CN105911285A - Kit for determining rheumatoid factors - Google Patents
Kit for determining rheumatoid factors Download PDFInfo
- Publication number
- CN105911285A CN105911285A CN201610359608.2A CN201610359608A CN105911285A CN 105911285 A CN105911285 A CN 105911285A CN 201610359608 A CN201610359608 A CN 201610359608A CN 105911285 A CN105911285 A CN 105911285A
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- Prior art keywords
- reagent
- rheumatoid factor
- mmol
- latex
- test kit
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Abstract
The invention relates to the technical field of medicine and biochemistry and particularly relates to a kit for determining rheumatoid factors, aiming at solving the problems in the prior art that a rheumatoid factor detection process is complicated to operate and the determination accuracy is low. Aiming at solving the technical problems above, the technical scheme provided by the invention is as follows: a kit for determining hyaluronic acid is provided, wherein the kit is composed of double liquid reagents R1 and R2, the reagent R1 is prepared from the following components: 30mmol/L-230mmol/L of a Tris buffering solution, 2.0mL/L-6.0mL/L of Tween-80, 5g/L-15g/L of polyethylene glycol-8000, 30mmol/L-50mmol/L of EDTA (Ethylene Diamine Tetraacetic Acid), 0.4g/L-1.0g/L of sodium azide and a solvent which is a purified water; the reagent R2 is prepared from the following components: 50mmol/L-150mmol/L of a Tris buffering solution, 5mmol/L-25mmol/L of glycerol, 0.4g/L-1.0g/L of sodium azide, 20g/L-60g/L of bovine serum albumin, 0.5%-4.0% of an emulsion-coated anti-human rheumatoid factor antibody and a solvent which is purified water. The kit for determining the rheumatoid factors has the advantages of high accuracy, convenience for operation and the like.
Description
Technical field
The present invention relates to medical science and technological field of biochemistry, a kind of reagent measuring rheumatoid factor
Box.
Background technology
Rheumatoid factor (rheumatoid factor, RF) can be divided into IgM, IgA, IgG, IgD, IgE five type, is class wind
For a class autoantibody of epitope, the more companion of rheumatoid factor positive patient in IgG Fc fragment in wet arthritis serum
There is extra-articular manifestation, such as subcutaneous nodule and vasculitis etc..IgM type RF positive rate is 60%-78%.
Rheumatoid factor (RF) is to find in rheumatoid arthritis (RA) patients serum, is that one is with degeneration IgG
The autoantibody of target antigen, is primarily present in serum and the joint fluid of patient with rheumatoid arthritis, and it is a kind of antitypy
The antibody of IgG, belongs to IgM type.Can be combined with IgG Fc section.Patient RA and Yue 50% healthy human body in all there are produce RF B
Cell clone, at degeneration IgG(or the IgG that is combined with antigen) or under Epstein-Barr virus directly acts on, RF can be synthesized in a large number.Healthy People produces
The cell clone of raw RF is less, and the soluble factor of mononuclear cell secretion can suppress the generation of RF, therefore is typically difficult to measure.RF
Predominantly IgM class autoantibody, but also have IgG class, IgA class, IgD class and IgE class.All kinds of RF clinical meanings are different.Survey
Determine IgG, IgA, IgM class RF and generally use ELISA indirect method, be i.e. coated Sptting plate micropore with thermal thermocoagulation rabbit igg, after adding sample,
Add anti-human igg, IgA, IgM enzymic-labelled antibody the most respectively, make after reaction again with substrate colour generation.For preventing each Ig classification RF the most dry
Disturbing, enzymic-labelled antibody uses the antibody F(ab of labelling) 2 fragments.
But enzyme-linked immunosorbent assay is the longest, operation complexity, operator is had higher professional skill requirement, and only
Can qualitative or half-quantitative detection.Latex agglutination tests qualitative detection only.
Summary of the invention
It is an object of the invention to solve rheumatoid factor detection process operation complexity and mensuration in prior art accurate
Spend low problem, it is provided that a kind of test kit measuring rheumatoid factor.
