CN101813700A - Kit for detecting beta2-microglobulin by nanometer microsphere immunoturbidimetry - Google Patents
Kit for detecting beta2-microglobulin by nanometer microsphere immunoturbidimetry Download PDFInfo
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- CN101813700A CN101813700A CN201010139410A CN201010139410A CN101813700A CN 101813700 A CN101813700 A CN 101813700A CN 201010139410 A CN201010139410 A CN 201010139410A CN 201010139410 A CN201010139410 A CN 201010139410A CN 101813700 A CN101813700 A CN 101813700A
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Abstract
The invention relates to a kit for measuring beta2-microglobulin in serum. The technical problem to be solved is to overcome the disadvantages of background technology and provide a kit for detecting the beta2-microglobulin by nanometer microsphere immunoturbidimetry, which has no need of dilution of a sample, simple operation, high accuracy and good repeatability, and is applicable to full automatic biochemical analyzers with diverse types. The technical scheme comprises that: the kit for detecting the beta2-microglobulin by the nanometer microsphere immunoturbidimetry comprises a reagent R1, a reagent R2 and a reference calibration material, wherein the reagent R1 comprises buffer solution, a preservative, a stabilizing agent, an electrolyte, a surfactant and the balance of purified water; the reagent R2 comprises the buffer solution, nanometer microspheres which is combined with an anti-human beta2-microglobulin antibody and has a diameter of between 50 and 150 nm and the preservative; and the reference calibration material comprises the buffer solution, the stabilizing agent, the preservative, a recombinant human beta2-microglobulin pure product in a proper amount determined by concentration requirement and the balance of the purified water.
Description
Technical field
The present invention relates to a kind of kit that is used to measure serum composition, particularly measure the kit of the B2M (β 2-MG) in the serum, can be widely used in medical science and technological field of biochemistry.
Background technology
β
2-microglobulin (β
2-MG) be the protein of a kind of low-molecular-weight (11800), in patient's urine of renal tubule nephritis, separate at first; β
2-MG is present in nearly all karyocyte surface, is the ingredient of humam leucocyte antigen (HLA) molecule light chain.As the product of HLA metabolism and decomposition, β
2-MG appears in serum, urine and other body fluid with the free form of low concentration.Free β
2-MG heavily absorbs by the glomerular filtration renal tubule and discharges and degraded.Because β
2Production rate is comparatively constant in vivo every day for-MG, and only by renal excretion, measures the index that blood β 2-MG can be used as glomerular filtration rate(GFR.
Known mensuration β
2-microglobulin (β
2-MG) method has SRID, immunoelectrophoresis, radio immunoassay, euzymelinked immunosorbent assay (ELISA) (ELISA method).These methods exist complex operation, need special equipment, and sample needs pre-service, can not carry out batch sample analysis and can not directly go up shortcoming such as automatic clinical chemistry analyzer detection.
Summary of the invention
Technical matters to be solved by this invention is the deficiency that overcomes the above-mentioned background technology, a kind of improvement of B2M detectable is provided, the reagent that is provided should have sample need not dilution, simple to operate, accuracy is high, good reproducibility, is applicable to that the Nano microsphere immunoturbidimetry of various types of automatic clinical chemistry analyzers detects the B2M kit.
Technical scheme provided by the invention is:
A kind of Nano microsphere immunoturbidimetry detects β
2-microglobulin kit comprises reagent R
1, reagent R
2And reference calibrations product; Wherein:
A, reagent R
1: a kind ofly make β in the sample
2-microglobulin antigen site fully exposes, and helps and anti-β
2The well-bound β of-microglobulin antibody reagent
2-microglobulin solution comprises stabilizing agent, 50-200mmol/L electrolyte, the 0.1-4mmol/L surfactant of 5-200mmol/L damping fluid, 0.1-5mmol/L antiseptic, 0.5-5mmol/L, and all the other are purified water;
B, reagent R
2: a kind of Nano microsphere solution that is combined with anti-people's B2M antibody, comprise the Nano microsphere of the anti-people's B2M antibody of being combined with of 5-200mmol/L damping fluid, 0.1%-5% weight ratio, an amount of antiseptic, the Nano microsphere diameter is 50-150nm;
C, reference calibrations product: a kind of being used for compared with sample, carry out the B2M solution that the result calculates, comprise damping fluid 100-200mmol/L, stabilizing agent 166-180mmol/, an amount of antiseptic and the pure product of determining according to the concentration needs of a certain amount of recombined human B2M, all the other are purified water.The pure product of recombined human B2M are added in the above-mentioned solution, obtain the β of desired concn
2-MG reference calibrations product (these B2M reference calibrations product for the human serum that adds the pure product of recombined human B2M or the liquid of other similar serum matrix).
