CN105606822A - Beta 2-microglobulin detection kit - Google Patents
Beta 2-microglobulin detection kit Download PDFInfo
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- CN105606822A CN105606822A CN201610050627.7A CN201610050627A CN105606822A CN 105606822 A CN105606822 A CN 105606822A CN 201610050627 A CN201610050627 A CN 201610050627A CN 105606822 A CN105606822 A CN 105606822A
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- buffer solution
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- microglobulin
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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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
- G01N33/531—Production of immunochemical test materials
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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
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Abstract
The invention discloses a beta 2-microglobulin detection kit which comprises a reagent R1 and a reagent R2, and the reagent R1 is a buffer solution; the reagent R2 is a mixture of beta 2-microglobulin antibody sensitization polystyrene latex particles and a buffer solution. The detection kit has the advantages that the detection is simple and rapid, the sensitivity is high, the accuracy is good, the disturbance resistance is high and the production cost is low. An adopted beta 2-microglobulin detection method is latex enhanced immunoturbidimetry, the beta 2-microglobulin detection is more economical, convenient and rapid with the method, and the beta 2-microglobulin detection kit is suitable for automatic biochemistry analyzers in vast majority of hospitals, and particularly suitable for realizing rapid quantitative detection on emergency treatment.
Description
Technical field
The present invention belongs to field of medical immunology, relates to a kind of immunologic function test reagent, furtherly, the present invention relates to β 2-Microglobulin detection kit.
Background technology
B2M is mainly used for renal functional evaluation and as non-specific tumor markers, urine concentration increasesSerum levels is normally because reabsorption reduces, and sees renal tubular disease as comprehensive in Fanconi syndrome, LoweDisease, chronic chromium poisoning, acute tubular necrosis, cystine bird disease, kidney transplant, acute or chronic pyelonephritis, and cystitis is justOften; Serum levels increases urine concentration normally because glomerulopathy filterability reduces, and sees acute or chronic nephritis; Serum waterGentle urine level concentration all raises to see to generate and increases and exceed reabsorption ability, sees malignant tumour as thin in liverBorn of the same parents' cancer, lung cancer, stomach and colon cancer, Huppert's disease, malignant lymphoma, chronic lymphocytic leukemia.
Diagnostic method: the mensuration of β 2MG generally adopts RIA method, ELISA method and CLlA method, latex immunoturbidimetry etc., butThe latex immunoturbidimetries that adopt clinically more
Serum (containing β 2mG)+reagent (the anti-human β 2mG of latex antibody) ━ ━ ━ ━ antigen antibody complex
Generation turbidity changes, and under certain wavelength, detects, and the variation of its turbidity becomes positive correlation with its content.
Antibody uses chemical coupling method conventionally with being connected of latex at present, and it is as R2 reagent, and very easy generation is coalescent,Reagent quality is declined along with the prolongation of resting period. Make the poor stability of reagent.
Summary of the invention
The present invention is not good according to R2 reagent stability, provides a kind of method to overcome the deficiencies in the prior art, makes itKeep steady quality, provide a kind of highly sensitive, the beta 2-microglobulin detecting kit of good stability.
For achieving the above object, the invention provides following technical scheme:
A kind of beta 2-microglobulin detecting kit, described kit comprises reagent R1, reagent R2, described reagent R1 isBuffer solution;
Described reagent R2 is the mixture of B2M antibody sensitized polystyrene latex particle and buffer solution, described examinationThe preparation process of agent R2 comprises:
Step (1): add ethyl dimethyl amine propyl carbodiimide diimine in the polystyrene latex particle with carboxyl, obtainThe latex particle activating;
Step (2): the latex particle that step (1) is activated and B2M antibody mix, and add Portugal after question response finishes againUnreacted radical on grape sugar sealing latex microsphere, the latex microsphere that obtains coupling antibody is B2M antiantibody sensitizationPolystyrene latex particle, is placed in buffer solution.
As present invention further optimization: described B2M antibody sensitized polystyrene latex particle is at reagentConcentration in R2 is 0.08～0.28 gram/100ml.