The technical scheme that the present invention solves above-mentioned technical problem and provides is: a kind of test kit measuring rheumatoid factor, bag
Include reagent R1 independent of each other and reagent R2 biliquid component, including composition and corresponding content be:
Reagent R1:
Tris buffer 30 ~ 230 mmol/L
Tween 80 2.0 ~ 6.0 mL/L
PEG-8 000 5 ~ 15 g/L
EDTA 30~50 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Its solvent is purified water.
Reagent R2:
Tris buffer 50 ~ 150 mmol/L
Glycerol 5 ~ 25 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Bovine serum albumin 20 ~ 60 g/L
Latex is coated anti-human rheumatoid factor antibodies 0.5 ~ 4.0 %
Its solvent is purified water.
As preferably, reagent R1 independent of each other and reagent R2 biliquid component, including composition and corresponding content be:
Reagent R1:
Tris buffer 130mmol/L
Tween 80 4.0 mL/L
PEG-8 000 10 g/L
EDTA 40 mmol/L
Sodium azide 0.7 g/L
Its solvent is purified water.
Reagent R2:
Tris buffer 100 mmol/L
Glycerol 15 mmol/L
Sodium azide 0.7 g/L
Bovine serum albumin 40g/L
Latex is coated anti-human rheumatoid factor antibodies 2.5 %
Its solvent is purified water.
As preferably, described latex is coated anti-human rheumatoid factor antibodies can be with anti-human rheumatoid factor antibodies for containing
The monoclonal complete antibody of specific binding Fab functional part or antibody fragment.
As preferably, the particle diameter of the anti-human rheumatoid factor antibodies of described latex is between 40 ~ 500nm.
As preferably, preparation method and the using method of the test kit of this mensuration rheumatoid factor comprise the following steps:
A () prepares R1 reagent according to following component content:
Tris buffer 30 ~ 230 mmol/L
Tween 80 2.0 ~ 6.0 mL/L
PEG-8 000 5 ~ 15 g/L
EDTA 30~50 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Its solvent is purified water.
B () prepares R2 reagent according to following component content:
Tris buffer 50 ~ 150 mmol/L
Glycerol 5 ~ 25 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Bovine serum albumin 20 ~ 60 g/L
Latex is coated anti-human rheumatoid factor antibodies 0.5 ~ 4.0 %
Its solvent is purified water.
C sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react, further preferred R1 and reagent R2
Volume ratio be 4:1, the volume ratio of the cumulative volume of sample to be tested and reagent R1 and reagent R2 is between 1:20 to 1:80；
D () measures reacted absorbance difference with automatic clinical chemistry analyzer；
E () calculates the value of rheumatoid factor in sample according to absorbance changing value.
As preferably, it is as follows that described latex is coated anti-human rheumatoid factor antibodies preparation process:
A particle diameter is the latex particle of 120nm by (), with the MES buffer diluted latex granule of 50 mmol/L to 2 g/L, every mL
Solution adds EDAC 1.0 mg, room temperature reaction 2 hours, is centrifuged 30 minutes under 15000 rpm rotating speeds in centrifuge, goes
Clearly, precipitation is suspended in the MES buffer of 50 mmol/L, ultrasonic disperse；
B the product of step (a) is centrifuged 30 minutes under 15000 rpm rotating speeds in centrifuge by (), abandon supernatant, precipitation be suspended in
In the MES buffer of 50 mmol/L so that latex particle ultimate density is 4%, ultrasonic disperse adds isopyknic while stirring
MES diluent containing anti-human rheumatoid factor antibodies, mix and blend, room temperature reaction 2 hours, latex particle ultimate density is 2 g/
L；
C the product of step (b) 15000 rpm rotating speeds in centrifuge are centrifuged 30 minutes by (), abandon supernatant, and precipitation is suspended in 50
Ultrasonic disperse in the MES buffer of mmol/L, adds BSA, closes overnight for 4 DEG C；Being centrifuged goes supernatant, prepared latex to be coated anti-human
Rheumatism factor antibody.
