CN106645745A - Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof - Google Patents
Homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and preparation and detection method thereof Download PDFInfo
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Abstract
The invention discloses a homogenous-phase fluorescent immune reagent for rapidly and quantitatively detecting trace albumin, and a preparation and detection method thereof. The reagent consists of anti-MAU marked by rare-earth element chelate and another anti-MAU marked by a near-infrared fluorescent compound, and a series of concentration MAU calibration products. By adopting the homogenous-phase fluorescent immune rapid detection technology, and utilizing the high-sensitivity characteristic of the fluorescence, the adverse influence of the non-homogeneity of pores of a nitrocellulose membrane in a colloidal gold or fluorescent MAU dry-type immune test paper on the detection accuracy and repetition can also be avoided, and the detection sensitivity and the linear range can be greatly improved; and the reagent provided by the invention is used for detecting trace albumin in human blood, serum or plasma, has the characteristics of simplicity and rapidness in operation, sensitivity, good specificity and the like, and has good clinical application prospect.
Description
Technical field
The invention belongs to field of medical examination, and in particular to a kind of homogeneous fluorescent of Quantitative detection microalbumin is exempted from
Epidemic disease reagent and preparation and detection method.
Background technology
Serum albumin moiety can be passed through by glomerulus, but almost all by reabsorption, when Glomerular lesions its
Filtrable volume increases, so that discharging from urine more than the reabsorption of renal tubule, just forms microdose urine protein (MAU).Urine is micro
Albumin refer in urine albuminous excretion rate it is per minute more than 20mg or 24h more than 30mg, concentration is 30-200mg/L, with often
Rule method cannot be detected.Numerous studies be proved the microdose urine protein positive be injury of kidney especially glomerular injury early stage mark
Will, its discharge rate number with GBM damage degree be proportionate, with type 1 diabetes, the prognosis of diabetes B
There is substantial connection.Numerous studies in recent years show that MAU is also simultaneously that hypertension, coronary heart disease, autoimmune disease etc. are caused
The early diagnosis index of injury of kidney, is clinically widely paid attention to by increasingly.
Diabetes (DM) illness rate increases year by year, and falling ill early stage, many concealments are asymptomatic, and late period jeopardizes because of severe complication
The health and lives of people, therefore, its early detection, diagnose and treat most important.Chemistry is respectively used to diabetic's urine
Test paper method carries out protein urine and Immunity transmission turbidity and carries out microdose urine protein quantitative determination, as a result finds, Urine proteins are fixed
Property test positive rate be only 8.46%, and the positive rate of microdose urine protein be 53.7%, illustrate that the detection of MAU more can be early
Ground prompting diabetic keratopathy injury of kidney.
In recent years, the research about essential hypertension is more and more deep, the target organ damage that particularly hypertension is caused
The lethal principal element for disabling of the elderly is become, it is proved that hypertension is also to increase the key factor of renal function exacerbation
One of, 25% ESRD is relevant with hypertension.The parteriole pathology that essential hypertension causes often involves Renal vascular, makes
Glomerular filtration membrane damage, causes the discharge rate for urinating MAU to increase.Hyperpietic is more universal with MAU, and with the age,
The order of severity of the course of disease and hypertension and raise.Have been reported that in High-normal blood pressure crowd and Hypertensive Population, the incidence of MAU
Respectively 15.O% and 26.2%, hence it is evident that higher than the 6.5% of normotensive subjects.Therefore, to the perspective study of hypertensive patient
Middle MAU is considered as the preferable early sign of a predicting cardiovascular M ＆ M.
Some researchs show that microalbuminuria can evaluate endothelial dysfunction and atherosclerotic as one
Reliability index, the danger that its appearance prompting occurs coronary heart disease will substantially increase.Determine ND patients with coronary heart disease urine
In from the content of albumen, find its MAU level apparently higher than control group patient, and with coronary artery pathological changes quantity and degree
Increase, the equal progressive of the positive rate of MAU raises, and points out MAU and atherosclerotic generation, development and lesion degree relation
Closely, MAU is probably the mark of coronary atherosclerosis.
