CN104833810A - Complement C3 detection method - Google Patents

Complement C3 detection method Download PDF

Info

Publication number
CN104833810A
CN104833810A CN201510226757.7A CN201510226757A CN104833810A CN 104833810 A CN104833810 A CN 104833810A CN 201510226757 A CN201510226757 A CN 201510226757A CN 104833810 A CN104833810 A CN 104833810A
Authority
CN
China
Prior art keywords
complement
reagent
latex
detection method
sample
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.)
Pending
Application number
CN201510226757.7A
Other languages
Chinese (zh)
Inventor
王贤俊
郑蓓蕾
郭二豪
江新涛
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to CN201510226757.7A priority Critical patent/CN104833810A/en
Publication of CN104833810A publication Critical patent/CN104833810A/en
Pending legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854Immunoglobulins

Landscapes

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

Abstract

The invention provides a complement C3 detection method, which is based on the latex-enhanced immune turbidimetry. In the detection method, two liquid reagents (reagent R1 and reagent R2) are used, the reagent R1 comprises a phosphate buffer solution (pH=6), tween 20, disodium ethylene diamine tetraacetic acid (EDTA-2Na), sodium chloride, polyethylene glycol (PEG6000) and sodium azide; and the reagent R2 is a solution containing complement C3 latex particles. The detection method is characterized in that chemical crosslinking is used, and through the water-soluble carbodiimide (EDC) and N-hydroxy succinimide (NHS), the goat anti-human complement C3 antiserum can carry out covalent crosslinking with carboxylated polystyrene latex to form a complement C3 latex reagent. The complement C3 latex reagent has the advantages of high sensitivity, strong specificity, and simple preparation, and thus is worthy to promote and use.

