CN102590526A - Beta 2-microglobulin detection kit - Google Patents
Beta 2-microglobulin detection kit Download PDFInfo
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Abstract
The invention provides a beta 2-microglobulin detection kit comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution, a preservative, a stabilizing agent, an electrolyte, a surfactant and the balance of purified water; and the reagent R2 comprises polystyrene latex grains sensitized by the beta 2-microglobulin antibody, a buffer solution, a preservative, a stabilizing agent and a residual amount of purified water. The polystyrene latex grains sensitized by the beta 2-microglobulin antibody are directionally coupled. A preparation method comprises the following steps of: activating the polystyrene latex grains; oxidizing the beta 2-microglobulin antibody; and coupling the beta 2-microglobulin antibody and the polystyrene latex grains. The kit provided by the invention has the advantages of high sensitivity, good specificity, good accuracy, strong anti-jamming capability and low production cost.
Description
Technical field
The invention belongs to the Medical Immunology field, relate to a kind of immunologic function test reagent, further, the present invention relates to a kind of B2M detection kit.
Background technology
B2M is a kind of micromolecule globulin that is produced by lymphocyte, blood platelet, polymorphonuclear leukocyte; Molecular mass is 11.8KD; It is the β chain (light chain) partly (being a single chain polypeptide) of the human lymphocyte antigens of cell surface (HLA); Extensively be present in blood plasma, urine, cerebrospinal fluid, saliva and first Ruzhong, content is very low.The synthetic ratio of normal person's B2M and the burst size from cell membrane are quite constant; B2M can freely filter from glomerulus; 99.9% in the absorption of near-end renal tubule, therefore measures the index that the blood B2M can be used as the reflection glomerular filtration rate(GFR.
The method of mensuration B2M commonly used has SRID, IE, radio immunoassay, ELISA, latex enhancing immune turbidimetry at present.Wherein using maximum is exactly the latex enhancing immune turbidimetry; Its ultimate principle is: with antibody sandwich on latex particle; After corresponding antigens generation immune response, form aggregated particle, under certain wavelength; Through measuring the turbidity that aggregation produced, can measure the content of checking matter in the sample.Be physisorphtion with the B2M antibody sandwich to the common method on the latex particle at present, this method less stable, antibody splits away off from latex particle easily; And receiving the interference of the rheumatoid factor (RF) and heterophil antibody easily, IgM, IgG type RF or heterophil antibody can directly combine with the Fc section of antibody on being coated on latex, thereby cause testing result false positive or false rising.
Summary of the invention
Technical matters to be solved by this invention is the deficiency to above-mentioned background technology, provide a kind of highly sensitive, specificity good, accuracy good, antijamming capability reach the low B2M detection kit of production cost by force.
The technical scheme that the present invention adopted is:
A kind of B2M detection kit, this kit comprise reagent R1, reagent R2, and said reagent R1 comprises damping fluid, antiseptic, stabilizing agent, electrolyte, surfactant, and all the other are purified water; Said reagent R2 comprises that all the other are purified water with B2M antibody sensitized polystyrene latex particle, damping fluid, antiseptic, stabilizing agent.
The volume ratio of reagent R1 and reagent R2 is 4: 1 in the mentioned reagent box of the present invention.
Reagent of the present invention 1 and damping fluid in the reagent 2 include but not limited to that PBS damping fluid, Tris damping fluid, glycine buffer, borate buffer solution, acetate buffer, citric acid-phosphate buffer, carbonate-bicarbonate buffer, 2-morpholino b acid (MES) damping fluid, ammonium chloride buffer and other have one or more in the damping fluid of similar quality.The used damping fluid of damping fluid that reagent R1 is used and reagent R2 is as long as all can for a kind of any collocation in the top damping fluid.
Reagent of the present invention 1 and antiseptic in the reagent 2 include but not limited to that potassium sorbate, Sodium Benzoate, Sodium azide, sodium nitrite, Proclin 300 and other have one or more in the antiseptic of similar quality.