Therefore, the detection of MAU not only has an important value to the diagnosis of kidney trouble, and with Non-renal disease such as glycosuria
Disease, angiocardiopathy etc. have close contact, therefore, using MAU as a conventional detection, the early stage of these diseases is examined
Disconnected, PD, prognosis and clinical guidance medication etc. have important clinical meaning.
Clinical labororatory frequently with radioimmunoassay (RIA), immunoturbidimetry (IT), FIA (FIA),
Enzyme immunoassay (EIA) (EIA), time-resolved fluorescent immunoassay (TRFIA), indirect latex agglutination test, Micral-Test and
Bromophenol blue (BPB) dye binding method determines urine MAU, and the reference range of these methods changes very big, from 5mg/L to exceeding
200mg/L.As people are for low-level MAU and the understanding of angiocardiopathy relation, the sensitivity of these assay methods is much
Requirement can not be met.The early diagnosis of disease and in time treatment it is critical that.But current detection method, or needing high
Expensive instrument and equipment and reagent, or simply semiquantitative manual method, working specification is poor.Therefore, when setting up a kind of detection
Between shorten as far as possible, and detect except can also be required to carry out bed side to detect laboratory is carried out in addition to, while can quantitative determine
MAU methods, are very necessary so as to provide accurate diagnosis basis for clinic.
Homogeneous fluorescent analytic approach (homogeneous fluoroimmunoassay, HFIA) is exempted from time-resolved fluorescence
A kind of new fluorescence formed on the basis of epidemic disease analysis (time-resolued fluoroimmunoassay, TRFIA) technology is exempted from
Epidemic disease analytical technology.The fluorescent material that TRFIA technologies are adopted is entirely different with traditional fluorescent dye, uses lanthanide series europium
(Eu), used as fluorescent material, sensitivity is very high, good stability for technetium (Tb) etc., and cryogenic conditions can be preserved 3 years, thus becomes two
The most popular immuno analytical method of eleventh century.
Homogeneous fluorescent immunodetection is to mark Eu respectively with two antibody of same antigen3+And fluorescent dye
Alexa647。Eu3+Labelled antibody is excited in free state by 340nm light, and only transmitting mean wavelength is 615nm fluorescence,
And when antigen, antibody complex are formed, there is energy transmission, excite fluorescent dye Alexa647 to launch 665nm fluorescence.Mark
Note antibody directly carries out antigen, antibody response with testing sample, if antigen, antibody complex can be formed, going out in 665nm can
Measure fluorescence signal.This method eliminates ELISA and is incubated repeatedly and the tedious operations step such as board-washing, and a few minutes are with regard to energy
Result is obtained, it is time saving and energy saving.Also, this method also accordingly avoid many manual operation factors and reagent, environment etc. it is extraneous because
Element interference, stability and repeatability it is all preferable, can more truly reflect the content of measured matter.Additionally, Eu3+And Alexa647
This differs larger between emission maximum optical wavelength to fluorescent material, and the background fluorescence value of antigen-antibody reaction does not occur just very
It is low.And 300～500nm fluorescence that non-specific material is produced in human serum, it is impossible to excite Alexa647 to launch fluorescence signal
650nm exciting lights.Therefore, non-specific fluorescence is very low.
Chinese patent document CN103115904A (application number：201310027507.1) microdose urine protein detection is disclosed
Method, system and kit.Including：(1) the different multiple solution samples of microdose urine protein concentration are prepared, wherein described in each
Cyanine dyes containing same concentrations in solution sample；(2) fluorescence intensity level at detection collection wavelength；(3) urine is obtained micro white
The calibration curve of protein concentration；(4) add cyanine dyes, the buffering containing potassium ion or sodium ion molten in liquid sample
Liquid, so that the concentration and pH value of the cyanine dyes in liquid sample are consistent with the solution sample in step (1), so as to obtain
Test solution, and record the ratio that liquid sample is diluted；(5) fluorescence intensity level at the collection wavelength is detected；(6)
The microdose urine protein concentration value of corresponding test solution is found in calibration curve, the urine Microalbunin of testing sample is calculated
White concentration；Scope of the wherein described collection wavelength in 550nm to 650nm.The method is to change micro- to determine according to shade
Amount albumin content, detection is vulnerable to the impact of some medicines and its metabolism product in urine, and specificity is poor.