β
2The concentration of-MG reference calibrations product can be high concentration single-point reference calibrations product, is diluted to the reference calibrations product of a plurality of variable concentrations in use with physiological saline; Also can directly be prepared into the reference calibrations product of a plurality of variable concentrations.Here have no particular limits, as long as the β that makes
2-MG reference calibrations product can compare with sample, can measure β in the sample
2The content of-MG gets final product.
Described β
2Also add reaction accelerator among the-microglobulin reagent R1, concentration is 3-10mmol/L.
Described stabilizing agent is one or more the mixing in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA) and the sodium chloride.
Described damping fluid is trishydroxymethylaminomethane or CAPSO.
Described antiseptic is a wide-spectrum bactericide.
The described preparation method who is combined with the Nano microsphere solution of anti-people's B2M antibody is: at room temperature diluting part by weight with damping fluid is 1: 1 anti-people β
2-MG antibody polyclonal antibody and Nano microsphere, with 2 hours (i.e. Chang Gui physisorphtion) of room temperature vibration absorption behind both mixings, add the damping fluid that contains 0.1%BSA and sealed the centrifugal supernatant that goes 1 hour, being diluted to concentration with damping fluid again is 0.3%, and adds an amount of antiseptic.
Anti-people β
2-MG antibody polyclonal antibody comprises the anti-people β of goat-anti
2The anti-people β of-MG antibody polyclonal antibody or rabbit
2The anti-people β of-MG antibody polyclonal antibody or mouse-anti
2-MG antibody polyclonal antibody.
Primary raw material required for the present invention
1, goat-anti people β
2-microglobulin polyclonal antibody; Buyable or entrust Zhejiang health biotechnology research and development centre of Erie to prepare according to a conventional method, this antibody only with people β
2The reaction of-microglobulin does not have immunological cross-reaction with other antigen, tires and can satisfy this reagent requirement.
2, Nano microsphere; There are many commercial Nano microspheres available on the market, comprise U.S. Sigma company, Merck KGaA company, Japanese UNF company; The Nano microsphere of optional majority kind diameter, it is the microballoon of 80～120nm that the present invention has adopted diameter, it is 600nm that reagent corresponding detects predominant wavelength.
3, the pure product of reorganization B2M; Buyable, or by the preparation of health biotechnology research and development centre of Zhejiang Erie, being used to prepare this reagent place needs the reference calibrations product.
The equal buyable of all the other biochemical raw materials.
The principle that the present invention measures sample is to utilize antigen-antibody reaction, adds reagent R1 (sample dilution) earlier, removes electronic shell and hydrated sheath around the antigen in the sample, and antigen site is fully exposed; Add reagent R then
2(anti-people β
2The Nano microsphere solution of-MG antibody), make β
2Corresponding β in the Nano microsphere of-MG antibody and the sample
2-MG antigen-reactive forms insoluble antigen-antibody complex, produces certain turbidity, the β in its turbidity height and the sample
2-MG content is directly proportional; Under provision wavelengths, measure the absorbance of this insoluble antigen-antibody complex, with the β of concentration known
2-MG reference calibrations product compare, and then can calculate β in the sample
2--the content of MG.
Nano microsphere can adopt physisorphtion or chemical crosslink technique preparation, preferred physisorphtion with combining of anti-people's B2M antibody among the reagent R2.
Reagent provided by the invention is liquid double reagent, and it is good to have specificity, highly sensitive, and accuracy reaches advantages such as antijamming capability is strong well, and simple to operate when measuring, sample need not dilute in advance, can be used for detecting β in the body fluid
2The concentration of-microglobulin is applicable to clinical automatic clinical chemistry analyzer.
Description of drawings
Fig. 1 measures gained β for adopting the embodiment of the invention 1
2The measured value of-MG and the β that passes through the radio immunoassay gained
2The mutual relationship synoptic diagram of-MG measured value.
Fig. 2 measures gained β for adopting the embodiment of the invention 2
2The measured value of-MG and the β that passes through the radio immunoassay gained
2The mutual relationship synoptic diagram of-MG measured value.
Fig. 3 measures gained β for adopting the embodiment of the invention 3
2The measured value of-MG and the β that passes through the radio immunoassay gained
2The mutual relationship synoptic diagram of-MG measured value.