The reaction principle of latex enhancing immune turbidimetry of the present invention is antigen antibody reaction, rheumatoid in sample
The factor (RF) can be combined generation agglutination by RF sensitization latex in reagent, produces certain turbidity.This turbidity height contains with RF's
Amount is directly proportional.Under certain wavelength, measure turbidity and the quantitative determination of rheumatoid factor can be carried out by multiple spot calibration curve.
Activity (the U/mL)=CS of rheumatoid factor (RF) in sample ×(U/mL)
In formula: the sample cell absorbance that Δ AT compares with blank tube absorbance
The calibration pipe absorbance that Δ AS compares with blank tube absorbance
The concentration of RF in CS calibration solution
Compared with prior art, the present invention has a following advantageous benefits: the present invention with the addition of in reagent R1 liquid Polyethylene Glycol-
8000, it is possible to accelerate reaction；Latex is used to be coated anti-human rheumatoid factor antibodies in reagent R2, the detection to rheumatoid factor
Sensitivity is higher, and detection accuracy is more preferable, and therefore accuracy in detection is high, additionally detects the most more convenient.
Accompanying drawing illustrates:
Fig. 1 is the testing result contrast of this test kit and chemoluminescence method.
Detailed description of the invention
Further illustrate below in conjunction with specific embodiment, but the present invention is not limited in these embodiments.
Embodiment 1
The test kit of the present invention includes reagent R1 independent of each other and reagent R2 biliquid component, wherein
Reagent R1:
Tris buffer 130mmol/L
Tween 80 4.0 mL/L
PEG-8 000 10 g/L
EDTA 40 mmol/L
Sodium azide 0.7 g/L
Its solvent is purified water.
Reagent R2:
Tris buffer 100 mmol/L
Glycerol 15 mmol/L
Sodium azide 0.7 g/L
Bovine serum albumin 40g/L
Latex is coated anti-human rheumatoid factor antibodies 2.5 %
Its solvent is purified water.
Embodiment 2
The preparation and application of test kit
1, it is the latex particle of 120nm by particle diameter, with the MES buffer diluted latex granule of 50 mmol/L to 2 g/L, every mL
Solution adds EDAC 1.0 mg, room temperature reaction 2 hours, is centrifuged 30 minutes under 15000 rpm rotating speeds in centrifuge, goes
Clearly, precipitation is suspended in the MES buffer of 50 mmol/L, ultrasonic disperse；Then in centrifuge under 15000 rpm rotating speeds
Centrifugal 30 minutes, abandon supernatant, precipitation is suspended in the MES buffer of 50 mmol/L so that latex particle ultimate density is
4%, ultrasonic disperse, add isopyknic MES diluent containing anti-human rheumatoid factor antibodies, mix and blend, room temperature while stirring
Reacting 2 hours, latex particle ultimate density is 2 g/L；Then it is centrifuged 30 minutes at 15000 rpm rotating speeds in centrifuge, abandons
Supernatant, precipitation is suspended in ultrasonic disperse in the MES buffer of 50 mmol/L, adds BSA, closes overnight for 4 DEG C；It is centrifuged and removes supernatant,
Prepared latex is coated anti-human rheumatoid factor antibodies.
2, reagent is prepared according to following component content:
A () prepares R1 reagent according to following component content:
Tris buffer 30 ~ 230 mmol/L
Tween 80 2.0 ~ 6.0 mL/L
PEG-8 000 5 ~ 15 g/L
EDTA 30~50 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Its solvent is purified water.
B () prepares R2 reagent according to following component content:
Tris buffer 50 ~ 150 mmol/L
Glycerol 5 ~ 25 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Bovine serum albumin 20 ~ 60 g/L
Latex is coated anti-human rheumatoid factor antibodies 0.5 ~ 4.0 %
Its solvent is purified water.
