CN110031631B - Stable high-specificity haptoglobin detection kit - Google Patents

Stable high-specificity haptoglobin detection kit Download PDF

Info

Publication number
CN110031631B
CN110031631B CN201910261110.6A CN201910261110A CN110031631B CN 110031631 B CN110031631 B CN 110031631B CN 201910261110 A CN201910261110 A CN 201910261110A CN 110031631 B CN110031631 B CN 110031631B
Authority
CN
China
Prior art keywords
reagent
haptoglobin
stable
kit
specificity
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
CN201910261110.6A
Other languages
Chinese (zh)
Other versions
CN110031631A (en
Inventor
甘宜梧
胡晓飞
李静
周东
罗维晓
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.)
Biobase Biodustry Shandong Co Ltd
Original Assignee
Biobase Biodustry Shandong 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 Biobase Biodustry Shandong Co Ltd filed Critical Biobase Biodustry Shandong Co Ltd
Priority to CN201910261110.6A priority Critical patent/CN110031631B/en
Publication of CN110031631A publication Critical patent/CN110031631A/en
Application granted granted Critical
Publication of CN110031631B publication Critical patent/CN110031631B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses a stable and strong-specificity haptoglobin detection kit, and relates to the technical field of biology. The detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: 10-100 mmol/L of buffer solution, 1 part of surfactant: 5-10 ml/L, 5-10 g/L of inorganic salt ions, 20-30 g/L of accelerator, 2: 2-20 g/L of isothiazolinone, 1-10 g/L of isothiazolinone and 0.5-1 g/L of preservative; the components and contents of the reagent R2 are as follows: 10-100 mmol/L of buffer solution, 5-10 mg/L of goat anti-human haptoglobin antibody, 5-10 g/L of bovine serum albumin, and polyether L-65: 0.5-1 g/L, 10-20 g/L of D-trehalose and 0.5-1 g/L of preservative. The invention has strong anti-interference capability, good stability and strong specificity.