As present invention further optimization: the buffer solution in described reagent R1 is PBS buffer solution, Tris buffer solution, sweetPropylhomoserin buffer solution, borate buffer solution, acetate buffer, citric acid-phosphate buffer, carbonate-buffered with bicarbonateIn liquid, MES buffer solution, ammonium chloride buffer at least one.
As present invention further optimization: the buffer solution in described reagent R2 is PBS buffer solution, Tris buffer solution, sweetPropylhomoserin buffer solution, borate buffer solution, acetate buffer, citric acid-phosphate buffer, carbonate-buffered with bicarbonateIn liquid, MES buffer solution, ammonium chloride buffer at least one.
As present invention further optimization: ethyl dimethyl amine propyl carbodiimide diimine in step (1): polystyrene colloidalThe weight ratio of breast particle is 0.5～5: 100.
As further preferred: described α 1-microglobulin antiantibody comprises polyclonal antibody or monoclonal antibody.
As further preferred: its average grain diameter of polystyrene latex particle in described step (1) is at 0.01 ~ 0.2mmBetween. The detection principle that kit of the present invention adopts is Latex-enhanced immunoturbidimetric assay, and its principle is β 2-microballoonThe latex particle of protein antibodies, after B2M generation immune response, forms aggregated particle, under certain wavelength, passes throughMeasure aggregation and form the turbidity producing, can measure the content of B2M.
Advantage of the present invention and beneficial effect:
Kit of the present invention have detect simple and quick, highly sensitive, accuracy is good, antijamming capability is strong and be produced intoThe advantage that this is low.
2. the B2M detection method that the present invention adopts is latex enhancing immune turbidimetry, and the method makes β 2-micro-The detection of globulin is more economical, convenient and quick, is applicable to the automatic biochemistry analyzer of most hospitals, particularly to urgencyExamine and can realize Quantitative detection.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, but the present invention is not only confined to following examples.
Embodiment 1: the preparation of beta 2-microglobulin detecting kit
It is as follows that kit of the present invention relates to the main material of reagent:
1. B2M antibody: polyclonal antibody (commercially available).
2. latex: adopting diameter is that 80～200nm tests with the polystyrene latex particle (commercially available) of carboxylic group.
Being formulated as follows of the main agents of the present embodiment:
Reagent R1: containing the phosphate buffer of 2.5%PEG6000 (Macrogol 6000), 95mmol/LNaCl, this reagent is nothingLook clear solution.
Reagent R2: the polystyrene latex particle that is 120nm with anti-human B2M antibody sensitized particle diameter. This reagent is breastWhite solution. Concrete steps are as follows:
1. get 1ml (100mg/ml) latex, with 0.02M (mol/L), the MES solution (MES buffer solution) of pH5.0 is washedWash ultrasonic dispersion 3 times;
2. then add 0.22ml 0.02M, (in the solution of preparation, EDAC's is dense for the freshly prepared EDAC of MES solution of pH5.0Degree is 10mg/ml) to mix completely, room temperature mixes 15min;
3. then use 0.05M, the MES solution washing of pH5.0 2 times, 0.1M, the phosphate solution washing of pH7.5 1 time, ultrasonic pointLoose for subsequent use;
4. get 2ml antibody (2mg/ml) solution, add 0.3ml 0.02M, the phosphate solution preparation of pH7.5, mixed at room temperature15min，
5. then add 0.32ml10%BSA (bSA) solution, 4 DEG C are mixed 4h;
6. add 0.4ml10% glucose solution, 4 DEG C of mixing are spent the night again;
7. then use 0.015M, the glycine solution washing of pH7.5 3 times, then add 0.015M, the glycine solution of pH7.5 (contains1％BSA，0.2％NaN3, 15% multitudinous sugar, 0.01%EDTA-Na2, the glycine buffer of 0.01%trtonX-100) and to latex (knotClosing the later weight of latex of antibody) final concentration is 0.18% (mass volume ratio i.e. 0.18 gram/100ml).
