CN105911291A - Urine trace protein detection kit and detection method thereof - Google Patents
Urine trace protein detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medical immunology and biology, in particular to a kit for joint detection of various trace proteins in urine and a detection method thereof. The detection kit is coupled with magnetic fluorescent microspheres of seven proteins and the like, enabling one-off joint detection of seven urine trace proteins, greatly improving detection efficiency and lowering detection cost; the kit is high in sensitivity and is advantageously beyond the methods such as immunonephelometry.
Description
Technical field
The present invention relates to Medical Immunology and biological technical field, be specifically related to a kind of test kit for urine lot of trace albumen joint-detection and detection method thereof.
Background technology
Kidney disease is the commonly encountered diseases that serious harm people's life is healthy.Under pathological conditions, glomerular filtration membrane permeability, the charged change of institute or reabsorption dysfunction, and the increase of specified protein synthesis all can cause in urine protein concentration to raise, body protein is primary nephropathy and other relevant diseases one of cause the most important Pathophysiology of nephropathy disorderly through homaluria abnormally.According to pathogenetic difference, albuminuria can be divided into overflow proteinuria, glomerular proteinuria, tubular proteinuria, mixed albuminuria, sense of organization albuminuria.
Urine micro protein refers in urine that the output of albumen exceedes normal limit, but the interstage of not up to clinical proteinuria, it is difficult to some protein detected by the qualitative or quantitative method of routine, is the sensitive indicator of Renal Injury.The lab testing of urine micro protein is respectively provided with important function to aspects such as early screening diagnosis, state of illness monitoring, observation of curative effect and the Index for diagnosis of kidney disease.
Microdose urine protein (microalbumin, MA), urinary metabolites (transferrin, TRF), B2M (β 2-microglobulin, β 2-MG), alpha2-macroglobulin (α 2-macroglobulin, α 2-MG), cystatin-C (Cystatin C), immunoglobulin G (immunoglobulin G, etc. IgG) being the most clinical more common urine trace egg project, early stage kidney injury point out value to obtain the affirmative of many documents by they.MA is detection diabetes, hypertension, cardiovascular disease and the important indicator of nephropathy blood vessel injury, early diagnosis, the early treatment of disease there are important reference value and clinical meaning, and the detection combining urine α 1-MG, TRF can point out position and the order of severity of kidney injury according to the size of protein molecular weight.Cystatin C is by the constant generation of all of nucleated cell, by metabolism after reabsorption after glomerular filtration, does not enter in urine, as acute injury of kidney (Acute kidney injury, AKI), time, the Cystatin C in urine raises, and early than the change of serum creatinine.B2M (β 2-microglobulin, β 2-MG) is concentration rising in urine when the injury of renal tubular that many reasons such as contacting nephrotoxicity material, operation on heart or renal transplantation causes, and more Zao than the increase of serum creatinine 4-5 days.
Nephropathy can be caused by many reasons, and there is the biggest individual variation, diagnose disease only according to an index, state of illness monitoring, observation of curative effect, Index for diagnosis are far from being enough, therefore, it is necessary that integrating Measuring Several Indexes carries out comprehensive assessment to patient, and provide more albuminuria index to come adjuvant clinical early diagnosis, early treatment, to improve the quality of life of patient.The urine micro protein detection method of traditional clinical application has immunodiffusion, immunoelectrophoresis, immunoturbidimetry, radioimmunity, enzyme linked immunological etc., it is small throughput detection, the detection cycle is long, and relies on substantially the detecting system of external import and supporting reagent is analyzed, and price is high.Some efficient detection technology (such as high performance liquid chroma-tography, capillary electrophoresis, mass spectrum etc.) are difficult to be promoted in clinic due to expensive equipment, detection of complex.Although urine protein detection project is extensively carried out in situation of all-level hospitals clinical laboratory at present, but being individual event and measure, the multi objective combined test kit that there is no maturation obtains State Food and Drug Administration's authentication registration.