Chinese patent document CN105301257A (application number：201510227240.X) disclose a kind of microdose urine protein
(mAlb) detection method, the detection method is based on latex enhancing immune turbidimetry, is liquid double reagent, including reagent R1 with
R2, the reagent R1 are reactant, and reagent R2 is the solution containing albumin immunity latex particle, and its feature is applied chemistry
Method is crosslinked, and by water-soluble carbodiimide (EDC) and N-hydroxy-succinamide (NHS) sheep anti-human albumin antibodies are made
(also referred to as goat-anti people mAlb antibody) and Carboxylated Polystyrene latex covalent cross-linking, forms mAlb antibody latex reagents.The method is received
The impact of the homogeneity of latex particle, differences between batches are larger.
The content of the invention
For the deficiency of existing MAU detection techniques, the present invention provides a kind of homogeneous fluorescent immunoreagent of quick detection MAU
And prepare and detection method.The present invention designs new former material according to immunofluorescence technique feature and MAU antigen-antibody system features
Material, reagent and technological process, the reagent detection MAU levels provided using the present invention, with simple, quickly, sensitive and specificity
Good the features such as, can simultaneous quantitative detection high level and low value sample, and cost performance is high, it is adaptable to clinical quick detection.
Technical scheme is as follows：
A kind of homogeneous fluorescent immunoreagent of Quantitative detection microalbumin, including rare earth chelate compound mark
The MAU calibration objects of anti-MAU, another anti-MAU of near infrared fluorescent compound mark and series concentration.
, according to the invention it is preferred to, described rare earth chelate compound is europium ion (Eu3+) chelate, further preferably
BHHCT-Eu3+、BHHBCB-Eu3+.The anti-MAU independent packagings of rare earth chelate compound mark.
, according to the invention it is preferred to, described near infrared fluorescent compound is AlexaOr
Series dyes, the dyestuff of further preferred Alexa Fluor 647, DyLight-DY647 or CF647.Near-infrared fluorescent chemical combination
Another anti-MAU independent packaging of substance markers.
, according to the invention it is preferred to, described MAU calibration objects are obtained with phosphate buffer dilution dissolving MAU sterlings；
It is further preferred that the concentration of phosphate buffer be 0.01mol/L, in phosphate buffer containing 0.01%-0.5%PEG,
1%-5%BSA, 0.01%-0.05% surfactant, is mass percentage content；Preferably, described surfactant
For polysorbas20.The MAU calibration object independent packagings of series concentration, in 4 DEG C of preservations.
, according to the invention it is preferred to, the concentration of described MAU calibration objects be respectively 5mg/L, 25mg/L, 50mg/L,
100mg/L、250mg/L。
According to the present invention, the preparation method of the anti-MAU of described rare earth chelate compound mark, including step is as follows：
After by the anti-human MAU solution dialysis of mouse, NaHCO is added3, adjust pH to 9.1, obtain antibody-solutions；By BHHCT or BHHBCB
Methanol solution be added drop-wise in antibody-solutions, stirring reaction, after being centrifuged off insoluble matter, chromatographic column separation marking protein and trip
From label；Ultraviolet/visible spectrophotometer detects the A of each collection liquid330Value, merges the solution containing labelled antibody；Add
Final mass percent concentration is the NaN of 0.1% BSA and 0.05%3, adjust pH to 6.2；Add EuC13Solution, obtains final product rare earth
The anti-MAU of element chelate labels.