Reagent of the present invention 1 comprises but is not limited to polyglycol, glycerine, propylene glycol, sucrose, trehalose, sorbierite, BSA and other to have in the stabilizing agent of similar quality one or several with stabilizing agent in the reagent 2.
Electrolyte in the reagent 1 of the present invention includes but not limited to that sodium chloride, potassium chloride, lime chloride and other have one or more in the electrolyte of similar quality.
Surfactant in the reagent 1 of the present invention comprises but is not limited to TWEEN series of surfactants, SPAN series of surfactants, TRITON series of surfactants and other to have in the surfactant of similar quality one or more.
Polystyrene latex particle of the present invention (this polystyrene latex particle is commercially available conventional products), its mean grain size are between 0.01～1.5 μ m, and particle diameter is during less than 0.01 μ m; Latex particle is assembled the optical density that produces and is changed too little; The result is difficult to reach the requirement of test susceptibility, in preparation reagent, needs the centrifugation time of overspending; Prolonged the time that reagent is produced, increased reagent cost and be unfavorable for repeatability.And particle diameter greater than the latex particle of 1.5 μ m when measuring high concentration and detect thing; It is assembled the optical density variation that produces and has surpassed detectability; The result be difficult to obtain to change corresponding to the optical density of analyte concentration, and particle too conference quicken self aggregation, cause dispersed the reduction.The latex particle particle diameter changes along with the change of test method and equipment, and therefore selected grain diameter is preferably between 0.05～0.5 μ m.
Antibody according to the invention includes but not limited to polyclonal antibody (sheep, rabbit, chicken) and monoclonal antibody.
B2M detection kit of the present invention also comprises the B2M calibration object except that comprising reagent 1 and reagent 2; Further, B2M content is 0～18mg/L in the described B2M calibration object.
Reagent R2 of the present invention is with B2M antibody sensitized polystyrene latex particle, places suitable damping fluid to form, and this reagent R2 preparation process comprises:
(1) activation of polystyrene latex particle: in having the polystyrene latex particle of carboxyl, add ethyl dimethyl amine propyl carbodiimide diimine (EDAC); Add adipic dihydrazide after the hybrid reaction; Continue hybrid reaction, the polystyrene latex particle that obtains activating;
(2) oxidation of B2M antibody: the non-binding active region of B2M antibody Fc terminal hydroxy group is oxidized to aldehyde radical with sodium metaperiodate;
(3) coupling of B2M antibody and polystyrene latex particle: with the polystyrene latex particle of step (1) activation and the B2M antibody hybrid reaction of step (2) oxidation; After question response finishes; Add unreacted radical on the glucose capped polystyrene latex microsphere again, obtain the polystyrene latex microballoon of coupling B2M antibody.
Material proportion is in each step of the above-mentioned preparation method of the present invention:
Ethyl dimethyl amine propyl carbodiimide diimine in the step (1): polystyrene latex particle=0.2～1: 100 (weight ratios); Adipic dihydrazide: polystyrene latex particle=20～50: 100 (weight ratios);
Step (2) meso-periodic acid sodium: B2M antibody=1: 2 (weight ratio);
Antibody in the step (3): polystyrene latex particle=10～20 of activation: 100 (weight ratios), glucose: polystyrene latex particle=25～50 of activation: 100.
The method of the used detection B2M of the present invention is the latex enhancing immune turbidimetry; Its principle is the present latex particulate that has encapsulated antigen or antibody; After corresponding antibodies in the sample or antigen generation immune response, form aggregated particle, under certain wavelength; Form the turbidity that is produced through measuring aggregation, can measure the content of checking matter in the sample.In the present invention, B2M in the serum and the anti-people's B2M antibodies that is combined in the polystyrene latex particle surface produce antigen-antibody reaction, the polystyrene latex particle is formed assemble; At 546nm, the 700nm wavelength is measured absorbance, and the reference standard curve can be obtained the content of B2M.