Luminex technology platform is a highly integrated, the preferable representative of high throughput testing and multifunctional bio technology platform.Luminex xMAP liquid chip is (also known as suspension array, streaming fluorescent technique) the uniform circular microsphere (a diameter of 5.6 μm) that made by polystyrene material of detecting system forms, two kinds of red fluorescence dyestuffs of cladding different proportion, produce the microsphere of 100 kinds of different proportion fluorescence colors, can according to different research purposes 100 kinds of probes of coupling (such as antigen, antibody, nucleic acid, enzyme, various receptors etc.), sample suspensions target and the specific combination of microsphere surface conjugated probes, thus realize 100 kinds of different target molecules in a sample to be tested being detected simultaneously.Microsphere by sense channel, is irradiated by while two bundle laser one by one, and fluorescent material in the first bundle laser excitation microsphere determines the classification (classification) of microsphere；Fluorescein on second bundle laser excitation reporter molecules, determines target molecule quantity (quantitatively).Liquid microarrays technology has that hybridization kinetics is fast, microarray applications flexibly, low cost, advantage easy and simple to handle.
In prior art report utilize having of Luminex detection of platform: Chinese patent application CN201510264780.5 discloses and a kind of detects test kit of lipoprotein phospholipase A2 concentration and preparation method thereof in sample, MAGPIX and Luminex that detecting instrument is Luminex 200,3D (Publication No. CN104820097A) used；Chinese patent application CN201510264522.7 discloses test kit of myeloperoxidase (MPO) (MPO) concentration and preparation method thereof in a kind of detection sample, and detecting instrument used is the MAGPIX (Publication No. CN104950111A) of Luminex；Chinese patent CN200510030338.2 discloses a kind of human papillomavirus's detection and genotyping method and test kit, and technology platform used is Luminex xMAP (Publication No. CN1948503A).
But, have not yet to see and realize the relevant report of lot of trace albumen joint-detection in urine based on Luminex technology platform.
Summary of the invention
It is an object of the invention to provide a kind of Urinary microprotein detection kit and detection method thereof.The advantages such as the detection kit of the present invention has highly sensitive, multiple urine micro protein one tubular type joint-detection.7 kinds of urine micro protein of the disposable joint-detection of this detection method, greatly improve detection efficiency, reduce testing cost, have the unrivaled superiority of the methods such as immunoturbidimetry.
A first aspect of the present invention, there is provided a kind of seven kinds of trace albumin combined detection kits of urine, specifically includes that
(1) Rhizoma Nelumbinis is associated with the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody；
(2) by biotin labeled seven kinds of trace albumin antigen mixed liquors；
(3) by the phycoerythrin of marked by streptavidin；
(4) calibration object, at least includes seven kinds of trace albumin mixed liquors；
(5) buffer；
Seven kinds of described trace albumin are MA, TRF, β 2-MG, α 2-MG, α 1-MG, Cystatin C, IgG；
Described buffer is made up of sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, bovine serum albumin and Proclin-300；Wherein, the concentration of sodium chloride is 6.0-10.0g/L, the concentration of disodium hydrogen phosphate is 2.5-3.5g/L, the concentration of sodium dihydrogen phosphate is 0.2-0.4g/L, the concentration of bovine serum albumin is 8.0-12.0g/L, the concentration of Proclin-300 be the concentration of 0.01-0.03ml/L, Triton X-100 be 0.1-0.3ml/L.
Described magnetic fluorescent microspheres, during its Rhizoma Nelumbinis connection, antibody concentration is: every 106Individual magnetic fluorescent microspheres, every kind of antibody consumption is 2-10ug.
Described magnetic fluorescent microspheres, prepares by the following method: by magnetic fluorescent microspheres activation processing 15-20 minute in the activation system being made up of MES, NHS and EDC, adds magnetic field, standing separation 5 minutes, adds antibody 2-10ug/10 after washing 3 times with MES6Individual fluorescent microsphere, after room temperature Rhizoma Nelumbinis joins 2 hours, washs 1 time with MES and removes free antibody, add 1%BSA solution, put 2-8 DEG C of refrigerator and close overnight, close after terminating, wash 1 time with 1%BSA solution, add Proclin 300,2-8 DEG C of preservation.
In described activation system, when microsphere quantity is 2.5 × 106Time, the volume of MES be 230ul, EDC be 10ul, NHS be 10ul；Or when microsphere quantity is 2.5 × 106Time, the volume of MES be 480ul, EDC be 10ul, NHS be 10ul.
Described Rhizoma Nelumbinis is associated with in the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody, and the concentration configuring each microsphere with buffer is 100/ul.
Described by biotin labeled seven kinds of trace albumin antigen mixed liquors, the mol ratio of every kind of proteantigen and biotin is 1:20-100, incubated at room 30 minutes, the proteantigen of labelling biotin is loaded bag filter, fully dialyse by PBS solution, to remove free biotin, biotinylated protein antigen adds 50% glycerol ,-20 DEG C of preservations.