According to the present invention, the preparation method of another anti-MAU that described near infrared fluorescent compound is marked, including
Step is as follows：
By anti-MAU monoclonal antibodies, diluted with sodium bicarbonate solution, add near infrared fluorescent compound lysate, stirred
It is even, incubation at room temperature；Column separating purification is crossed, the fluorescent compound label antibody for having marked is collected, it is mixed with phosphate buffer dilution
It is even, obtain final product another anti-MAU of near infrared fluorescent compound mark.
According to the present invention, the preparation method of the MAU calibration objects of described series concentration, including step is as follows：
With being 0.01%-0.5%PEG1000,1%-5%BSA, 0.01%-0.05% surface containing mass percent concentration
The phosphate buffer of activating agent, according to the concentration dilution of 5mg/L, 25mg/L, 50mg/L, 100mg/L, 250mg/L MAU is dissolved
Sterling, is well mixed, and obtains final product the MAU calibration objects of series concentration.
According to the present invention, using the method for above-mentioned homogeneous fluorescent immunoreagent Quantitative detection microalbumin, including
Step is as follows：
First rare earth chelate compound mark anti-MAU solution is added in reaction micropore, near-infrared fluorescent chemical combination is added
Another anti-MAU solution of substance markers, is eventually adding MAU calibration objects, after 37 DEG C are reacted 20 minutes, with homogeneous fluorescent immunity
Analyzer fluorescence intensity, according to relative intensity of fluorescence data, makes the calibration curve of MAU concentration-fluorescence intensity；
Clinical detection sample is replaced into MAU calibration objects, repeats said process, according to relative intensity of fluorescence data correspondence standard
Curve, obtains microalbumin content.
The principle of the present invention：
The homogeneous fluorescent immunization of the Quantitative detection MAU that the present invention is provided, its reaction principle is double antibody sandwich method
Homogeneous fluorescent immunization.The Eu of testing sample and proper proportion3+Fully mix in liquid phase homogeneous medium with FLA
With it is uniform, in the process the MAU in sample can in specific manner with Eu3+Anti- MAU antibody is marked fully to combine, also can be with fluorescence
Mark MAU antibody fully reacts, the Eu of formation3+The immunity of-anti-MAU1-MAU-anti-MAU2-Alexa Fluor 647 is multiple
Compound, fluorescence intensity can be quantitative determined with homogeneous fluorescent immunoassay instrument, and fluorescence intensity is directly proportional to MAU concentration in sample.
Beneficial effects of the present invention are as follows：
1st, the present invention, using the highly sensitive feature of fluorescence, is equally also avoided using homogeneous fluorescent immunity Fast Detection Technique
Nitrocellulose filter itself hole heterogeneity characteristic in collaurum or fluorescence MAU dry type immune test papers is to accuracy in detection
With the harmful effect of repeatability.Because sample and FLA overall process are all complete in the liquid phase in homogeneous fluorescent immune detection
Face contacts, and reaction is abundant, therefore can increase substantially detection sensitivity and the range of linearity, and LDL is up to 0.5mg/L, line
Property scope be 5-250mg/L.Simultaneous reactions carry out also increasing the extension rate of sample in liquid phase, eliminate the matrix effect of sample
Should affect, make quantitative result have good repeatability, improve the preci-sion and accuracy of quantitative result, clinic can be met and examined
The requirement of disconnected extensive detection.
2nd, MAU levels in the reagent detection human body that the application present invention is provided, it is with low cost, it is simple to operate, quick, sensitive,
And specificity is good, it is only necessary to supporting special homogeneous fluorescent immune detector, therefore medical inspection places at different levels are can be widely applied to,
Especially basic medical unit, can carry out including health clinics in towns and townships etc..
3rd, the present invention can simultaneously detect high level and low value MAU, especially low value MAU, and it occurs for cardiocerebrovasculaevents events
Prevention have particularly important meaning.
Description of the drawings
Fig. 1 is immune detection theory structure schematic diagram of the present invention.