Embodiment
β
2The preparation embodiment of-MG detectable:
Embodiment one
1, β
2-MG solution (reagent R
1)
TRIS buffer 50mmol/L
Tween-20 (surfactant) 0.5mmol/L
NaCL (electrolyte) 100mmol/L
PEG-6000 (reaction accelerator) 4mmol/L
Wide-spectrum bactericide (antiseptic) 3mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 5mmol/L
All the other are purified water
2, anti-people β
2-MG antibody-solutions (reagent R
2)
At room temperature dilute goat-anti people β with 100mmol/L TRIS buffer (pH 7.4)
2-MG antibody polyclonal antibody and 80nm Nano microsphere (part by weight of antibody and microballoon is 1: 1), with 2 hours (being the known physisorphtions of those skilled in the art) of room temperature vibration absorption behind both mixings, add confining liquid (containing the amino four alkane damping fluids of 0.1%BSA trihydroxy methyl) sealing 1 hour, the centrifugal supernatant that goes, being diluted to concentration with the 100mmol/L TRIS buffer is 0.3%, and adds an amount of antiseptic; Promptly obtain β
2-microglobulin detection kit reagent (R
2).
3, serotype β
2-MG solution (reference calibrations product)
CAPSO (damping fluid) 100mmol/L
Wide-spectrum bactericide (antiseptic) 2mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 2mmol/L
Bovine serum albumin(BSA) (stabilizing agent) 3mmol/L
Sodium chloride (stabilizing agent) 166mmol/L
All the other are purified water
According to needed β
2-MG reference calibrations product concentration is with the recombinant beta of respective amount
2The pure product 40mg/L of-MG adds in the above-mentioned solution, is prepared into the β of 40mg/L concentration
2-MG reference calibrations product.
Adopt present embodiment sample to be measured gained β
2The measured value of-MG and the β that passes through the radio immunoassay gained
2-MG measured value compares (referring to Fig. 1, the line retrace analysis of going forward side by side); Know correlativity r=0.9995 (y=1.007x+0.0236); Demonstrate present embodiment and radio immunoassay and have good correlativity.
Embodiment two
1, β
2-MG solution (reagent R
1)
Trishydroxymethylaminomethane (damping fluid) 150mmol/L
Tween-20 (surfactant) 4mmol/L
NaCL (electrolyte) 60mmol/L
PEG-6000 (reaction promoter) 8mmol/L
Wide-spectrum bactericide (antiseptic) 0.1mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 0.5mmol/L
All the other are purified water
2, anti-people β
2-MG antibody-solutions (reagent R
2)
At room temperature dilute goat-anti people β with 150mmol/L TRIS buffer (pH7.4)
2-MG antibody polyclonal antibody and 100nm Nano microsphere (part by weight of antibody and microballoon is 1: 1), with 2 hours (being the known physisorphtions of those skilled in the art) of room temperature vibration absorption behind both mixings, add the amino four alkane damping fluids sealing of the trihydroxy methyl that contains 0.1%BSA 1 hour, the centrifugal supernatant that goes, being diluted to concentration with the 100mmol/L TRIS buffer is 4%, and adds an amount of antiseptic; Promptly obtain β
2-microglobulin detection kit reagent (R
2).
3, serotype β
2-MG solution (reference calibrations product)
CAPSO (damping fluid) 150mmol/L
Wide-spectrum bactericide (antiseptic) 5mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 3mmol/L
Bovine serum albumin(BSA) (stabilizing agent) 3mmol/L
Sodium chloride (stabilizing agent) 166mmol/L
All the other are purified water
According to needed β
2-MG reference calibrations product concentration is with the recombinant beta of respective amount
2The pure product 60mg/L of-MG adds in the above-mentioned solution, is prepared into the β of 60mg/L concentration
2-MG reference calibrations product.
Adopt present embodiment sample to be measured gained β
2The measured value of-MG and the β that passes through the radio immunoassay gained
2-MG measured value compares (referring to Fig. 2, the line retrace analysis of going forward side by side); Know correlativity r=0.9985 (y=1.0264x+0.0149); Demonstrate present embodiment and radio immunoassay and have good correlativity.