Described biotin is succinimide ester, and the mol ratio of every kind of proteantigen and biotin is 1:50.
In described biotin labeled seven kinds of trace albumin antigen mixed liquors, each proteantigen buffer dilution ratio is as follows:
Preferably, each proteantigen buffer dilution ratio is as follows:
It is 2mg/L that the phycoerythrin buffer of described marked by streptavidin configures its concentration.
Described calibration object, including: seven kinds of trace albumin mixed liquor S1-S6, in mixed liquor S1, the concentration of seven kinds of trace albumin is respectively as follows: the Cystatin C of α 1-MG, 3mg/L of α 2-MG, 70mg/L of β 2-MG, 90mg/L of TRF, 2.5mg/L of MA, 50mg/L of 60mg/L, and the IgG of 100mg/L；Mixed liquor S2-S6 is according to 1:2 or 1:3 gradient dilution by mixed liquor S1；Also include buffer S7, as blank reference.
In described buffer, the concentration of sodium chloride is 8.0g/L, and the concentration of disodium hydrogen phosphate is 2.9g/L, the concentration of sodium dihydrogen phosphate is 0.296g/L, the concentration of bovine serum albumin be the concentration of 10.0g/L, Proclin-300 be the concentration of 0.02ml/L, Triton X-100 be 0.2ml/L.
Described test kit also includes cleaning buffer solution, and composition is 0.1M PBS, 0.05%Tween-20,0.1%Proclin 300.
A second aspect of the present invention, it is provided that the method using above-mentioned Urinary microprotein detection kit detection Urinary microprotein, including:
A. in ELISA Plate micropore, add 10ul urine specimen, take simultaneously each 10ul of calibration object S1-S6 as standard with reference to and buffer S7 10ul as blank reference；
B. take biotin labeled seven kinds of trace albumin antigen mixed liquor 10ul and add in ELISA Plate micropore, mix homogeneously；
C. take Rhizoma Nelumbinis to be associated with in the magnetic fluorescent microspheres mixed liquor 10ul addition ELISA Plate micropore of seven kinds of trace albumin antibody, hatch 30 minutes in 37 DEG C of first times after concussion mixing, wash 3 times after standing 2-5 clock on Magneto separate plate after hatching end；
D. the phycoerythrin 20ul taking marked by streptavidin adds in ELISA Plate micropore, and mixing is placed on 37 DEG C of second time and hatches 20 minutes, washs 3 times after standing 2-5 clock after hatching end on Magneto separate plate；
E. add the buffer of 50ul, Luminex 200 instrument reads average fluorescent strength, records the content of seven kinds of urine micro protein.
The present invention utilizes Luminex 200 liquid microarrays technology platform feature the quickest, high-throughout, develop method and the technology of lot of trace albumen joint-detection in urine, prepare the magnetic fluorescent microspheres that the capture of MA, TRF, β 2-MG, α 2-MG, α 1-MG, Cystatin C and IgG 7 species specificity is antibody linked, after adding urine specimen, the determinand in sample forms competitive reaction with biotin labeled antigen.After reaction terminates, upper machine-readable it is averaged fluorescence intensity, passes through fluorescence intensity--the linear equation that dose curve is set up, obtain the measurement result of 7 species specificity urine micro protein simultaneously.7 kinds of urine micro protein of the disposable joint-detection of the method, greatly improve detection efficiency, reduce testing cost, have the unrivaled superiority of the methods such as immunoturbidimetry.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of microsphere coupled antibody of the present invention；
Fig. 2 is albumin standard canonical plotting；
Fig. 3 is transferrins standard substance canonical plotting；
Fig. 4 is IgG standard substance canonical plotting；
Fig. 5 is cystatin c standard substance canonical plotting；
Fig. 6 is alpha2-macroglobulin standard substance canonical plotting；
Fig. 7 is B2M standard substance canonical plotting；
Fig. 8 is α 1-microglobulin standard substance canonical plotting.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words.Additionally, any method similar or impartial to described content and material all can be applicable in the present invention.Preferable implementation described in literary composition only presents a demonstration with material and is used.
Reagent source is described as follows:
1, Sulfo-NHS: Chinese: N-hydroxy thiosuccinimide, English full name: N-Hydroxysul fosuccinimide sodium salt, purchased from Thermo SCIENTIFIC company
2, EDC: Chinese: 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, English full name: N-Hydroxysulfosuccinimide sodium salt, purchased from Thermo SCIENTIFIC company
3, the buffer related in embodiment, unless otherwise noted, is the buffer formulation in test kit of the present invention.