Wherein, 1：Eu3+Mark anti-MAU；2：Alexa647 marks anti-MAU；3：In calibration object or sample to be tested
MAU；4：Eu3+- anti-MAU-MAU-anti-MAU-Alexa647 immune complexs.
Fig. 2 is the calibration curve of MAU concentration in test example of the present invention 1.
Fig. 3 is MAU correlation analysis curves in test example of the present invention 2.
Specific embodiment
With reference to specific embodiment, the invention will be further described, all according to done by the disclosure of invention
This area equivalent, belongs to protection scope of the present invention.
" % " is mass percent in embodiment.
Embodiment 1：
The preparation method of quantitative determination microalbumin (MAU) homogeneous fluorescent immunoreagent, comprises the steps：
1st, the mark preparation of anti-MAU：
From the anti-MAU monoclonal antibodies of the gene engineering expression of purifying.Eu3+Mark with anti-MAU monoclonal antibodies commodity
Numbering is 19C7；It is 16A11 and 560 that fluorescein is marked with anti-MAU monoclonal antibodies goods number.
2nd, rare earth chelate compound marks the preparation of anti-MAU：
The anti-human MAU monoclonal antibodies 19C7 solution (3mg/mL) of mouse is dialysed in 4 DEG C twice with 3L 0.9%NaCl, each 24hr.Plus
Water degree of thickening is to 1.5mg/mL.The 0.6mL antibody-solutions are taken, 1mL NaHCO are added3(0.2mol/L), and 1mol/L NaOH are used
Adjust pH to 9.1.20 μ l BHHCT methanol solutions (30 μ g/mL) are added drop-wise in the antibody-solutions under stirring, and it is anti-to continue stirring
Answer lhr.Centrifugation (10000rpm, 10min) is removed after insoluble matter, and upper SephadexG-50 posts use 0.05mol/LNH4HCO3(pH
=8.0) elute, separation marking protein and free label.Ultraviolet/visible spectrophotometer detects the A of each collection liquid330
Value, merges the solution containing labelled antibody.Add final concentration of 0.1% BSA and 0.05% NaN3, adjusted with 1mol/LHCl
PH to 6.2.- 20 DEG C store for future use after packing.Before immunoassay, EuC1 is added3Solution (BHHCT and Eu3+Equimolar is dense
Degree).During for immunoassay, used with label diluted, 2-8 DEG C of packing is preserved.
3rd, the preparation of Alexa647 labelled antibodies：
By anti-MAU monoclonal antibodies 16A11,560, be diluted to 1mg/mL with 0.1mol/L sodium bicarbonate solutions respectively, respectively
5mL antibody-solutions are taken, 30mg fluorescein Alexa647 lysates are separately added into, is stirred evenly, be incubated at room temperature 1 hour, every 15 minutes
Mix once.Finally column separating purification is crossed with G25 gel columns, collect the fluorescein labelled antibody for having marked, with containing 0.01%
PEG, 1%BSA, 5% glycerine, the 0.01mol/L phosphate buffers dilution of 0.01% surfactant, use plastic bottle sealed bundle
Dress, in 4 DEG C of preservations.
4th, the preparation of series concentration MAU calibration object：
With containing 0.08%PEG1000,4.5%BSA, 0.045% surfactant 0.01mol/L phosphate buffers,
Concentration dilution according to 5mg/L, 25mg/L, 50mg/L, 100mg/L, 250mg/L dissolves MAU sterlings, mixes after 4 DEG C of preservations.