Description

Stable high-specificity haptoglobin detection kit
Technical Field
The invention relates to the technical field of biology, in particular to a stable and strong-specificity haptoglobin detection kit.
Background
Haptoglobin (Hp) is an acidic glycoprotein with a molecular weight of 85000, widely exists in serum and other body fluids of human beings and many mammals, and is located in an alpha 2 zone in CAM electrophoresis and agarose gel electrophoresis, and two pairs of peptide chains (alpha chain and beta chain) in a molecule form a tetramer of alpha 2 beta 2. Haptoglobin is mainly synthesized in the liver, and is degraded in the liver, and the half-life period is about 3.5-4 days.
Hp and CRP belong to the same acute-phase reaction protein, and the main function of the protein is to form an Hp-Hb compound by combining with free hemoglobin (Hb), and the Hp-Hb compound is quickly cleared away by scavenger receptor CD163 mediated by a mononuclear-macrophage system, so that the oxidative damage of Hb to tissues is prevented, the Hb is prevented from being filtered from glomerulus, and the damage of Hb to renal tubules is avoided. Hp has antioxidant activity, antiinflammatory, antibacterial, and angiogenesis promoting effects.
The normal haptoglobin content in serum is 0.5-2.0 g/L, and if the haptoglobin level is changed, the occurrence of diseases can be marked or the development of diseases can be resisted. The increase is seen in pregnancy, chronic infection, etc.; reduce the symptoms of various hemolysis, liver diseases or megaloblastic anemia. Proteomics has become a new tool for diagnosing diabetic nephropathy. Among them, haptoglobin is closely related to the occurrence and development of diabetic nephropathy, so that it becomes a new biomarker for predicting renal function decline in early stage of diabetes.
The current methods for clinical detection of haptoglobin are hemoglobin binding and immunoturbidimetry. The hemoglobin binding method is characterized in that a rocket electrophoresis method is adopted for detection according to the equimolar binding characteristics of binding globin and hemoglobin, namely, excessive hemoglobin is added into detected serum, the binding globin and the hemoglobin in the serum are combined to generate a compound, two zones of the hemoglobin and the compound are separated after rocket electrophoresis, and the concentration of HP is calculated through a series of operations. Compared with a hemoglobin binding method, the immunoturbidimetry method has the advantages of simplicity in operation and small detection result error, but the existing immunoturbidimetry and globin detection kit is poor in anti-interference capability, stability and specificity and needs to be further improved.
Disclosure of Invention
The invention provides a stable and strong-specificity haptoglobin detection kit which is strong in anti-interference capability, good in stability and strong in specificity.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a stable and strong-specificity kit for detecting haptoglobin, which comprises a reagent R1 and a reagent R2, wherein:
the components and contents of the reagent R1 are as follows:
Figure BDA0002015301600000021
the components and contents of the reagent R2 are as follows:
Figure BDA0002015301600000022
further, the components and contents of the reagent R1 are as follows:
Figure BDA0002015301600000023
Figure BDA0002015301600000031
the components and contents of the reagent R2 are as follows:
Figure BDA0002015301600000032
further, the buffer solution is one or more of PBS buffer solution, glycine buffer solution and Tris buffer solution, the temperature of the buffer solution in the reagent R1 and the reagent R2 is 25 ℃, and the pH value is 7.4.
Further, the surfactant 1 is Triton X-100, and the surfactant 2 is polyoxyethylene lauryl ether.
Further, the inorganic salt ions are one or more of sodium chloride, potassium chloride and magnesium chloride.
Further, the accelerator is one or a combination of polyethylene glycol-2000, polyethylene glycol-6000 and polyethylene glycol-8000.
Further, the preservative is PC-300.
Further, the ratio of the reagent R1 to the reagent R2 is 4: 1.
Compared with the prior art, the invention has the following beneficial effects:
1) the repeatability is good: the variation coefficient CV of the haptoglobin kit is less than 5 percent, and the kit has better repeatability;
2) the stability and the anti-interference performance are high: the invention adopts a new buffer system and a stabilizer and adds a plurality of anti-interference agents, can effectively resist the interference of factors such as VC, bilirubin, hemoglobin, triglyceride and the like, and obviously improves the stability and anti-interference of the reagent;