Embodiment 1 is by the interference between cross reaction 7 kinds of albumen of checking
1, in order to verify these albumen, purpose: 7 kinds of albumen are in MA, TRF, β 2-MG, α 2-MG, α 1-MG, Cystatin C, IgG, detects whether that existence interferes, has carried out cross reaction experiment.
2, scheme: whether with antibody coupling microsphere and corresponding biotin protein, testing it can be with other albumino reaction.Its MFI value being compared with blank MFI value, if thinking there is cross reaction with ratio less than 90%, otherwise the most there is not cross reaction.
3, method:
1) preparation Rhizoma Nelumbinis is associated with the magnetic fluorescent microspheres mixed liquor of antibody: Rhizoma Nelumbinis is associated with the magnetic fluorescent microspheres of 7 kinds of different antibodies, joins in buffer, make the microsphere concentration of various antibody be 100/ul after dilution；
2) preparation biotinylated protein antigen mixed liquor: be diluted with buffer, each proteantigen diluted concentration is MA (1:5000), TRF (1:2000), IgG (1:50000), Cys-C (1:40000) and α 1-MG (1:40000), α 2-MG (1:20000) and β 2-MG (1:60000)；
3) taking 7 kinds of proteantigen buffer compound concentrations respectively is the protein solution of 100ug/ml；
4) at microwell plate, above-mentioned albumen sample is added separately in different micropore, sample-adding amount is 10ul, biotinylated protein antigen mixed liquor 10ul is added again in all micropores, magnetic fluorescent microspheres mixed liquor 10ul is added after concussion mixing, mixing, put 37 DEG C to hatch 30 minutes, wash 3 times after standing 2-5 clock on Magneto separate plate after hatching end；
5) with buffer dilution Streptavidin phycoerythrin (SAPE), concentration is 2ug/ml；
6) in micropore, SAPE 20ul is added, concussion mixing, hatch 20 minutes for 37 DEG C, after standing 2-5 clock on Magneto separate plate after hatching end, wash plate 3 times.Add buffer 50ul, reading on Luminex 200 instrument.Record MFI value and be shown in Table 1；The MFI value of table 1 being compared with blank MFI value, result of calculation is shown in Table 2.
Table 1
Table 2
4, conclusion: 7 kinds of albumen have been carried out hybrid detection, result proves that it does not exist cross reaction, does not therefore have the situation interfered in actual joint inspection.
The screening of antibody type
Antibody type screening technique:
1, as described in table 1, table 2, should there is not cross reaction in selected antibody, is specificity, effectiveness to the target antigen in hybrid antigen complex.
2, between the antibody of different company, in the standard substance of antigen gradient dilution, antibody titer the superior is the standard selected, i.e. the MFI value in Luminex platform final detection result is maximum.
According to both the above principle, the selection result of antibody is as shown in table 3:
Table 3
The optimization of embodiment 2 antibody consumption
1, purpose: in 7 kinds of albumen, the antibody corresponding to every kind of albumen, carry out the optimal screening of antibody consumption.
2, scheme: the coupling method provided according to Luminex company, the workbook provided with reference to Luminex company, entitled xMAP Cookbook A collection of methods and protocols for developing multiplex assays with xMAP Technology.
(http://cdn2.hubspot.net/hubfs/128032/Cookbook/BR574.0915.xMAP.Cookboo k.3rdEdition.WR.pdf？T=1458937217219, page15-19), antagonist consumption factor carries out process optimization.
3, method: the coupling method provided according to Luminex company, during experimental antibodies amount, 2.5 × 106Being separately added into antibody 5ug, 10ug, 15ug, 20ug, 25ug in magnetic bead, other process conditions are consistent, compare its MFI value of test.
As a example by protein I gG, concrete technology condition and result are as shown in table 4:
Table 4
4, conclusion: the amount of antibody MFI value when 2-10ug does not has notable difference, i.e. antibody usage amount 2-10ug/106Magnetic bead is inconspicuous on experimental result impact.
The optimization of embodiment 3 microsphere priming reaction system
1, purpose: in 7 kinds of albumen, the antibody that every kind of albumen is corresponding, carry out the optimal screening of microsphere priming reaction system volume.
2, scheme: the coupling method provided according to Luminex company, is optimized microsphere priming reaction volume.