Embodiment 2：
The preparation method of the present embodiment is substantially the same manner as Example 1, and difference is：
In step 2, the preparation method of rare earth chelate compound mark anti-MAU is：It is saturating in 4 DEG C with 3L 0.9%NaCl
Analyse the anti-human MAU solution (3mg/mL) of mouse twice, each 24hr.Degree of thickening add water to 1.5mg/mL.The 0.6mL antibody-solutions are taken,
Add 1mL NaHCO3(0.2mol/L), and with 1mol/L NaOH pH to 9.1 is adjusted.By 20 μ L BHHBCB methanol solutions (30 μ g/
ML) it is added drop-wise in the antibody-solutions under stirring, and continues stirring reaction lhr.Centrifugation (10000rpm, 10min) removes insoluble matter
Afterwards, upper SephadexG-25 posts, use 0.05mol/L NH4HCO3(pH=8.0) wash-out, separation marking protein and free mark
Note thing.Ultraviolet/visible spectrophotometer detects the A of each collection liquid330Value, merges the solution containing labelled antibody.Add final concentration
BSA and 0.05% NaN for 0.1%3, with 1mol/L HCl pH to 6.2 is adjusted.- 20 DEG C store for future use after packing.For immunity
Before analysis, EuC1 is added3Solution (BHHBCB and Eu3+Equimolar concentration).During for immunoassay, label diluted is used
Use, 2-8 DEG C of packing is preserved.
Embodiment 3：
The preparation method of the present embodiment is substantially the same manner as Example 1, and difference is：
In step 3, by anti-MAU monoclonal antibodies 16A11,560, be diluted to 0.1mol/L sodium bicarbonate solutions respectively
1mg/mL, respectively takes 5mL antibody-solutions, is separately added into 40mg fluorescein DyLight-DY647 lysates, stirs evenly, incubation at room temperature 1.5
Hour, mixed once every 15 minutes.Finally column separating purification is crossed with G25 gel columns, collect the fluorescein mark for having marked anti-
Body, with containing 0.05%PEG600,3.5%BSA, 10% glycerine, the 0.01mol/L phosphate buffers of 0.05% surfactant
Dilution, is packed with plastic bottle, in 4 DEG C of preservations.
Embodiment 4：
The preparation method of the present embodiment is substantially the same manner as Example 1, and difference is：
In step 3, by anti-MAU monoclonal antibodies 16A11,560, be diluted to 0.1mol/L sodium bicarbonate solutions respectively
1mg/mL, respectively takes 5mL antibody-solutions, is separately added into 50mg fluorescein CF647 lysates, stirs evenly, and is incubated at room temperature 2 hours, every
Mix once within 15 minutes.Finally cross column separating purification with G25 gel columns, collect the fluorescein labelled antibody for having marked, with containing
0.03%PEG, 5%BSA, 10% glycerine, the 0.01M phosphate buffers dilution of 0.05% surfactant, it is close with plastic bottle
Package is filled, in 4 DEG C of preservations.
Embodiment 5：
In clinical detection, experimental procedure is：
First the rare earth chelate compound mark anti-MAU solution of 50 μ L is added in reaction micropore, the near of 50 μ L is added
Another anti-MAU solution of infrared fluorescent compounds mark, is finally separately added into the MAU calibration objects of 50 μ L, 37 DEG C of reactions 20
After minute, with homogeneous fluorescent immunity analysis instrument fluorescence intensity, according to relative intensity of fluorescence data, MAU concentration-fluorescence is made
The calibration curve of intensity；
Clinical detection sample is replaced into MAU calibration objects, repeats said process, according to relative intensity of fluorescence data correspondence standard
Curve, obtains microalbumin content.Sentence read result is detected with homogeneous fluorescent immunity analysis instrument.
Test example 1：
With homogeneous fluorescent immunity analysis instrument fluorescence intensity, each concentration calibration product testing result is as follows：
| MAU concentration (mg/L) | 5 | 25 | 50 | 100 | 250 |
| Relative intensity of fluorescence | 418 | 876 | 1537 | 2892 | 6168 |
According to relative intensity of fluorescence data, the calibration curve of MAU is made, as shown in Fig. 2 the range of linearity is 5-250mg/L.
The calibration curve computing formula of MAU is Y=23.51X+355.7, R2=0.997.Series is carried out for 5mg/L calibration objects to concentration
Dilution detection, this method LDL is up to 0.5mg/L.
