrins detection kit of width detection range
CN109613257A (en) * 2018-12-01 2019-04-12 山东博科生物产业有限公司 A kind of transferrins (TRF) detection kit
CN109813910A (en) * 2018-12-29 2019-05-28 宁波普瑞柏生物技术股份有限公司 The method and kit for eliminating hook effect are reacted using competitive immunization
CN110133270A (en) * 2018-02-09 2019-08-16 长沙文瀚生物技术有限责任公司 A kind of enhancing immunosupress is than turbid antibody screening reagent and its making and use method
CN110596405A (en) * 2019-09-20 2019-12-20 上海捷门生物技术有限公司 Kit for detecting content of heart-type fatty acid binding protein by latex enhanced immunoturbidimetry
CN111766381A (en) * 2020-06-11 2020-10-13 武汉生之源生物科技股份有限公司 Determination kit based on latex immunoturbidimetry and application thereof
CN112129934A (en) * 2020-09-25 2020-12-25 上海捷门生物技术有限公司 Ferritin detection kit and preparation method thereof
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

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5522192A (en) * 1978-08-02 1980-02-16 Hoffmann La Roche Immunine reagent
US4342739A (en) * 1979-01-09 1982-08-03 Fuji Photo Film Co., Ltd. Novel material for immunological assay of biochemical components and a process for the determination of said components
WO2010011315A2 (en) * 2008-07-25 2010-01-28 Viral Genetics, Inc. Proteins for use in diagnosing and treating infection and disease
CN102128924A (en) * 2010-01-12 2011-07-20 上海景源医疗器械有限公司 Agent kit for measuring urinary transferrin by latex-enhanced immunoturbidimetry and preparation method thereof
WO2012123419A1 (en) * 2011-03-11 2012-09-20 Vib Vzw Molecules and methods for inhibition and detection of proteins
CN102628864A (en) * 2011-12-30 2012-08-08 北京九强生物技术股份有限公司 Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay
CN102628867A (en) * 2011-12-30 2012-08-08 北京九强生物技术股份有限公司 Double antibody latex enhanced retinol binding protein detection kit
CN103954766A (en) * 2014-01-24 2014-07-30 武汉生之源生物科技有限公司 Transferring receptor detection kit and preparation method thereof
CN104215770A (en) * 2014-08-14 2014-12-17 上海睿康生物科技有限公司 Two-particle-based retinol binding protein detection kit
CN105044352A (en) * 2015-07-01 2015-11-11 南京普朗医疗设备有限公司 Urinary transferrin immuno-turbidimetry reagent and preparation method thereof
CN106918708A (en) * 2015-12-28 2017-07-04 北京九强生物技术股份有限公司 A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin
CN106932588A (en) * 2015-12-30 2017-07-07 上海复星长征医学科学有限公司 Detection α1Kit of-microglobulin and preparation method thereof
CN105675887A (en) * 2016-04-13 2016-06-15 柏荣诊断产品（上海）有限公司 High-performance human urine microalbumin detection kit
CN106053838A (en) * 2016-07-12 2016-10-26 安徽伊普诺康生物技术股份有限公司 Kit for determining transferrin and preparation method thereof
CN107643282A (en) * 2016-07-20 2018-01-30 长春市诺克因生物科技有限公司 A kind of kit of Chemiluminescence quantitative immunoassay detection urinary metabolites and preparation method thereof
CN106324239A (en) * 2016-07-28 2017-01-11 武汉景川诊断技术股份有限公司 preparation method and application of C-reactive protein immuno latex microsphere
CN107727867A (en) * 2017-09-30 2018-02-23 安徽伊普诺康生物技术股份有限公司 A kind of preparation method of Complement C_3 detection kit
CN110133270A (en) * 2018-02-09 2019-08-16 长沙文瀚生物技术有限责任公司 A kind of enhancing immunosupress is than turbid antibody screening reagent and its making and use method
CN108761070A (en) * 2018-05-03 2018-11-06 柏荣诊断产品（上海）有限公司 A kind of urine transferrins detection kit of width detection range
CN108593940A (en) * 2018-07-20 2018-09-28 迪瑞医疗科技股份有限公司 A kind of urine transferrins detection kit
CN109613257A (en) * 2018-12-01 2019-04-12 山东博科生物产业有限公司 A kind of transferrins (TRF) detection kit
CN109813910A (en) * 2018-12-29 2019-05-28 宁波普瑞柏生物技术股份有限公司 The method and kit for eliminating hook effect are reacted using competitive immunization
CN110596405A (en) * 2019-09-20 2019-12-20 上海捷门生物技术有限公司 Kit for detecting content of heart-type fatty acid binding protein by latex enhanced immunoturbidimetry
CN111766381A (en) * 2020-06-11 2020-10-13 武汉生之源生物科技股份有限公司 Determination kit based on latex immunoturbidimetry and application thereof
CN112129934A (en) * 2020-09-25 2020-12-25 上海捷门生物技术有限公司 Ferritin detection kit and preparation method thereof
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Dissociation of Hepatitis B Surface Antigen (HBsAg) from Immobilised Anti—HBsAg Antibody in a Two Site Immunoradiometric Assay: Its Relevance to Hook Effect and Recycled Assay;S L Ch\'ng 等;J Immunoassay;20061206;8(2-3);全文 *
双抗体夹心ELISA法检测嗜酸性粒细胞阳离子蛋白;赵俊芳;王学谦;李桂珍;李会强;;临床检验杂志;20110815(05);全文 *
血清和尿液中黄醇结合蛋白胶乳增强免疫比浊试剂盒的制备及性能评价;方亮;王芬;刘献文;邬期望;;国际检验医学杂志;20180115(01);全文 *