Advantage of the present invention:
1. the present invention is through improving the latex particle method for coating, makes that this kit is highly sensitive, specificity good, accuracy is high, antijamming capability is strong, production cost is low.
2. reagent R2 in the kit of the present invention preparation is a kind of antigen-antibody reaction test method, and special feature is, the present invention adopt directed coupling method with the B2M antibody sandwich to the polystyrene latex particle; The present invention is coupled to the Fc end orientation of the non-binding active region of B2M antibody on the polystyrene latex particle; The testing result false positive that interference caused or the false rising of the rheumatoid factor (RF) and heterophil antibody can not take place; The B2M antibody coupling of the directed coupling of this method to the polystyrene latex particle after, its antigen binding site points to moving phase, the antibody coupling position is the Fc fragment; Therefore the situation of antigen binding capacity loss can not take place; The vigor that has kept antibody has significantly reduced the consumption of antibody, has reduced production cost.
3. directed chemical coupling method is adopted in the R2 preparation in the kit of the present invention; The sensitizing latex granule stability that obtains is better; Antibody can not come off from latex particle, and because in the chemical coupling process, structural change has taken place the Fc section; Therefore can not receive the interference of the rheumatoid factor (RF) and heterophil antibody, promote the accuracy and the credibility of testing result greatly.
Embodiment
Through embodiment the present invention is described in further detail below, but the present invention not only is confined to following examples.
The preparation of embodiment 1 B2M detection kit
It is following that kit of the present invention relates to the main raw material(s) of reagent:
1. B2M antibody: adopting indirect elisa method to measure and tiring is 1: 100000.
2. latex: the only exemplary employing diameter of the present invention is that the polystyrene latex particle of 200～300nm band carboxylic group experimentizes.
The preparation of the main agents of present embodiment is following:
Reagent R1: contain 1.0%PEG6000 (Macrogol 6000), 0.09% Sodium azide, 0.2%BSA, 0.8%NaCl, the 0.2M ammonium chloride buffer of 0.3% polysorbas20.This reagent is colourless transparent solution.
Contain 0.2% B2M antibody sensitized polystyrene latex particle, 1%BSA in the reagent R2:0.1M Tris damping fluid, 0.2%NaN
3This reagent is milky white solution.Concrete steps are following:
1. get 1ml (100mg/ml) latex, with 0.1M (mol/L), the MES solution of pH5.0 (2-morpholino b acid damping fluid) washing 3 times disperses;
2. add 0.05ml and use 0.1M, the freshly prepared EDAC of MES solution (10mg/ml) of pH5.0 mixes fully, mixed 1 hour of room temperature;
3. add 0.5ml and use 0.1M, the freshly prepared adipic dihydrazide of MES solution (100mg/ml) of pH5.0,4 ℃ of reactions are spent the night;
4. use 0.1M, the MES solution washing of pH5.0 2 times, 0.1M, the phosphate solution washing of pH7.5 1 time disperses subsequent use;
5. get 1.5ml antibody (10mg/ml) solution, add 0.5ml and use 0.1M, the sodium periodate solution (10mg/ml) of the phosphate solution preparation of pH7.5, mixed at room temperature 15min,
6. well-oxygenated antibody in the step 5 is joined in the latex solution that has activated in the step 4,4 ℃ of reactions are spent the night;
7. add 0.32ml 10%BSA (bSA) solution, 4 ℃ are mixed 4h;
8. add 0.4ml 10% glucose solution, 4 ℃ of mixing are spent the night;
9. use 0.1M, pH7.5Tris solution washing 3 times adds 0.1M, and pH7.5Tris solution (contains 1%BSA, 0.2%NaN
3The Tris damping fluid) to the latex final concentration be 0.2%.
B2M calibration object: the B2M albumen that in 0.9% physiological saline, adds different content; Aseptic filtration; B2M content is controlled between 0～18mg/L in the described B2M calibration object, and these calibration objects are colourless transparent liquid.