Description

A kind of Complement C_3 detection method
Technical field:
The invention belongs to biological technical field, a kind of Complement C_3 detection method is provided, has highly sensitive, high specificity, the simple feature of preparation of reagents, be worth further genralrlization to use.
Background technology:
C3 is the complement component that in serum, content is the highest, a kind of beta Globulin synthesized by macrophage, monocyte, lymphoid tissue, marrow, peritonaeum and liver etc., under the effect of C3 convertase, be cracked into C3a and C3b two fragments, all play a significant role in complement Classical pathway and alternative activation pathway.
C3 is the one that in each composition of complement, content is the highest, and is most important link in complement activation pathway, therefore the mensuration of its content is extremely important.C3 increases and substantially conforms to complement activity with minimizing, but more responsive.Acute glomerulonephritis, Lupus Nephritis Patients change of serum C 3 content of about 70%-80% reduce, and can recover normal, therefore the mensuration of C3 not only contribute to diagnosis after disease amelioration, can also observe the curative effect and monitoring prognosis.C3 reduction is also shown in autoimmune disease, respiratory distress syndrome of newborn, bacteremia, histologic lesion and chronic hepatitis.
The detection method of Complement C_3 has simple immunodiffusion method, ELISA method, Immunity transmission turbidity etc.Simple immunodiffusion method is time-consuming, and variation is large, and result is not easily observed, and measures diameter out of true; ELISA method repeatability is bad, easily occurs false positive, has the greatest impact by temperature and time; The first two method that Immunity transmission turbidity is better than relatively, but there is the large shortcoming of required antiserum amount.The present invention adopts latex enhancing immune turbidimetry, not only highly sensitive, and accuracy is good, not time-consuming advantage, and required sample size is few, saves more economically.
Summary of the invention:
The object of the invention is to, prepare Complement C_3 emulsion reagent in a large number, to improve Complement C_3 detection efficiency, strengthen detection accuracy.
Technical scheme of the present invention: applied chemistry method is cross-linked, make goat-anti human complement c 3 antiserum and Carboxylated Polystyrene latex covalent cross-linking by water-soluble carbodiimide (EDC) and N-hydroxy-succinamide (NHS), form Complement C_3 emulsion reagent.Concrete operations are as follows:
The preparation of Complement C_3 emulsion reagent:
To get particle diameter be 100nm concentration is 5% latex microsphere 1ml, add 100mmol/L, the phosphate buffer 9ml of PH6.0, mix rear 5mg water-soluble carbodiimide (EDC) and the N-hydroxy-succinamide (NHS) of adding respectively, stirring at room temperature 20min, 2-8 DEG C of centrifugal 30min (rotating speed 12000), with 100mmol/L, the phosphate buffer of PH6.0 washs three times, removing supernatant, get precipitation 100mmol/L, the phosphate buffer 1 ml of PH6.0 is resuspended, and add 200 μ l goat-anti human complement c 3 antiserums, stirring at room temperature 5h, 2-8 DEG C of centrifugal 30min (rotating speed 12000), precipitation is with containing 0.05% Tween-20 (Tween 20), 100mmol/L, the phosphate buffer of PH6.0 washs three times, removing supernatant, gained precipitation 100mmol/L, the phosphate buffer of PH6.0 dilutes, 2-8 DEG C of sealing is preserved.
This detection side ratio juris be by antibody linked for goat-anti human complement c 3 in present latex particulate, with the Complement C_3 generation antigen-antibody reaction in testing sample, cause microparticle agglutination, form certain turbidity, under 340nm wavelength, by the calibration object contrast processed equally, quantitatively detect the content of Complement C_3 in sample.
Accompanying drawing illustrates:
Fig. 1 adopts reagent of the present invention and commercial reagent A respectively, adopts Olympus 400 automatic clinical chemistry analyzer, to 50 increments this (comprising normal and exceptional sample), measures, and carry out correlation analysis to measured value by each autoregressive parameter.The measured value of what wherein X-axis represented is reagent of the present invention, the measured value of what Y-axis represented is commercial reagent A.Related coefficient: r 2=0.9948, linear equation is: y=1.041x-0.054.
Fig. 2: adopt Olympus 400 automatic clinical chemistry analyzer, the reagent of 5 gradient concentrations is measured, and correlation analysis is carried out to measured value.What wherein X-axis represented is dilute concentration, and what Y-axis represented is measured value average.Related coefficient: r 2=0.9963, linear equation is: y=1.0272x-0.0007.
Embodiment:
Embodiment
The inventive method is mixed with kit, carries out carrying out Performance comparision with commercial reagent box:
The concrete composition that reagent of the present invention is made into kit is as follows:
Complement C_3 detects the use of reagent:
1) detecting instrument: the Biochemical Analyzer with 340nm wavelength, 37 DEG C of thermostats.
2) sample to be tested: fresh not haemolysis serum, 2-8 DEG C of Absorbable organic halogens seven days.-20 DEG C of Absorbable organic halogens one month.
3) concrete trace routine:
4) result of calculation: Complement C_3 concentration (g/L)=Δ AT/ Δ AS × Cs
Δ AT: sample hose absorbance
Δ AS: calibration tube absorbance
Cs: calibration object concentration value
5) reference range: 0.9 ~ 1.5g/L
6) precision: CV≤4% in batch; Relative extreme difference≤6% between batch.
7) accuracy: relative deviation controls in ± 10%.
8) range of linearity: within the scope of 0.1 ~ 3g/L, correlation coefficient r >=0.990.
The commercially available import reagent A of contrast, its information is as follows:
Name of product: Complement C_3 detection kit (immunoturbidimetry)
Cleaning Principle: the C3 antibody in human serum in Complement C_3 and antiserum reagent forms antigen antibody complex, and make reactant liquor occur turbidity, the concentration recording Complement C_3 in serum is compared in the change detecting absorbance under 340nm with standard items.
Main composition:
Reagent R1:TRIS damping fluid: 100mmol/L, pH 8.0
Polyglycol: 3.0%
Antiseptic
Reagent R2: anti-human C3/C3b/C3c antibody (goat): according to titre
TRIS damping fluid: 33mmol/L
Antiseptic
Commercial reagent A by specification operation.
Example 1 reagent of the present invention compares with the performance index of commercial reagent A:
1) precision measures: in same sample, continuous drawing measures for 20 times, calculates the mean of measured value, standard deviation and the coefficient of variation, CV = SD X ‾ × 100 %
Table 1 precision testing result
Coefficient of variation CV is generally used for the precision of a measurement assay method, and CV value is less, represents that the result precision of this assay method is better.For clinical chemistry test project, CV be less than 5% method precision generally acknowledge be acceptable.In table 1, the CV value of reagent of the present invention is less than commercial reagent A, shows that the precision of the inventive method is slightly better than commercial reagent A.
2) linear determination: adopt reagent of the present invention and commercial reagent A respectively, adopt Olympus 400 automatic clinical chemistry analyzer, to 50 increments this (comprising normal and exceptional sample), measure by each autoregressive parameter, and correlation analysis is carried out to measured value (the results are shown in Figure 1, what X-axis represented is the measured value that the present invention tries, the measured value of what Y-axis represented is commercial reagent A).Related coefficient: r 2=0.9948, linear equation is: y=1.041x-0.054, and result shows that this reagent and commercial reagent correlativity are good.
Table 2 linear correlation detection result
Example 2 preparation of reagents of the present invention becomes kit correlated performance to assess
1) precision measures: in same sample, continuous drawing measures for 20 times, calculates the mean of measured value, standard deviation and the coefficient of variation, CV = SD X ‾ × 100 %
Table 3 precision testing result
Coefficient of variation CV is generally used for the precision of a measurement assay method, and CV value is less, represents that the result precision of this assay method is better.For clinical chemistry test project, CV be less than 5% method precision generally acknowledge be acceptable.In table 3, CV value is less than 3%, shows that the inventive method has excellent precision.
2) accuracy determination: use same quality-control product, replication 3 times, averages, should in ± 10% scope with quality-control product target value relative deviation.
Table 4 accuracy testing result
Relative deviation CB=-2.00% in table 4, be in ± 10% scope in, show that the inventive method has excellent accuracy.
3) linear determination: use deionized water reagent dilutions to be become 5 gradient concentrations, each gradient concentration detects 3 times, averages, and does regretional analysis to measured value and desired value, calculates r value and relative deviation (the results are shown in Figure 2, unit g/L).
Table 5 linear correlation detection result
Related coefficient is obtained: r by table 5 2=0.9963, linear equation is: y=1.0272x-0.0007, and result shows that this reagent correlativity is good.
4) Stability Determination: the present invention is detected reagent and be placed on room temperature and 4 DEG C of refrigerators respectively, substitute sample with freshly prepared 3.0g/L human complement c 3 standard items, measured 1 time every 1 month, and aggegation required time appears in record, the results are shown in Table 6.Result shows, kit is placed at 4 DEG C of refrigerators and do not had obvious loss of activity in more than at least 6 months, but should not deposit in room temperature.
Table 6 Detection of Stability result
Under room temperature There is the time (min) of aggegation At 4 DEG C There is the time (min) of aggegation
New preparation 1 New preparation 1
Deposit 1 month 2 Deposit 1 month 1
Deposit 2 months Not aggegation Deposit 2 months 1
Deposit 3 months Not aggegation Deposit 3 months 1
Deposit 4 months Not aggegation Deposit 4 months 1
Deposit 5 months Not aggegation Deposit 5 months 1
Deposit 6 months Not aggegation Deposit 6 months 1
5) specific assay: choose 3 kinds of potential interference things and carry out interference experiment mensuration, equal unrestraint effect, show that this kit has good specificity, result is as shown in table 7:
Table 7 specific detection result