3, method: the coupling method provided according to Luminex company, during test experiments reaction volume, other process conditions are consistent, the only reaction volume when activating microsphere is set to 250ul, 500ul, 750ul, 1000ul, 1250ul, compare final detection result MFI value.
As a example by protein I gG, concrete technology condition and result are as shown in table 5:
Table 5
4, conclusion: be 2.5 × 10 in microsphere quantity6Time, when activation volume is 250 microlitres or 500 microlitre, result is optimal.
The process optimization of reaction system during embodiment 4 detection
1, scheme: test kit uses competitive mechanism to be designed, the experiment of reaction system to incubation temperature, incubation time, sample-adding amount, hatch four aspects of mode and test, the reaction system optimal to obtain condition.
2, method: use above-mentioned Urinary microprotein detection kit, add the urine specimen of sample size in ELISA Plate micropore, be subsequently added into the biotinylated protein of 10ul, adds Rhizoma Nelumbinis and is associated with the magnetic fluorescent microspheres of antibody after mixing, carry out hatching for the first time.After hatching end for the first time, wash 3 times after Magneto separate plate stands 2-5 clock, add the Streptavidin phycoerythrin SAPE of 20ul, carry out second time after mixing and hatch；Hatch and wash 3 times after standing 2-5 clock on Magneto separate plate after end, add the buffer of 50ul, upper machine-readable be averaged fluorescence intensity, record the content of 7 kinds of urine micro protein.
1) incubation temperature (controlling twice incubation temperature identical): take 7 example urine specimens, contrast room temperature and 37 DEG C of two reaction conditions, control identical incubation time (60 minutes for the first time and for the second time 20 minutes), on Luminex platform, detect that final MFI value is as shown in table 6:
Table 6
Note: A represents under 37 DEG C of incubation conditions, Luminex detection of platform sample gained MFI value, and B represents under room temperature incubation conditions, and Luminex detection of platform sample gained MFI value, A/B-1 institute value is under two kinds of incubation conditions, the diversity factor of MFI value.
Conclusion: under conditions of incubation temperature is respectively room temperature and 37 DEG C, MFI value does not has notable difference, it is contemplated that the incubation conditions of 37 DEG C is the most stable, and subsequent experimental all uses 37 DEG C of incubation conditions.
2) incubation time and the experiment of sample-adding amount: for 1 example urine specimen, contrast difference incubation time 30min+10min, 30min+20min, 40min+10min, 40min+20min, 50min+10min；And compare sample-adding amount be 10ul and 20ul sample-adding (each sample-adding amount parallel laboratory test 2 times)；Control identical incubation temperature 37 DEG C, on Luminex platform, detect that final MFI value is as shown in table 7:
Table 7
Conclusion: from the point of view of the MFI value of table 7, the MFI value of incubation time 30min+20min the most just reaches maximum, and therefore, choice experiment reaction pattern is 30min+20min；For sample-adding amount, the MFI value of 20ul sample-adding is significantly greater than the MFI value of 10ul sample-adding, and in order to expand the detection range of linearity, sample-adding amount selects 10 microlitre sample-addings.
3) mode is hatched: take 7 example urine specimens, according to above-mentioned experimental technique, shake the need of to Sptting plate before the reaction, carry out experiment to compare, incubation time is first time 30min+ second time 20min, and incubation temperature 37 DEG C, on Luminex platform, final detection result, MFI value is as shown in table 8:
Table 8
Conclusion: experimental data shows, whether shaking the MFI value on sample during hatching has significantly impact, but by MFI value matched curve inverse concentration of specimens, then both do not have notable difference, therefore, for the simplification in view of experimental implementation, take do not shake to hatch mode.