Test example 2：
Using the inventive method reagent, with homogeneous fluorescent immunity analysis instrument 43 clinical patients with coronary heart disease serum samples are detected
This, the synchronous electrochemical process MAU reagent using Roche companies of Switzerland carries out contrasting detection, correlation analysis is carried out, such as Fig. 3 institutes
Show.
From the figure 3, it may be seen that the present invention is consistent with commercialized product testing result, with clinical equivalent.
Claims (10)
1. a kind of homogeneous fluorescent immunoreagent of Quantitative detection microalbumin, it is characterised in that the reagent includes rare earth
The MAU of the anti-MAU, another anti-MAU of near infrared fluorescent compound mark and series concentration of element chelate labels
Calibration object.
2. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
Described rare earth chelate compound is europium ion (Eu3+) chelate.
3. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 2, it is characterised in that
Described europium ion (Eu3+) chelate be BHHCT-Eu3+、BHHBCB-Eu3+。
4. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
Described near infrared fluorescent compound is AlexaOrSeries dyes, preferred Alexa Fluor
647th, the dyestuff of DyLight-DY647 or CF647.
5. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
Described MAU calibration objects are obtained with phosphate buffer dilution dissolving MAU sterlings；
Preferably, the concentration of phosphate buffer be 0.01mol/L, in phosphate buffer containing 0.01%-0.5%PEG,
1%-5%BSA, 0.01%-0.05% surfactant, is mass percentage content.
6. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
The concentration of described MAU calibration objects is respectively 5mg/L, 25mg/L, 50mg/L, 100mg/L, 250mg/L.
7. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
The preparation method of the anti-MAU of described rare earth chelate compound mark, including step is as follows：
After by the anti-human MAU solution dialysis of mouse, NaHCO is added3, adjust pH to 9.1, obtain antibody-solutions；By the first of BHHCT or BHHBCB
Alcoholic solution is added drop-wise in antibody-solutions, stirring reaction, after being centrifuged off insoluble matter, chromatographic column separation marking protein and free
Label；Ultraviolet/visible spectrophotometer detects the A of each collection liquid330Value, merges the solution containing labelled antibody；Add final
Mass percent concentration is the NaN of 0.1% BSA and 0.05%3, adjust pH to 6.2；Add EuC13Solution, obtains final product rare earth element
The anti-MAU of chelate labels.
8. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
The preparation method of another anti-MAU of described near infrared fluorescent compound mark, including step is as follows：
By anti-MAU monoclonal antibodies, diluted with sodium bicarbonate solution, add near infrared fluorescent compound lysate, stirred evenly,
Incubation at room temperature；Column separating purification is crossed, the fluorescent compound label antibody for having marked is collected, is mixed with phosphate buffer dilution,
Obtain final product another anti-MAU of near infrared fluorescent compound mark.
9. the homogeneous fluorescent immunoreagent of Quantitative detection microalbumin according to claim 1, it is characterised in that
The preparation method of the MAU calibration objects of described series concentration, including step is as follows：
With being 0.01%-0.5%PEG1000,1%-5%BSA, 0.01%-0.05% surface-active containing mass percent concentration
The phosphate buffer of agent, the concentration dilution dissolving MAU according to 5mg/L, 25mg/L, 50mg/L, 100mg/L, 250mg/L is pure
Product, are well mixed, and obtain final product the MAU calibration objects of series concentration.
10. using the side of the homogeneous fluorescent immunoreagent Quantitative detection microalbumin described in any one of claim 1-9
Method, including step is as follows：
First rare earth chelate compound mark anti-MAU solution is added in reaction micropore, near infrared fluorescent compound mark is added
Another anti-MAU solution of note, is eventually adding MAU calibration objects, after 37 DEG C are reacted 20 minutes, uses homogeneous fluorescent immunoassay
Instrument fluorescence intensity, according to relative intensity of fluorescence data, makes the calibration curve of MAU concentration-fluorescence intensity；
Clinical detection sample is replaced into MAU calibration objects, repeats said process, it is bent according to relative intensity of fluorescence data correspondence standard
Line, obtains microalbumin content.
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