Claims (4)

1. the invention provides a kind of Complement C_3 detection method, described detection method is based on latex enhancing immune turbidimetry, for liquid double reagent, comprise reagent R1 and R2, described reagent R1 contains PH6.0 phosphate buffer, Tween-20 (Tween 20), disodium ethylene diamine tetraacetate (EDTA-2Na), sodium chloride, polyglycol (PEG6000), Sodium azide; Reagent R2 is the solution containing Complement C_3 latex particle, its feature is that applied chemistry method is cross-linked, make goat-anti human complement c 3 antiserum and Carboxylated Polystyrene latex covalent cross-linking by water-soluble carbodiimide (EDC) and N-hydroxy-succinamide (NHS), form Complement C_3 emulsion reagent.
2. Chemical Crosslinking Methods prepares Complement C_3 emulsion reagent according to claim 1, it is characterized in that Carboxylated Polystyrene latex particle size used is 100nm.
3. Chemical Crosslinking Methods prepares Complement C_3 emulsion reagent according to claim 1, it is characterized in that adding water-soluble carbodiimide (EDC) and N-hydroxy-succinamide (NHS) in Carboxylated Polystyrene latex and goat-anti people C3 antiserum covalent cross-linking process respectively.
4. according to claim 1, it is characterized in that the computing formula of mAlb in sample is:
Complement C_3 concentration (g/L)=Cs × Δ A in sample t/ Δ A s
In formula: Δ A twith the sample hose absorbance of blank tube absorbance for contrast;
Δ A swith the calibration tube absorbance of blank tube absorbance for contrast;
C sthe concentration of Complement C_3 in calibration solution.
CN201510226757.7A 2015-05-02 2015-05-02 Complement C3 detection method Pending CN104833810A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510226757.7A CN104833810A (en) 2015-05-02 2015-05-02 Complement C3 detection method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510226757.7A CN104833810A (en) 2015-05-02 2015-05-02 Complement C3 detection method