Also Published As

Publication number Publication date
CN114047338A (en) 2022-02-15

Similar Documents

Publication Publication Date Title
US4615972A (en) Stabilization of indicators for detecting enzyme activity
Lubran The measurement of total serum proteins by the Biuret method
JP3523878B2 (en) Cyanide-free reagent and method for determination of hemoglobin
CN102998445B (en) Reagent and preparation method for determining glycocholic acid
Loontiens et al. Binding of 4-methylumbelliferyl α-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric concanavalin A: equilibrium studies
CN101377492A (en) Bladder chalone C determining reagent kit
CN102621332A (en) Retinol binding protein assay kit based on latex particle coating
CN112526134A (en) Chitinase 3-like protein 1 determination kit
CN107907691A (en) A kind of myoglobin assay kit and its application method
CN106932594A (en) A kind of preferred cystatin C detection kit and preparation method thereof
CN112946255A (en) Reagent or kit for detecting magnetic particle chemiluminescence of sialorrhea liquefied sugar chain antigen and application of reagent or kit
CN103954766B (en) Transferring receptor detection kit and preparation method thereof
CN107228940B (en) A kind of detection reagent and method of cystatin C
CN108593940B (en) Urine transferrin detect reagent box
CN110383067A (en) Antihuman hemoglobin monoclonal antibody or antibody kit, antihuman hemoglobin monoclonal antibody immobilization examination with insoluble carrier particle and measurement reagent or measuring method using them
CN107942068B (en) β2Microglobulin assay kit
CN114047338B (en) Urine transferrin detection kit and detection method thereof
CN111721934B (en) Improved specific growth factor detection kit and application thereof
CN111190003A (en) Retinol binding protein detection kit and preparation method thereof
US3667915A (en) Colorimetric methods and compositions for determining iron in blood
CN109959797A (en) A kind of Procalcitonin detection kit and preparation method thereof
CN106443004A (en) Time-resolved fluorescence immunochromatographic reagent for rapidly, quantitatively and simultaneously detecting cTnI and H-FABP, and preparation method thereof
CN115343483A (en) Kit for detecting autoimmune diabetes and preparation method thereof
US4211531A (en) Colorimetric cholesterol assay
CN110702892A (en) Special reagent for measuring hemoglobin concentration for blood cell analyzer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CB03 Change of inventor or designer information

Inventor after: Chen Xiao

Inventor before: Chen Yitao

Inventor before: Mao Xiaobo

Inventor before: Gu Lili

Inventor before: Wang Lijiao

CB03 Change of inventor or designer information