The preparation of embodiment 5 Urinary microprotein detection kit
1, preparation buffer:
Buffer is made up of sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, casein and Proclin-300.Described sodium chloride concentration is 8.0g/L, and disodium hydrogen phosphate concentration is 2.9g/L, and phosphate dihydrogen sodium concentration is 0.296g/L, casein concentration be 40.0g/L, Proclin-300 percent by volume be 0.02ml/L, Triton X-100 percent by volume be 0.2ml/L.Buffer is prepared according to formula purified water,
2, Rhizoma Nelumbinis is associated with the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody
With albumin antibody coupling 2.5 × 106As a example by individual fluorescent microsphere:
(microsphere concentration is 12.5 × 10 to take the magnetic fluorescent microspheres solution of 200ul6Individual/ml), join the centrifuge tube of 2ml, centrifuge tube is put on magnetic separator 1-2 minute, suck supernatant with pipet, add the MEST buffer (the MES buffer of the Tween20 containing 0.05%, pH=5.0) of 1.0ml, the vortex 10-20 second, the ultrasonic 10-20 second.It is put into again on magnetic separator 1-2 minute, supernatant is sucked with pipet, repeated washing 3 times, with the MES buffer dispersed magnetic fluorescent microsphere again of 480ul, adds the EDC solution 10ul of Sulfo-NHS solution 10ul and 40mM of 50mM, activate 20 minutes, it is put on magnetic separator 1-2 minute, sucks supernatant with pipet, add the MES buffer of 1.0ml, the vortex 10-20 second, the ultrasonic 10-20 second.It is put into again on magnetic separator 1-2 minute, sucks supernatant with pipet；With the MES buffer dispersed magnetic fluorescent microsphere again of 480ul, add albumin antibody 20ul (antibody concentration is 2mg/ml), after room temperature Rhizoma Nelumbinis joins 2 hours, centrifuge tube is put on magnetic separator 1-2 minute, supernatant is sucked with pipet, add the confining liquid (MES solution containing 1%BSA) of 1.0ml, put 2-8 DEG C of refrigerator and close overnight.After closing terminates, wash 1 time with 1%BSA solution, add Proclin 300,2-8 DEG C of preservation.
Remaining 6 kinds of antibody carries out coupling in the same manner, it is associated with the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody with buffer configuration Rhizoma Nelumbinis after preparation, specifically comprise the following steps that the microsphere of various coupled antibody, equal-volume mixes, and in final mixed liquor, the concentration of every kind of microsphere is 100 microspheres of every microlitre.
3, biotin labeled seven kinds of trace albumin antigen mixed liquors
As a example by with albumin antigen labelling:
Mix for 1:50 in molar ratio with biotin succinimide ester after albumin antigen is dialysed, incubated at room 30 minutes.The albumin antigen of labelling biotin being loaded bag filter, fully dialyses by PBS solution, to remove free biotin, biotin labeling albumin antigen adds 50% glycerol ,-20 DEG C of preservations.
Remaining 6 kinds of antigen carries out biotin succinimide ester labelling in the same manner, biotin labeled seven kinds of trace albumin antigen mixed liquors are configured with buffer after preparation, specifically comprise the following steps that according to the antigen diluent ratio indicated in table 9, set final mixed liquor cumulative volume, carry out the dilution of each antigen.
Table 9 each biotin labeling antigen diluent concentration
| Biotin labeling antigen | Dilution ratio | Biotin labeling antigen | Dilution ratio |
| MA: | 1:5000 | α 1-MG: | 1:40000 |
| TRF: | 1:2000 | α 2-MG: | 1:20000 |
| IgG: | 1:50000 | β 2-MG: | 1:60000 |
| Cystatin C: | 1:40000 |
4, the mixed liquor calibration object S1-S7 of seven kinds of trace albumin
Albumen is added in buffer, being configured to concentration is: MA (60mg/L), TRF (50mg/L), β 2-MG (2.5mg/L), α 2-MG (90mg/L), α 1-MG (70mg/L), Cystatin C (3mg/L) and the mixed liquid of protein of IgG (100mg/L), as calibration object S1, becoming 6 concentration with this buffer in 1:2 ratio gradient dilution again, S7 is buffer.In 6 calibration objects, each protein concentration is as shown in table 10:
Table 10 antigen standard concentrations control table (unit: mg/L)
| Project | S1 | S2 | S3 | S4 | S5 | S6 |
| Albumin | 60 | 20.000 | 6.667 | 2.222 | 0.741 | 0.247 |
| Transferrins | 50 | 16.667 | 5.556 | 1.852 | 0.617 | 0.206 |
| IgG | 100 | 33.333 | 11.111 | 3.704 | 1.235 | 0.412 |
| Bladder chalone C | 3 | 1.000 | 0.333 | 0.111 | 0.037 | 0.012 |
| Alpha2-macroglobulin | 90 | 30.000 | 10.000 | 3.333 | 1.111 | 0.370 |
| B2M | 2.5 | 0.833 | 0.278 | 0.093 | 0.031 | 0.010 |
| α 1-microglobulin | 70 | 23.333 | 7.778 | 2.593 | 0.864 | 0.288 |
The equation of 7 kinds of standard substance standard curves is as follows, and standard curve is shown in Fig. 2-8.