3, automatic clinical chemistry analyzer parameter is arranged
(a) detection temperature: 37 DEG C；
(b) detection wavelength: dominant wavelength 600nm；
(c) response time: 10min, wherein, incubation time 5min, measure immediately after adding reagent R2 and read absorbance
Read absorbance A 2 after A1,5min, calculate absorbance changes delta A=A2-A1；
(d) the Direction of Reaction: positive reaction；
4, detecting step
A () takes 224 μ l reagent R1 and the mixing of 4 μ l samples to be tested；
B solution after mixing is hatched 5min under conditions of 37 DEG C by ()；
C () adds 56 μ l reagent R2, measure immediately and read absorbance A 1, reads absorbance A 2 after 5min, calculates absorbance change
Δ A=A2-A1；
Activity (the U/mL)=CS of rheumatoid factor (RF) in (d) sample ×(U/mL) the class wind in sample is calculated
The activity of the wet factor.
Referring to the drawings 1, accompanying drawing 1 is for repeatedly measuring with a kind of test kit measuring rheumatoid factor obtained by embodiment 1
The measurement result of rheumatoid factor in different samples, and under equal conditions with chemiluminescence determination result to score
Analysis.From dependency experimental result, this test kit is fine with chemiluminescence detection results relevance, dependent equation y=
0.9693x+1.755, R2=0.9907, this kit results accuracy is reliable as can be seen from the results, and dependency is good, permissible
It is applied to the detection of rheumatoid factor.
Claims (8)
1. measuring a test kit for rheumatoid factor, including reagent R1 independent of each other and reagent R2 biliquid component, it is special
Levy and be: described reagent R1 independent of each other and reagent R2 biliquid component, including composition and corresponding content be:
Reagent R1:
Tris buffer 30 ~ 230 mmol/L
Tween 80 2.0 ~ 6.0 mL/L
PEG-8 000 5 ~ 15 g/L
EDTA 30~50 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Its solvent is purified water；
Reagent R2:
Tris buffer 50 ~ 150 mmol/L
Glycerol 5 ~ 25 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Bovine serum albumin 20 ~ 60 g/L
Latex is coated anti-human rheumatoid factor antibodies 0.5 ~ 4.0 %
Its solvent is purified water.
A kind of test kit measuring rheumatoid factor the most according to claim 1, it is characterised in that: include independent of each other
Reagent R1 and reagent R2 biliquid component, including composition and corresponding content be:
Reagent R1:
Tris buffer 130mmol/L
Tween 80 4.0 mL/L
PEG-8 000 10 g/L
EDTA 40 mmol/L
Sodium azide 0.7 g/L
Its solvent is purified water；
Reagent R2:
Tris buffer 100 mmol/L
Glycerol 15 mmol/L
Sodium azide 0.7 g/L
Bovine serum albumin 40g/L
Latex is coated anti-human rheumatoid factor antibodies 2.5 %
Its solvent is purified water.
A kind of test kit measuring rheumatoid factor the most according to claim 1, it is characterised in that: described latex is coated
Anti-human rheumatoid factor antibodies is the monoclonal containing Fab functional part that can be specific binding with anti-human rheumatoid factor antibodies
Complete antibody or antibody fragment.
A kind of test kit measuring rheumatoid factor the most according to claim 1, it is characterised in that: described latex is anti-human
The particle diameter of rheumatoid factor antibodies is between 40 ~ 500nm.
A kind of test kit measuring rheumatoid factor the most according to claim 1, it is characterised in that: the preparation side of test kit
Method and using method comprise the following steps:
A () prepares R1 reagent according to following component content:
Tris buffer 30 ~ 230 mmol/L
Tween 80 2.0 ~ 6.0 mL/L
PEG-8 000 5 ~ 15 g/L
EDTA 30~50 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Its solvent is purified water；
B () prepares R2 reagent according to following component content:
Tris buffer 50 ~ 150 mmol/L
Glycerol 5 ~ 25 mmol/L
Sodium azide 0.4 ~ 1.0 g/L
Bovine serum albumin 20 ~ 60 g/L
Latex is coated anti-human rheumatoid factor antibodies 0.5 ~ 4.0 %
Its solvent is purified water；
C sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react；
D () measures reacted absorbance difference with automatic clinical chemistry analyzer；
E () calculates the value of rheumatoid factor in sample according to absorbance changing value.