3) the specificity is strong: the goat anti-human haptoglobin antibody in the haptoglobin detection kit can be specifically combined with HP, so that the accuracy is improved, the use is convenient, and the clinical requirements can be completely met.
Drawings
FIG. 1 is a graph showing the correlation between the kit of example 1 and the kit of a control group;
FIG. 2 is a graph showing the correlation between the kit of example 2 and the kit of a control group;
FIG. 3 is a graph showing the correlation between the kit of example 3 and the kit of a control group;
FIG. 4 is a graph showing the stability of the kits of examples 1 to 3 and a control group after treatment at 2 to 8 ℃;
FIG. 5 is a graph showing the stability of the kits of examples 1 to 3 and a control group after treatment at 37 ℃.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is made with reference to specific embodiments and accompanying drawings.
Example 1:
the stable and high-specificity haptoglobin detection kit comprises a reagent R1 and a reagent R2, wherein:
the components and contents of the reagent R1 are as follows:
Figure BDA0002015301600000041
the components and contents of the reagent R2 are as follows:
Figure BDA0002015301600000051
the detection method comprises the following steps: the determination is carried out by a two-point endpoint method at a main wavelength of 340nm using a full-automatic biochemical analyzer (such as Hitachi 7180 full-automatic analyzer, OLYMPUS AU 640). Placing the reagent on the corresponding reagent position, placing the distilled water, the calibrator and the sample on the corresponding position of the sample tray, and operating as the following table 1:
table 1 example 1 reagent detection method
Figure BDA0002015301600000052
And (3) calculating: sample bound globin content (g/L) ═ Δ a assay ÷ Δ a calibration) × C calibration.
Example 2:
the stable and high-specificity haptoglobin detection kit comprises a reagent R1 and a reagent R2, wherein:
the components and contents of the reagent R1 are as follows:
Figure BDA0002015301600000053
Figure BDA0002015301600000061
the components and contents of the reagent R2 are as follows:
Figure BDA0002015301600000062
the detection method was the same as in example 1.
Example 3:
the stable and high-specificity haptoglobin detection kit comprises a reagent R1 and a reagent R2, wherein:
the components and contents of the reagent R1 are as follows:
Figure BDA0002015301600000063
the components and contents of the reagent R2 are as follows:
Figure BDA0002015301600000064
Figure BDA0002015301600000071
the detection method was the same as in example 1.
The control group adopts a haptoglobin detection kit of a company which is commonly accepted by the national food and drug administration on the market.
And (3) repeatability test:
control tests were performed using the reagents of example 1, example 2, example 3 and the control group, and the quality control samples (target value 1.52g/L) were each tested and repeated 10 times, and the coefficient of variation was calculated, and the test results are shown in Table 2.
TABLE 2 kit repeatability test results
Figure BDA0002015301600000072
As can be seen from Table 7, the reagents obtained in examples 1, 2 and 3 have a repetitive CV value of less than 2%, and the reagent in example 1 has a repetitive CV value of only 0.84%, which is far superior to that of the control reagent.
And (3) interference resistance test:
fresh mixed serum was divided into 4 aliquots, each aliquot was then subdivided into 5 aliquots, and different interferents were added to achieve the serum concentrations as specified in Table 3. Then, the content of haptoglobin in serum was simultaneously determined by comparison using the binding protein assay kits of example 1, example 2, example 3 and control group, respectively, and the determination results are shown in Table 3.
TABLE 3 interference test results
Figure BDA0002015301600000081
Relative deviation (%) - (measurement mean of example-measurement mean of control group)/measurement mean of control group × 100%.
As can be seen from Table 3, the reagent of example 1 does not significantly interfere with the test results when triglyceride is less than or equal to 50mg/dL, bilirubin is less than or equal to 1mg/dL, hemoglobin is less than or equal to 50mg/dL, and VC is less than or equal to 1 mg/dL; the anti-interference performance of the reagents of the embodiment 2 and the embodiment 3 is slightly weaker than that of the embodiment 1, but not more than 5%; the control reagent is significantly interfered with in the presence of the interfering substance at the above-mentioned concentration. The anti-interference performance of the haptoglobin detection kit is obviously higher than that of the haptoglobin detection kit in the existing market.
And (3) correlation test:
control tests were performed using the reagents of example 1, example 2, example 3 and the control group, and 20 clinical serum samples were simultaneously tested, and the test results are shown in table 4, and correlation curves between the kits of examples 1 to 3 and the kits of the control group, respectively, were obtained (as shown in fig. 1 to 3).
TABLE 4 correlation test results
Figure BDA0002015301600000091
Relative deviation (%) - (measurement mean of example-measurement mean of control group)/measurement mean of control group × 100%.
As can be seen from table 4 and fig. 1, the correlation coefficients of the pre-control groups of the reagents obtained in the above examples 1, 2 and 3 are 0.9999, 0.9999 and 0.9998, respectively, which illustrates that the haptoglobin assay kit of the present invention has great correlation with the haptoglobin assay kit of a certain company recognized by the national food and drug administration in the existing market, and the accuracy of the haptoglobin assay kit of the present invention is not significantly different from the accuracy of the haptoglobin assay kit of a certain company recognized by the national food and drug administration in the existing market, further indicating that the antibody of the haptoglobin assay kit of the present invention can be effectively combined with an antigen, and the kit has strong specificity and can meet clinical requirements.
And (3) stability detection:
the reagents of example 1, example 2 and example 3 were uniformly divided into 26 groups, and the reagents of the 26 control groups were obtained as controls. Wherein 13 groups are placed in a refrigerator at 2-8 deg.C, 13 groups are placed in a water bath at 37 deg.C, a group of reagent detection quality control articles (target value is 1.52g/L) are taken out at the same time every day, and the detection results are shown in Table 5, Table 6, attached figure 4 and attached figure 5:
TABLE 5 test results of the kit after 2-8 deg.C treatment
Figure BDA0002015301600000101
Figure BDA0002015301600000111
TABLE 6 detection results of the kit after 37 deg.C treatment
Figure BDA0002015301600000112
As can be seen from Table 5, the reagent stored at 2-8 ℃ in the kit of example 1 hardly decayed any more, and the kits of examples 2 and 3 decayed a little, but not more than 22%, after a lapse of 4 months. But the control agent decayed 11% at day 30 and had decayed 35% after 4 months.
As can be seen from table 6, under the condition of 37 degrees, the attenuation of the kit of example 1 was 7.9% at 30 days, and was approximately 50% after 4 months, and the attenuation of the kits of example 2 and example 3 was approximately 11% at 30 days, and was approximately 54% after 4 months. Whereas the control agent had decayed 8.5% at day 9 and around 85% after 4 months. The results shown in FIG. 2 and FIG. 3 show that the reagent of example 1 is more stable than the conventional HP detection kit in the market under the storage conditions of 2-8 ℃ and 37 ℃, but still has the defects.
In conclusion, the stable and high-specificity haptoglobin detection kit has better repeatability, stability, specificity and anti-interference performance.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1. A stable, highly specific kit for detecting haptoglobin, comprising reagent R1 and reagent R2, wherein:
the components and contents of the reagent R1 are as follows:
Figure FDA0002885773260000011
the components and contents of the reagent R2 are as follows:
Figure FDA0002885773260000012
2. the stable, highly specific haptoglobin assay kit of claim 1 wherein reagent R1 comprises the following components and amounts:
Figure FDA0002885773260000013
the components and contents of the reagent R2 are as follows:
Figure FDA0002885773260000021
3. the stable, highly specific haptoglobin assay kit of claim 1 or 2 wherein the buffer is one or a combination of PBS buffer, glycine buffer and Tris buffer and the buffers in reagents R1 and R2 are at 25 ℃ and PH 7.4.
4. The stable, highly specific haptoglobin assay kit of claim 1 or 2 wherein the inorganic salt ion is one or more of sodium chloride, potassium chloride and magnesium chloride in combination.
5. The stable, highly specific haptoglobin assay kit of claim 1 or 2, wherein the accelerator agent is one or a combination of polyethylene glycol-2000, polyethylene glycol-6000 and polyethylene glycol-8000.
6. The stable, highly specific haptoglobin assay kit of claim 1 or 2 wherein the preservative is PC-300.
7. The stable, highly specific haptoglobin assay kit of claim 1 or 2 wherein reagent R1 and reagent R2 are present in a ratio of 4: 1.
CN201910261110.6A 2019-04-02 2019-04-02 Stable high-specificity haptoglobin detection kit Active CN110031631B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910261110.6A CN110031631B (en) 2019-04-02 2019-04-02 Stable high-specificity haptoglobin detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910261110.6A CN110031631B (en) 2019-04-02 2019-04-02 Stable high-specificity haptoglobin detection kit