Albumin: y=-1.1011x+0.1731, R2=0.9908
Transferrins: y=-0.8965x-0.4437, R2=0.9909
IgG:y=-1.211x+0.5533, R2=0.9813
Cystatin c:y=-1.3192x-1.0848, R2=0.997
Alpha2-macroglobulin: y=-0.8205x+0.3195, R2=0.9989
B2M: y=-1.0953x-0.4606, R2=0.9952
α 1-microglobulin: y=-0.7219x+0.2032, R2=0.9934
In note: Fig. 2 to Fig. 8, abscissa be the calibration object of detection target protein after gradient dilution successively, the concentration value of each concentration dilution point is taken the logarithm value, i.e. LogCi, and Ci is each albumen each dilution point concentration value.Vertical coordinate isWherein Ai is the every kind of target protein calibration object each gradient dilution point reading MFI value at Luminex platform, and A0 is the blank product reading MFI value at Luminex platform of corresponding each gradient calibration object.
5, the phycoerythrin SAPE of marked by streptavidin
SAPE buys from Lifetechnologies company, article No.: S866, SAPE buffer is diluted to 2ug/ml, is working concentration.
Embodiment 6 uses Urinary microprotein detection kit to detect urine specimen
The test kit using embodiment 5 preparation carries out the detection of Urinary microprotein:
(1) before using, first test kit is taken out, place and balance at room temperature to room temperature；
(2) in microwell plate, add 10ul sample to be tested, take each 10ul of calibration object S1-S7 as standard reference simultaneously；Sample to be tested is to collect from Changhai hospital on October 20th, 2015, redeterminates 7 testing results through Xinhua Hospital；
(3) add the biotin labeled seven kinds of trace albumin antigen mixed liquors of 10ul, shake mix homogeneously；
(4) Rhizoma Nelumbinis adding 10ul is associated with the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody, and concussion is placed on 37 DEG C and hatches 30 minutes；
(5), after hatching end, microwell plate is placed in Magneto separate and washes washing 3 times on trigger；
(6) adding the concentration 2mg/L Streptavidin phycoerythrin SAPE solution of 20ul, mixing is placed on 37 DEG C and hatches 20 minutes；
(7), after hatching end, microwell plate is placed in Magneto separate and washes washing 3 times on trigger；
(8) add buffer 50ul, on Luminex 200 instrument, read average fluorescent strength after concussion mixing, record the content of 7 kinds of urine micro protein.
The data recorded through above-mentioned detection method are compared with Clinical detection result, and result is as shown in table 11:
Testing result in table in comparison, the detection method of the present invention is as follows compared to the superiority of traditional detection mode, detection technique and method:
1, can disposable 7 kinds of urine micro protein of joint-detection, greatly improve detection efficiency, reduce testing cost.
2, pattern detection is highly sensitive, has the unrivaled superiority of the methods such as immunoturbidimetry.
Below preferred embodiment to the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make modification or the replacement of all equivalents on the premise of the invention spirit, and modification or the replacement of these equivalents are all contained in the application claim limited range.