Embodiment three
1, β
2-MG solution (reagent R
1)
TRIS buffer 200mmol/L
Tween-20 (surfactant) 2mmol/L
NaCL (electrolyte) 200mmol/L
PEG-6000 (reaction promoter) 5mmol/L
Wide-spectrum bactericide (antiseptic) 3mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 2mmol/L
All the other are purified water
2, anti-people β
2-MG antibody-solutions (reagent R
2)
At room temperature dilute goat-anti people β with 200mmol/L TRIS buffer (pH7.4)
2-MG antibody polyclonal antibody and 120nm Nano microsphere (part by weight of antibody and microballoon is 1: 1), with 2 hours (being the known physisorphtions of those skilled in the art) of room temperature vibration absorption behind both mixings, add the amino four alkane damping fluids sealing of the trihydroxy methyl that contains 0.1%BSA 1 hour, the centrifugal supernatant that goes, being diluted to concentration with the 100mmol/L TRIS buffer is 2%, and adds an amount of antiseptic; Promptly obtain β
2-microglobulin detection kit reagent (R
2).
3, serotype β
2-MG solution (reference calibrations product)
CAPSO (damping fluid) 200mmol/L
Wide-spectrum bactericide (antiseptic) 2mmol/L
Disodium ethylene diamine tetraacetate (stabilizing agent) 0.5mmol/L
Bovine serum albumin(BSA) (stabilizing agent) 5mmol/L
Sodium chloride (stabilizing agent) 166mmol/L
All the other are purified water
According to needed β
2-MG reference calibrations product concentration is with the recombinant beta of respective amount
2The pure product 80mg/L of-MG adds in the above-mentioned damping fluid, is prepared into the β of 80mg/L concentration
2-MG reference calibrations product.
Adopt present embodiment sample to be measured gained β
2The measured value of-MG and the β that passes through the radio immunoassay gained
2-MG measured value compares (referring to Fig. 3, the line retrace analysis of going forward side by side); Know correlativity r=0.9992 (y=1.0062x+0.0285); Demonstrate present embodiment and radio immunoassay and have good correlativity.
Condition determination of the present invention and step:
Result's calculating:
In the formula:
The average per minute absorbance of Δ AT testing sample changing value
The average per minute absorbance of Δ AS calibration solution changing value
β in the CS calibration solution
2The concentration of-MG
Still need and additional be: all herein ratios of not indicating especially are weight ratio.
Claims (5)
1. a Nano microsphere immunoturbidimetry detects β
2-microglobulin kit comprises reagent R
1, reagent R
2And reference calibrations product; Wherein:
A, reagent R
1: a kind ofly make β in the sample
2-microglobulin antigen site fully exposes, and helps and anti-β
2The well-bound β of-microglobulin antibody reagent
2-microglobulin solution comprises stabilizing agent, 50-200mmol/L electrolyte, the 0.1-4mmol/L surfactant of 5-200mmol/L damping fluid, 0.1-5mmol/L antiseptic, 0.5-5mmol/L, and all the other are purified water;
B, reagent R
2: a kind of Nano microsphere solution that is combined with anti-people's B2M antibody, comprise the Nano microsphere of the anti-people's B2M antibody of being combined with of 5-200mmol/L damping fluid, 0.1%-5% weight ratio, an amount of antiseptic, the Nano microsphere diameter is 50-150nm;
C, reference calibrations product: a kind of being used for compared with sample, carry out the B2M solution that the result calculates, comprise damping fluid 100-200mmol/L, stabilizing agent 166-180mmol/, an amount of antiseptic 2mmol/L and the pure product of determining according to the concentration needs of a certain amount of recombined human B2M, all the other are purified water.
2. a kind of Nano microsphere immunoturbidimetry according to claim 1 detects β
2-microglobulin kit is characterized in that described β
2Also add reaction accelerator among the-microglobulin reagent R1, concentration is 3-10mmol/L.
3. a kind of Nano microsphere immunoturbidimetry according to claim 1 and 2 detects β
2-microglobulin kit is characterized in that described stabilizing agent is one or more the mixing in disodium ethylene diamine tetraacetate, bovine serum albumin(BSA) and the sodium chloride.
4. a kind of Nano microsphere immunoturbidimetry according to claim 1 and 2 detects β
2-microglobulin kit is characterized in that described damping fluid is trishydroxymethylaminomethane or CAPSO.
5. the described a kind of Nano microsphere immunoturbidimetry of claim 1 detects β
2-microglobulin kit is characterized in that the described preparation method who is combined with the Nano microsphere solution of anti-people's B2M antibody is: at room temperature diluting part by weight with damping fluid is 1: 1 anti-people β
2-MG antibody polyclonal antibody and Nano microsphere with room temperature vibration absorption behind both mixings 2 hours, add the damping fluid that contains 0.1%BSA and sealed 1 hour, the centrifugal supernatant that goes, and being diluted to concentration with damping fluid again is 0.3%, and adds an amount of antiseptic.
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