Publications (1)

Publication Number Publication Date
CN104833810A true CN104833810A (en) 2015-08-12

Family

ID=53811822

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510226757.7A Pending CN104833810A (en) 2015-05-02 2015-05-02 Complement C3 detection method

Country Status (1)

Country Link
CN (1) CN104833810A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106483299A (en) * 2016-10-03 2017-03-08 王贤俊 A kind of method for detecting superoxide dismutase content in human serum
CN107490676A (en) * 2017-08-10 2017-12-19 迈克生物股份有限公司 A kind of Complement C_3 detection kit and detection method
CN109752332A (en) * 2017-11-07 2019-05-14 重庆中元汇吉生物技术有限公司 A kind of C1Q detection kit
CN111693697A (en) * 2020-07-07 2020-09-22 上海怡珏生物科技有限公司 Application of C3C antibody in preparation of detection kit

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106483299A (en) * 2016-10-03 2017-03-08 王贤俊 A kind of method for detecting superoxide dismutase content in human serum
CN107490676A (en) * 2017-08-10 2017-12-19 迈克生物股份有限公司 A kind of Complement C_3 detection kit and detection method
CN109752332A (en) * 2017-11-07 2019-05-14 重庆中元汇吉生物技术有限公司 A kind of C1Q detection kit
CN111693697A (en) * 2020-07-07 2020-09-22 上海怡珏生物科技有限公司 Application of C3C antibody in preparation of detection kit

Similar Documents

Publication Publication Date Title
CN101769932B (en) Full-range C-reactive protein detection kit
Evans Manual and automated methods for measuring urea based on a modification of its reaction with diacetyl monoxime and thiosemicarbazide.
CN102253217B (en) Detection kit of latex particle enhanced neutrophil gelatinase-associated lipid transfer protein
CN102621332B (en) Retinol binding protein assay kit based on latex particle coating
CN102507918B (en) Kit for determining anti-cyclic citrullinated peptide (Anti-CCP) antibody
CN101699287B (en) Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof
CN103592445A (en) Kit for detecting procalcitonin
CN105510604A (en) Method for improving sensitivity and linearity of latex reagent
CN102393456B (en) Kit for detecting Hepassocin (HPS)
JP6749337B2 (en) Prostate antigen standards and their use
CN104849473A (en) Microalbuminuria detection kit and preparation thereof
CN110862881A (en) Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof
CN104215770A (en) Two-particle-based retinol binding protein detection kit
CN104034893B (en) A kind of melamine rapid detection method based on latex and kit
CN104833810A (en) Complement C3 detection method
CN105203748A (en) Full-range quantitative detection kit for C-reactive protein
CN105277717A (en) Magnetic particle separation chemiluminescence immunoassay method for thyroglobulin
CN102243241A (en) Homogeneous phase aerosol particle-type neutrophile granulocyte gelatinase-related lipid carrier protein determination kit and preparation method thereof
CN105353139A (en) Parathyroid hormone quantitative determination kit
CN105929166A (en) Magnetic particle-based quantitative chemiluminescent assay kit for anti-LKM-1 antibody IgG, and preparation and detection methods thereof
CN107247138B (en) It is a kind of for measuring the method for coating of the Chemiluminescent plate of d-dimer content
CN105699665A (en) Detection kit of lipocalin related to neutrophil gelatinase
CN108663526B (en) Secondary antibody competes immunoturbidimetry assay kit and its making and use method
CN102866256A (en) Detection method and detection reagent for hypersensitive C reactive protein
CN104849224A (en) Complement C3 detection kit and preparation thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
EXSB Decision made by sipo to initiate substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20150812