Claims (12)
1. seven kinds of trace albumin combined detection kits of urine, including:
(1) Rhizoma Nelumbinis is associated with the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody；
(2) by biotin labeled seven kinds of trace albumin antigen mixed liquors；
(3) by the phycoerythrin of marked by streptavidin；
(4) calibration object, at least includes seven kinds of trace albumin mixed liquors；
(5) buffer；
Seven kinds of described trace albumin be MA, TRF, β 2-MG, α 2-MG, α 1-MG, Cystatin C,
IgG；
Described buffer by sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, bovine serum albumin and
Proclin-300 forms；Wherein, the concentration of sodium chloride is 6.0-10.0g/L, and the concentration of disodium hydrogen phosphate is
2.5-3.5g/L, the concentration of sodium dihydrogen phosphate is 0.2-0.4g/L, and the concentration of bovine serum albumin is
The concentration of 8.0-12.0g/L, Proclin-300 is that the concentration of 0.01-0.03ml/L, Triton X-100 is
0.1-0.3ml/L。
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature exists
In: described magnetic fluorescent microspheres, during its Rhizoma Nelumbinis connection, antibody concentration is: every 106Individual magnetic fluorescent microspheres, often
Planting antibody consumption is 2-10ug.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1 and 2, it is special
Levy and be: described magnetic fluorescent microspheres, prepare by the following method: by magnetic fluorescent microspheres by MES,
Activation processing 15-20 minute in the activation system of NHS and EDC composition, adds magnetic field, standing separation 5 points
Clock, adds antibody 2-10ug/10 after washing 3 times with MES6Individual fluorescent microsphere, after room temperature Rhizoma Nelumbinis joins 2 hours,
Wash 1 time with MES and remove free antibody, add 1%BSA solution, put 2-8 DEG C of refrigerator and closed
At night, close after terminating, wash 1 time with 1%BSA solution, add Proclin 300,2-8 DEG C of preservation.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 3, its feature exists
In: in described activation system, when microsphere quantity is 2.5 × 106Time, the volume of MES is 230ul, EDC
It is 10ul for 10ul, NHS；Or when microsphere quantity is 2.5 × 106Time, the volume of MES is 480ul,
EDC be 10ul, NHS be 10ul.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature exists
In: described Rhizoma Nelumbinis is associated with in the magnetic fluorescent microspheres mixed liquor of seven kinds of trace albumin antibody, various antibody microspheres
Concentration be 100/ul.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature exists
In: in described biotin labeled seven kinds of trace albumin antigen mixed liquors, every kind of proteantigen and biotin
Mol ratio be 1:20-100.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 6, its feature exists
In: described biotin is succinimide ester, and the mol ratio of every kind of proteantigen and biotin is 1:50.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature exists
In: in described biotin labeled seven kinds of trace albumin antigen mixed liquors, the concentration of various proteantigens is such as
Under:
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature exists
In: the concentration of the phycoerythrin of described marked by streptavidin is 2mg/L.
A kind of seven kinds of trace albumin combined detection kits of urine the most according to claim 1, its feature
It is: described calibration object, including:
Seven kinds of trace albumin mixed liquor S1, wherein the concentration of seven kinds of trace albumin is respectively as follows: 60mg/L's
The α 1-MG of α 2-MG, 70mg/L of β 2-MG, 90mg/L of TRF, 2.5mg/L of MA, 50mg/L,
The IgG of the Cystatin C, 100mg/L of 3mg/L；
Seven kinds of trace albumin mixed liquor S2-S6, by described mixed liquor S1 according to 1:2 or 1:3 gradient dilution after
Series of calibration product；And,
Buffer S7, as blank reference.
11. a kind of seven kinds of trace albumin combined detection kits of urine according to claim 1, its feature
Being: in described buffer, the concentration of sodium chloride is 8.0g/L, and the concentration of disodium hydrogen phosphate is 2.9g/L,
The concentration of sodium dihydrogen phosphate is 0.296g/L, and the concentration of bovine serum albumin is 10.0g/L, Proclin-300
The concentration that concentration is 0.02ml/L, Triton X-100 be 0.2ml/L.
12. sides using the test kit seven kinds of trace albumin of joint-detection urine described in any one of claim 1-11
Method, including:
A. in ELISA Plate micropore, add 10ul urine specimen, take each 10ul of calibration object as standard reference simultaneously,
Wherein buffer is as blank reference；
B. taking biotin labeled seven kinds of trace albumin antigen mixed liquor 10ul and add in ELISA Plate micropore, mixing is all
Even；
C. take Rhizoma Nelumbinis and be associated with the magnetic fluorescent microspheres mixed liquor 10ul addition ELISA Plate micropore of seven kinds of trace albumin antibody
In, hatch 30 minutes in 37 DEG C of first times after concussion mixing, stand on Magneto separate plate after hatching end
Wash 3 times after 2-5 clock；
D. the phycoerythrin 20ul taking marked by streptavidin adds in ELISA Plate micropore, and mixing is placed on 37 DEG C the
Secondary is hatched 20 minutes, washs 3 time after standing 2-5 clock after hatching end on Magneto separate plate；
E. add the buffer of 50ul, Luminex 200 instrument reads average fluorescent strength, records seven kinds of urine
The content of trace albumin.
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| CN107328933A (en) * | 2017-09-06 | 2017-11-07 | 深圳蓝韵生物技术有限公司 | Magnetic particle enhancing immunoturbidimetry detection box and its application |
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| CN109001463A (en) * | 2018-07-09 | 2018-12-14 | 广州华澳生物科技有限公司 | A kind of gastric cancer risk indicator Internet of Things close quantitative testing test paper and preparation method |
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