Embodiment 2: the mensuration of B2M
Testing tool: Hitachi's 7060 type automatic analyzers.
Analytical method: Two point end assay; Dominant wavelength: 570nm, commplementary wave length: 700nm: sample size: 2ul (microlitre); R1:200ul; R2:50ul; The Direction of Reaction: rise; Measure temperature: 37 DEG C; After sample and R1 mix, read absorbance in the 10th secondA1; In the time of 3～5min, add R2, after 5min, read absorbance A 2. The difference that is calculated as A2 and A1 of reaction absorbance.
Computational methods: multiple spot calibration, make dosage/response curve according to the value of absorbance and reference serum, sample size canCalculate on dosage/response curve according to its absorbance.
Embodiment 3: beta 2-microglobulin detecting kit performance evaluation
1. sensitivity for analysis assessment
Adopt 5% bovine serum albumin solution as dummy, dummy should not contain measured object. On Biochemical Analyzer, connectContinuous detection 20 times, calculates average and the standard deviation SD of 20 results. Add twice standard deviation (+2SD) as report using blank averageThe detectability of method. As seen from Table 1, sensitivity is 0.053mg/L.
Table 1
2. the linear assessment of high value
By 1 part of low value serum (0.5mg/L) and 1 part high value serum (25mg/L) by 9: 1,4: 1,2: 1,1: 1,1: 2,1: 3,1: 4,1: 9 (sample order is numbered 1～No. 6), prepares 6 variable concentrations samples, and each sample replication 2 times, can from table 2See, the highest detection scope of detection kit of the present invention can reach 20.1mg/L, judgment basis r2 >=0.990.
Table 2
3. precision assessment
Use the human serum sample of 2 different B2M content, measure the withinrun precision of detection kit of the present invention.Use 3 lot number kits to measure respectively 1 human serum sample 20 times, calculate detection kit of the present invention batch between antipodePoor. Result shows, the withinrun precision of detection kit of the present invention is 3.8% and 1.38% (in table 3), and batch between extreme difference relativelyBe 2.43% (in table 4).
Table 3
Table 4
4. accuracy (recovery experiment) assessment
Select the conventional detection sample of suitable concn, be divided into identical 3 parts of volume. In 2 increments bases, add difference to measure thereinDeterminand standard, makes the recovery sample that 2 differences add concentration, calculates the concentration of the determinand adding. At another increment originallyIn add the solvent without measured object of same amount, make basic sample. Carry out replication 3 times to reclaiming sample and basic sampleAnalyze, get its average and calculate. (in table 5)
Table 5
5. interference test
Add respectively the interfering material of different content in a human serum sample after, measure. Add after interfering materialMeasured value is divided by adding the measured value before interfering material to be the rate of recovery, and result of the test shows bilirubin, hemoglobin, glycerine threeBelow the variable concentrations of ester and the rheumatoid factor time, testing result is disturbed (with the rate of recovery of B2M without remarkableBetween 90%～110%, be as the criterion). (in table 6).
Table 6
By above-mentioned test, advantage of the present invention and beneficial effect:
Kit of the present invention have detect simple and quick, highly sensitive, accuracy is good, antijamming capability is strong and be produced intoThe advantage that this is low.
2. the B2M detection method that the present invention adopts is latex enhancing immune turbidimetry, and the method makes β 2-micro-The detection of globulin is more economical, convenient and quick, is applicable to the automatic biochemistry analyzer of most hospitals, particularly to urgencyExamine and can realize Quantitative detection.
The above is only the preferred embodiment of the present invention, and protection scope of the present invention is also not only confined to above-mentioned enforcementExample, all technical schemes belonging under thinking of the present invention all belong to protection scope of the present invention. It should be pointed out that for the artThose of ordinary skill, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications alsoShould be considered as protection scope of the present invention.