Publications (2)

Publication Number Publication Date
CN110031631A CN110031631A (en) 2019-07-19
CN110031631B true CN110031631B (en) 2021-04-23

Family

ID=67237186

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910261110.6A Active CN110031631B (en) 2019-04-02 2019-04-02 Stable high-specificity haptoglobin detection kit

Country Status (1)

Country Link
CN (1) CN110031631B (en)

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101726579B (en) * 2008-10-17 2014-06-18 深圳迈瑞生物医疗电子股份有限公司 Blood test reagent and method
CN102507957A (en) * 2011-11-01 2012-06-20 北京利德曼生化股份有限公司 Method and kit for determining apolipoprotein A2 by using immunity transmission turbidimetric method
CN103694970B (en) * 2014-01-14 2016-06-08 河南金马石油科技有限责任公司 A kind of paraffin wax emulsions and preparation method thereof
CN104215632B (en) * 2014-08-28 2017-12-19 宁波瑞源生物科技有限公司 A kind of fatty enzyme reagent kit of stabilization
CN105548573A (en) * 2016-02-02 2016-05-04 潍坊三维生物工程集团有限公司 Kit and method for detecting content of KAPPA light chain and application
CN106053444A (en) * 2016-04-28 2016-10-26 安徽伊普诺康生物技术股份有限公司 A kit for measuring a specific growth factor and a preparing method thereof
CN106053839A (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 haptoglobin and preparation method thereof

Also Published As

Publication number Publication date
CN110031631A (en) 2019-07-19

Similar Documents

Publication Publication Date Title
Kouzuma et al. Study of glycated amino acid elimination reaction for an improved enzymatic glycated albumin measurement method
Wild et al. 12 Investigation of abnormal haemoglobins and thalassaemia
US8105840B2 (en) Urine pretreatment agent for urinary protein quantitation, urine pretreatment method, and urinary protein quantitation method
John et al. Enzyme immunoassay--a new technique for estimating hemoglobin A1c
JP5568636B2 (en) Analysis and assay of glycated hemoglobin by capillary electrophoresis, buffer composition and kit for capillary electrophoresis
CN109239059B (en) Glycated serum protein assay kit and preparation method and application thereof
Daniel et al. Quantification of hemoglobin A2 by tandem mass spectrometry
US20130236977A1 (en) Compositions and methods for plasma peptide analysis
HUT70463A (en) Immunological method for determination of hemoglobin-derivate
US8026078B2 (en) Method of quantifying glycosylated protein using redox reaction and quantification kit
US7361513B2 (en) Cellular controls for glycated hemoglobin Hb A1c
EP3264083B1 (en) L-fabp immunoassay method
EP0455225B1 (en) Method for measuring the percentage of glycation of a particular protein
HU188211B (en) Process and reagent for determining glycolyzed hemoglobine
CN110031631B (en) Stable high-specificity haptoglobin detection kit
Ikemoto et al. A useful ELISA system for human liver-type arginase, and its utility in diagnosis of liver diseases
Speeckaert et al. Evolution of vitamin D binding protein concentration in sera from cardiac surgery patients is determined by triglyceridemia
JPWO2020066722A1 (en) Hemoglobin measurement reagents, measurement kits and measurement methods
WO2020167411A1 (en) Calibrators and controls for the determination of percent glycated hemoglobin in a patient's liquid test sample
US20210381935A1 (en) Preservative solution for heme protein, and method for stabilizing heme protein
EP3421600B1 (en) Liver-type fatty acid-binding protein preparation and use thereof
EP3264084A1 (en) Immunoassay method and assay reagent used in said method
CN113125759A (en) Glycosylated hemoglobin detection kit
JP4896022B2 (en) Method and kit for immunochemical measurement of cytochrome c
JP7508494B2 (en) Hemoglobin measurement reagent, measurement kit and measurement method

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
PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Stable and specific binding Globin detection kit

Effective date of registration: 20230628

Granted publication date: 20210423

Pledgee: Bank of Communications Ltd. Shandong branch

Pledgor: BIOBASE BIODUSTRY (SHANDONG) Co.,Ltd.

Registration number: Y2023980045843

PC01 Cancellation of the registration of the contract for pledge of patent right
PC01 Cancellation of the registration of the contract for pledge of patent right

Granted publication date: 20210423

Pledgee: Bank of Communications Ltd. Shandong branch

Pledgor: BIOBASE BIODUSTRY (SHANDONG) Co.,Ltd.

Registration number: Y2023980045843