The mensuration of embodiment 2 B2Ms
Testing tool: Hitachi's 7060 type automatic analyzers.
Analytical approach: 2 end-point methods; Predominant wavelength: 546nm, commplementary wave length: 700nm: sample size: 3.0ul; Reagent R1:240ul; Reagent R2:60ul; Calibrating mode: splines Spline; The Direction of Reaction: rise; Measure temperature: 37 ℃; Behind sample and the reagent R1 mixing, read absorbance A in the 30th second
1In the time of 5 minutes, add reagent R2, in the time of 5 minutes, read absorbance A
2React the A that is calculated as of absorbance
2With A
1The calibration difference.
Computing method: the multiple spot calibration, as computation schema, make dosage/response curve with splines according to the value of absorbance and reference serum, sample size can be calculated on dosage/response curve according to its absorbance.
Term of reference: 1.0～3.0mg/L
Embodiment 3 B2M detection kit each item testing performance indexes
1. sensitivity test
Measure the sample of 6 kinds of different B2M content, each sample is surveyed 10 times, and through computation of mean values and SD value (standard difference), visible from table 1, the sensitivity of detection kit of the present invention is 0.08mg/L.
Table 1
| β2-MG(mg/L) | Measure number of times | Average | 2.6SD | M+2.6SD | M-2.6SD |
| 0 | 10 | 0.02 | 0.031 | 0.05 | -0.02 |
| 0.04 | 10 | 0.05 | 0.044 | 0.10 | 0.01 |
| 0.08 | 10 | 0.10 | 0.031 | 0.13 | 0.07 |
| 0.12 | 10 | 0.15 | 0.058 | 0.21 | 0.10 |
| 0.16 | 10 | 0.19 | 0.044 | 0.23 | 0.14 |
| 0.20 | 10 | 0.22 | 0.032 | 0.25 | 0.19 |
2. high value linear determination
Measure the sample of 10 different B2M content, each sample is surveyed 2 times, and visible from table 2, the highest detection scope of detection kit of the present invention can reach 18mg/L, judgment basis r
2>=0.990.
Table 2
| β 2-MG theoretical value (mg/L) | β 2-MG actual value (mg/L) | |
| 1 | 0 | 0.03 |
| 2 | 2 | 2.08 |
| 3 | 4 | 3.91 |
| 4 | 6 | 5.89 |
| 5 | 8 | 8.15 |
| 6 | 10 | 10.09 |
| 7 | 12 | 11.85 |
| 8 | 14 | 14.22 |
| 9 | 16 | 16.29 |
| 10 | 18 | 18.30 |
3. precision test
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 1 human serum sample 20 times respectively, calculate detection kit of the present invention batch between extreme difference relatively.The result shows that the withinrun precision of detection kit of the present invention is 1.36% and 1.62% (seeing table 3), and relative extreme difference is 4.03% (seeing table 4) between criticizing.
Table 3
| Sample 1 | Sample 2 | |
| Measure number of times | 20 | 20 |
| Mean value (mg/L) | 1.430 | 11.318 |
| Minimum value (mg/L) | 1.400 | 10.840 |
| Maximal value (mg/L) | 1.460 | 11.560 |
| Standard deviation (SD) | 0.0194 | 0.1840 |
| The coefficient of variation (CV%) | 1.36 | 1.62 |
Table 4
4. interference test
Measure after in a human serum sample, adding the interfering material of different content respectively.The measured value that adds behind the interfering material is contributive rate divided by the measured value that adds before the interfering material; Test findings shows unconjugated bilirubin; Combined with bilirubin, the concentration of haemoglobin and chylomicron be respectively at 200mg/dl, 200mg/dl; When 5000mg/dl and 21000FTU were following, they all (were seeing table 5) below 2% to the interference of measuring the result.