A kind of test kit measuring rheumatoid factor the most according to claim 1, it is characterised in that: described latex is coated
Anti-human rheumatoid factor antibodies preparation process is as follows:
A particle diameter is the latex particle of 120nm by (), with the MES buffer diluted latex granule of 50 mmol/L to 2 g/L, every mL
Solution adds EDAC 1.0 mg, room temperature reaction 2 hours, is centrifuged 30 minutes under 15000 rpm rotating speeds in centrifuge, goes
Clearly, precipitation is suspended in the MES buffer of 50 mmol/L, ultrasonic disperse；
B the product of step (a) is centrifuged 30 minutes under 15000 rpm rotating speeds in centrifuge by (), abandon supernatant, precipitation be suspended in
In the MES buffer of 50 mmol/L so that latex particle ultimate density is 4%, ultrasonic disperse adds isopyknic while stirring
MES diluent containing anti-human rheumatoid factor antibodies, mix and blend, room temperature reaction 2 hours, latex particle ultimate density is 2 g/
L；
C the product of step (b) 15000 rpm rotating speeds in centrifuge are centrifuged 30 minutes by (), abandon supernatant, and precipitation is suspended in 50
Ultrasonic disperse in the MES buffer of mmol/L, adds BSA, closes overnight for 4 DEG C；Being centrifuged goes supernatant, prepared latex to be coated anti-human
Rheumatism factor antibody.
The preparation method of a kind of test kit measuring rheumatoid factor the most according to claim 5 and using method, it is special
Levying and be: in step (c), the volume ratio of described reagent R1 and reagent R2 is 4:1.
The preparation method of a kind of test kit measuring rheumatoid factor the most according to claim 5 and using method, it is special
Levy and be: in step (c), the volume ratio of the cumulative volume of described sample to be tested and reagent R1 and reagent R2 1:20 to 1:80 it
Between.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610359608.2A CN105911285A (en) | 2016-05-27 | 2016-05-27 | Kit for determining rheumatoid factors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610359608.2A CN105911285A (en) | 2016-05-27 | 2016-05-27 | Kit for determining rheumatoid factors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105911285A true CN105911285A (en) | 2016-08-31 |
Family
ID=56741463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610359608.2A Pending CN105911285A (en) | 2016-05-27 | 2016-05-27 | Kit for determining rheumatoid factors |
Country Status (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406796A (en) * | 2018-12-11 | 2019-03-01 | 迈克生物股份有限公司 | Rheumatoid factor detection reagent box and its detection method |
| CN112485435A (en) * | 2020-11-07 | 2021-03-12 | 山东博科生物产业有限公司 | Stable rheumatoid factor detection kit for slowing down hook effect |
| CN117214428A (en) * | 2023-11-07 | 2023-12-12 | 宁波美康盛德生物科技有限公司 | Rheumatoid factor detection kit and detection method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406796A (en) * | 2018-12-11 | 2019-03-01 | 迈克生物股份有限公司 | Rheumatoid factor detection reagent box and its detection method |
| CN112485435A (en) * | 2020-11-07 | 2021-03-12 | 山东博科生物产业有限公司 | Stable rheumatoid factor detection kit for slowing down hook effect |
| CN112485435B (en) * | 2020-11-07 | 2023-08-29 | 山东博科生物产业有限公司 | Stable rheumatoid factor detection kit capable of relieving hook effect |
| CN117214428A (en) * | 2023-11-07 | 2023-12-12 | 宁波美康盛德生物科技有限公司 | Rheumatoid factor detection kit and detection method |
| CN117214428B (en) * | 2023-11-07 | 2024-02-02 | 宁波美康盛德生物科技有限公司 | Rheumatoid factor detection kit and detection method |
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Application publication date: 20160831 |