Claims (7)
1. a beta 2-microglobulin detecting kit, described kit comprises reagent R1, reagent R2, described reagent R1 isBuffer solution;
Described reagent R2 is the mixture of B2M antibody sensitized polystyrene latex particle and buffer solution, described examinationThe preparation process of agent R2 comprises:
Step (1): add ethyl dimethyl amine propyl carbodiimide diimine in the polystyrene latex particle with carboxyl, obtainThe latex particle activating;
Step (2): the latex particle that step (1) is activated and B2M antibody mix, and add Portugal after question response finishes againUnreacted radical on grape sugar sealing latex microsphere, the latex microsphere that obtains coupling antibody is B2M antiantibody sensitizationPolystyrene latex particle, is placed in buffer solution.
2. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: described B2M antibodyThe concentration of sensitization polystyrene latex particle in reagent R2 is 0.08～0.28 gram/100ml.
3. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: the buffering in described reagent R1Liquid is PBS buffer solution, Tris buffer solution, glycine buffer, borate buffer solution, acetate buffer, citric acid-phosphateIn buffer solution, carbonate-bicarbonate buffer, MES buffer solution, ammonium chloride buffer at least one.
4. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: the buffering in described reagent R2Liquid is PBS buffer solution, Tris buffer solution, glycine buffer, borate buffer solution, acetate buffer, citric acid-phosphateIn buffer solution, carbonate-bicarbonate buffer, MES buffer solution, ammonium chloride buffer at least one.
5. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: ethyl dimethyl in step (1)Amine propyl carbodiimide diimine: the weight ratio of polystyrene latex particle is 0.5～5: 100.
6. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: described α 1-microglobulin is anti-Body comprises polyclonal antibody or monoclonal antibody.
7. beta 2-microglobulin detecting kit according to claim 1, is characterized in that: the polyphenyl in described step (1)Its average grain diameter of ethylene latex particle is between 0.01 ~ 0.2mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610050627.7A CN105606822A (en) | 2016-01-26 | 2016-01-26 | Beta 2-microglobulin detection kit |
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| CN201610050627.7A CN105606822A (en) | 2016-01-26 | 2016-01-26 | Beta 2-microglobulin detection kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645753A (en) * | 2016-12-30 | 2017-05-10 | 广州华弘生物科技有限公司 | Rapid detection kit of beta 2-microglobulin and application of rapid detection kit |
| CN106771228A (en) * | 2016-11-17 | 2017-05-31 | 安徽同致生物工程股份有限公司 | A kind of microglobulins of β 2 determine kit and preparation method thereof |
| CN108872590A (en) * | 2018-06-29 | 2018-11-23 | 宁波海壹生物科技有限公司 | The kit of β2-microglobulin in serum and urine specimen can be detected simultaneously |
| CN109575133A (en) * | 2018-12-28 | 2019-04-05 | 江苏众红生物工程创药研究院有限公司 | Anti-human β2- MG antibody and its application |
| CN109975550A (en) * | 2018-12-29 | 2019-07-05 | 宁波普瑞柏生物技术股份有限公司 | Eliminate the method and kit of hook effect in β2-microglobulin detection |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771228A (en) * | 2016-11-17 | 2017-05-31 | 安徽同致生物工程股份有限公司 | A kind of microglobulins of β 2 determine kit and preparation method thereof |
| CN106645753A (en) * | 2016-12-30 | 2017-05-10 | 广州华弘生物科技有限公司 | Rapid detection kit of beta 2-microglobulin and application of rapid detection kit |
| CN108872590A (en) * | 2018-06-29 | 2018-11-23 | 宁波海壹生物科技有限公司 | The kit of β2-microglobulin in serum and urine specimen can be detected simultaneously |
| CN109575133A (en) * | 2018-12-28 | 2019-04-05 | 江苏众红生物工程创药研究院有限公司 | Anti-human β2- MG antibody and its application |
| CN109575133B (en) * | 2018-12-28 | 2022-04-05 | 江苏众红生物工程创药研究院有限公司 | Anti-human beta2-MG antibodies and uses thereof |
| CN109975550A (en) * | 2018-12-29 | 2019-07-05 | 宁波普瑞柏生物技术股份有限公司 | Eliminate the method and kit of hook effect in β2-microglobulin detection |
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Application publication date: 20160525 |