Table 5
5. stability test
Under 2-8 ℃ of storage requirement, 0 month, April, August, Dec and 14 months same serum sample to be measured respectively, each sample is surveyed 5 times, gets average (result sees table 6).The result shows, April, August, compares with 0 month Dec and 14 months, and measured value difference is very little, explains that detection kit of the present invention can stablize 1 year under 2-8 ℃ of storage requirement.
Table 6
| Project | 0 month | April | August | Dec | 14 months |
| The reagent blank absorbance A 0 | 0.7871 | 0.7780 | 0.7809 | 0.7951 | 0.7885 |
| The sample measured value | 2.463 | 2.49 | 2.505 | 2.52 | 2.523 |
| CV (%) in crowd | 1.30 | 1.32 | 1.25 | 1.42 | 1.57 |
But from the B2M detection kit of the foregoing description knowledge capital invention have highly sensitive, specificity good, accuracy good, antijamming capability reach the low advantage of production cost by force.
Claims (10)
1. B2M detection kit, it is characterized in that: comprise reagent R1, reagent R2, said reagent R1 comprises damping fluid, antiseptic, stabilizing agent, electrolyte, surfactant, and all the other are purified water; Said reagent R2 comprises that all the other are purified water with B2M antibody sensitized polystyrene latex particle, damping fluid, antiseptic, stabilizing agent.
2. B2M detection kit according to claim 1 is characterized in that: the volume ratio of said reagent R1 and reagent R2 is 4: 1.
3. B2M detection kit according to claim 1 is characterized in that: the damping fluid in described reagent 1 and the reagent 2 is one or more in PBS damping fluid, Tris damping fluid, glycine buffer, borate buffer solution, acetate buffer, citric acid-phosphate buffer, carbonate-bicarbonate buffer, 2-morpholino b acid damping fluid, the ammonium chloride buffer.
4. B2M detection kit according to claim 1 is characterized in that: the mean grain size of described polystyrene latex particle is 0.01～1.5 μ m.
5. B2M detection kit according to claim 4 is characterized in that: the mean grain size of described polystyrene latex particle is 0.05～0.5 μ m.
6. B2M detection kit according to claim 1 is characterized in that: described B2M antibody is anti-people's polyclonal antibody of goat-anti people polyclonal antibody or rabbit or mouse-anti human monoclonal antibodies.
7. B2M detection kit according to claim 1 is characterized in that: also comprise the B2M calibration object.
8. B2M detection kit according to claim 7 is characterized in that: the content of B2M is 0～18mg/L in the said B2M calibration object.
9. B2M detection kit according to claim 1 is characterized in that: the preparation process of said reagent R2 comprises:
(1) activation of polystyrene latex particle: in having the polystyrene latex particle of carboxyl, add ethyl dimethyl amine propyl carbodiimide diimine, add adipic dihydrazide after the hybrid reaction, continue hybrid reaction, the polystyrene latex particle that obtains activating;
(2) oxidation of B2M antibody: the non-binding active region of B2M antibody Fc terminal hydroxy group is oxidized to aldehyde radical with sodium metaperiodate;
(3) coupling of B2M antibody and polystyrene latex particle: with the polystyrene latex particle of step (1) activation and the B2M antibody hybrid reaction of step (2) oxidation; After question response finishes; Add unreacted radical on the glucose capped polystyrene latex particle again, obtain the polystyrene latex particle of coupling B2M antibody.
10. B2M detection kit according to claim 9 is characterized in that: ethyl dimethyl amine propyl carbodiimide diimine in the said step (1): polystyrene latex particle=0.2～1: 100 (weight ratios); Adipic dihydrazide: polystyrene latex particle=20～50: 100 (weight ratios); Step (2) meso-periodic acid sodium: B2M antibody=1: 2 (weight ratio); B2M antibody in the step (3): polystyrene latex particle=10～20 of activation: 100 (weight ratios), glucose: polystyrene latex particle=25～50 of activation: 100 (weight ratios).
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